阅读说明：本技术 产生莨菪烷生物碱(ta)的非植物宿主细胞及其制备和使用方法 (Non-plant host cells that produce Tropane Alkaloids (TA) and methods of making and using the same ) 是由 克里斯蒂娜·D·斯默克 普拉桑特·斯里尼瓦桑 于 2020-03-06 设计创作，主要内容包括：本文尤其提供了一种工程化的非植物细胞,其产生莨菪烷生物碱产物、莨菪烷生物碱产物的前体,或莨菪烷生物碱产物的衍生物。还描述了一种用于利用细胞产生莨菪烷生物碱、莨菪烷生物碱产物的前体或莨菪烷生物碱产物的衍生物的方法。(Provided herein, inter alia, is an engineered non-plant cell that produces a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product. Also described is a method for producing a tropane alkaloid, a precursor of a tropane alkaloid product or a derivative of a tropane alkaloid product using a cell.)

1. An engineered non-plant cell that produces a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product, wherein said engineered non-plant cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes within a pathway for producing said precursor of a tropane alkaloid product, said tropane alkaloid product, or said derivative of a tropane alkaloid product;

Wherein the cell comprises one or more alterations to one or more endogenous metabolic pathways or regulatory mechanisms selected from the group consisting of: endogenous arginine metabolism, endogenous phenylalanine and phenylalanine metabolism, endogenous polyamine regulatory mechanisms and metabolism, endogenous acetate metabolism, and endogenous glycoside metabolism.

2. The cell of claim 1, wherein the cell comprises one or more alterations to one or more endogenous metabolic pathways or regulatory mechanisms selected from the group consisting of: endogenous arginine metabolism, endogenous phenylalanine and phenylalkane metabolism, endogenous polyamine regulatory mechanisms and metabolism, and endogenous acetate metabolism.

3. The cell of claim 1, wherein the cell comprises one or more alterations to endogenous glycoside metabolism.

4. The cell of claims 1-3, wherein the cell is a microbial cell.

5. The cell of claim 4, wherein the cell is a fungal cell.

6. The cell of claims 1-5, wherein the engineered cell comprises one or more heterologous coding sequences for one or more enzymes, wherein at least one of the enzymes is selected from the group consisting of: arginine decarboxylase, agmatine urea hydrolase, agmatine enzyme, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylalanine ammonia lyase, tyrosine ammonia lyase, phenylpyruvate reductase, 4-coumaric acid-CoA ligase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, ritoprene synthase, ritoprinosine mutase, hyoscyamine dehydrogenase, hyoscyamine 6 β -hydroxylase/dioxygenase, and cocaine synthase.

7. The cell of any one of claims 1-6, wherein endogenous arginine metabolism in the cell is altered by modifying one or more coding sequences for one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of: glutamate N-acetyltransferase, acetylglutamate kinase, N-acetyl-gamma-glutamyl-phosphate reductase, acetylornithine aminotransferase, ornithine acetyltransferase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase and arginase.

8. The cell of any one of claims 1-7, wherein endogenous phenylalanine and phenylalanine metabolism in the cell is altered by modifying one or more coding sequences for one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of: pentafunctional AROM polypeptides, chorismate synthase, chorismate mutase, prephenate dehydratase, aromatic aminotransferase, and phenylacrylate decarboxylase.

9. The cell of any one of claims 1-8, wherein endogenous polyamine regulatory mechanisms in the cell are altered by modifying one or more coding sequences of one or more endogenous proteins, wherein at least one of the proteins is selected from the group consisting of: methionine adenosine phosphorylase, ornithine decarboxylase antitase, polyamine oxidase, spermidine synthase, spermine synthase, polyamine transporter, and polyamine permease.

10. The cell of any one of claims 1-9, wherein endogenous acetate metabolism in the cell is altered by modification of one or more coding sequences for one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of an alcohol dehydrogenase and an aldehyde dehydrogenase.

11. The cell of any one of claims 1-10, wherein endogenous glycoside metabolism in the cell is altered by modifying one or more coding sequences for one or more endogenous enzymes, wherein at least one of the enzymes is selected from the group consisting of glucan 1, 3-beta-glucosidase and sterol-beta-glucosidase.

12. The cell of any one of claims 6-11, wherein the modification of one or more coding sequences is selected from the group consisting of: a feedback inhibition mitigating mutation in a biosynthetic enzyme or regulatory protein gene native to the cell, a transcriptional regulatory modification of a biosynthetic enzyme gene native to the cell, and an inactivating mutation in an enzyme or protein native to the cell.

13. The cell of any one of claims 1-12, wherein the engineered cell comprises one or more heterologous coding sequences encoding one or more enzymes comprising one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain to enable functional expression of the acyltransferase domain in a subcellular compartment of the engineered cell.

14. The cell of any one of claims 1-13, wherein the cell produces a precursor of a tropane alkaloid product selected from the group consisting of: agmatine, N-carbamoylputrescine, N-methylputrescine, 4-methylaminobutyraldehyde, N-methylpyrrolidinium, 4- (1-methyl-2-pyrrolidinyl) -3-oxobutanoic acid, tropinone, tropine, pseudotropine, ecgonine, methylecgonine, coenzyme a covalently bonded to phenyllactic acid through a thioester bond, or a sugar covalently bonded to cinnamic acid, ferulic acid, coumaric acid, or phenyllactic acid through a glycosidic bond.

15. The cell of any one of claims 1-14, wherein the cell produces a tropane alkaloid product selected from the group consisting of: hyoscyamine, atropine, anisodamine, scopolamine, calycanthin, cocaine, or non-natural tropane alkaloids.

16. The cell of any one of claims 1-15, wherein the cell produces a derivative of a tropane alkaloid product selected from the group consisting of: p-hydroxy atropine, p-hydroxy scopolamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methyl scopolamine, N-butyl scopolamine, N-acetyl scopolamine, and N-acetyl scopolamine.

17. The cell of claim 15, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, or scopolamine.

18. The cell of any one of claims 1-17, wherein transport of one or more TA, one or more TA precursors and/or one or more TA derivatives across the intracellular membrane or across the plasma membrane is improved in the cell.

19. The cell of claim 18, wherein improved transport is achieved by one or more heterologous coding sequences encoding one or more transporters, wherein at least one of the transporters is selected from the group consisting of: multidrug and toxin efflux transporters, nitrate/peptide family transporters, ATP-binding cassette transporters, and multi-effect drug-resistant transporters.

20. An engineered non-plant cell that produces a tropane alkaloid product or a derivative of a tropane alkaloid product, wherein said engineered non-plant cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes within a pathway for producing the tropane alkaloid product or the derivative of a tropane alkaloid product.

21. The cell of claim 20, wherein the cell is a microbial cell.

22. The cell of claim 21, wherein the cell is a fungal cell.

23. The cell of claims 20-22, wherein the engineered cell comprises one or more heterologous coding sequences for one or more enzymes, wherein at least one of the enzymes is selected from the group consisting of: arginine decarboxylase, agmatine urea hydrolase, agmatine enzyme, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylalanine ammonia lyase, tyrosine ammonia lyase, phenylpyruvate reductase, 4-coumaric acid-CoA ligase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, ritoprene synthase, ritoprinosine mutase, hyoscyamine dehydrogenase, hyoscyamine 6 β -hydroxylase/dioxygenase, and cocaine synthase.

24. The cell of any one of claims 20-23, wherein the engineered cell comprises one or more heterologous coding sequences encoding one or more enzymes comprising one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain to enable functional expression of the acyltransferase domain in a subcellular compartment of the engineered cell.

25. The cell of any one of claims 20-24, wherein the cell produces a tropane alkaloid product selected from the group consisting of: hyoscyamine, atropine, anisodamine, scopolamine, calycanthin, cocaine, or non-natural tropane alkaloids.

26. The cell of any one of claims 20-25, wherein the cell produces a derivative of a tropane alkaloid product selected from the group consisting of: p-hydroxy atropine, p-hydroxy scopolamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methyl scopolamine, N-butyl scopolamine, N-acetyl scopolamine, and N-acetyl scopolamine.

27. The cell of claim 25, wherein the cell produces a tropane alkaloid product selected from the group consisting of hyoscyamine, atropine, or scopolamine.

28. The cell of any one of claims 20-27, wherein transport of one or more TA, one or more TA precursors and/or one or more TA derivatives across the intracellular membrane or across the plasma membrane is improved in the cell.

29. The cell of claim 28, wherein improved transport is achieved by one or more heterologous coding sequences encoding one or more transporters, wherein at least one of the transporters is selected from the group consisting of: multidrug and toxin efflux transporters, nitrate/peptide family transporters, ATP-binding cassette transporters, and multi-effect drug-resistant transporters.

30. A method for producing a tropane alkaloid product, a precursor of a tropane alkaloid product or a derivative of a tropane alkaloid product, comprising

(a) Culturing the cell of any one of claims 1-29 under conditions suitable for protein production;

(b) adding a starter compound to the cell culture; and

(c) recovering the tropane alkaloid product, the precursor of a tropane alkaloid product, or the derivative of a tropane alkaloid product from the culture.

31. The method of claim 30, wherein the cell is cultured in a fed-batch or batch fermentation.

32. The method of claims 30-31, wherein the starting compound added to the cell culture is a sugar or a substrate containing one or more sugars or converted to one or more sugars during microbial fermentation.

33. The method of claims 30-31, wherein the starting compound added to the cell culture is an amino acid or a mixture comprising one or more amino acids, or a substrate that is converted to one or more amino acids during microbial fermentation.

34. The method of claims 30-31, wherein the starting compound added to the cell culture is a precursor to a tropane alkaloid product.

35. The method of claims 30-34, wherein the precursor of a tropane alkaloid product, the tropane alkaloid product, or the derivative of a tropane alkaloid product is recovered by a process comprising liquid-liquid extraction, chromatographic separation, distillation, or recrystallization.

Background

Tropane Alkaloids (TAs) are a class of anticholinergic secondary metabolites produced by plants of the Solanaceae family (Solanaceae). Several TAs, including atropine, hyoscyamine and scopolamine, are listed as basic drugs by the World Health Organization (World Health Organization) for the treatment of various nervous system disorders, such as organophosphorous and nerve toxic agents poisoning, gastrointestinal spasms and arrhythmias, and to control the symptoms of parkinson's disease. Therefore, it is of interest to have a sufficient and stable supply of these TA molecules so that researchers and physicians can use them. The current supply chain for medicinal TA relies on extraction from a single cultivated plant that is not sustainable and geographically limited, where TA is only 0.2-4% dry weight and the plant is susceptible to pests, land use changes and climate. Due to the difficulties posed by TA stereochemistry, the total chemical synthesis of TA from simple starting materials has not proven to be economical enough for industrial use. Furthermore, poor economies of scale and long generation times have made engineering transgenic plants or plant cultures with increased TA yield, to date, an unfeasible strategy for obtaining these compounds. Therefore, methods for the preparation of TA are of interest.

Disclosure of Invention

The present invention includes non-plant organisms engineered for the production of various Tropane Alkaloids (TAs) from precursors and sugars. For example, the present invention includes engineered microbial strains for the production of pharmaceutically acceptable TAs, defined herein as naturally occurring TAs of established use in current medical practice, including hyoscyamine, atropine, anisodamine and scopolamine, as well as precursors and derivatives thereof. The present invention also includes engineered microbial strains for the production of non-medicinal TA, defined herein as naturally occurring TA that has not been determined for use in current medical practice but may have a biological activity of pharmaceutical interest, including calycanthine (calystegine), cocaine, and precursors and derivatives thereof. The invention also includes engineered microbial strains for producing non-native TA, defined herein as TA not produced by an unmodified organism, such as TA produced by esterification of unesterified acyl donor and acyl acceptor compounds in a naturally occurring organism, including derivatives of pharmaceutically acceptable TA and derivatives of non-pharmaceutically acceptable TA. Examples of the schemes involved in the present invention are detailed in fig. 1-3.

The invention includes methods of producing pseudotropine and alkaloids derived from pseudotropine (e.g., calystegine) using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst. The invention also includes methods of using microorganisms engineered to express at least one heterologous enzyme as a microbial catalyst to produce various compounds that can be used as acyl donors for biosynthesis of TA scaffolds. The invention also includes methods of esterifying an acyl donor and acceptor for producing a TA scaffold using a microorganism engineered to express at least one heterologous enzyme as a microbial catalyst. The invention also includes methods of modifying and culturing engineered microbial strains for the production of pharmaceutically acceptable TAs such as hyoscyamine and scopolamine, non-pharmaceutically acceptable TAs such as calycanthin, and non-native TAs such as those derived from esterification of a trope with an acyl donor compound other than 3-phenyllactic acid (PLA).

Host cells engineered to produce Tropane Alkaloids (TAs) of interest, such as scopolamine and scopolamine, are provided. TA of interest can include TA precursors, TA, and modifications (modifications) of TA, including derivatives of TA. The host cell may have one or more modifications selected from: a feedback inhibition mitigating mutation in an enzyme gene; transcriptional regulatory modifications of biosynthetic enzyme genes; an inactivating mutation in the enzyme; and heterologous coding sequences. Also provided are methods of using the host cells to produce the TA of interest, as well as compositions, e.g., kits, systems, etc., that can be used in the methods of the invention.

One aspect of the invention provides a method for forming a product stream having a Tropane Alkaloid (TA) product. The method comprises providing engineered non-plant cells having at least one modification selected from the group consisting of: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; and inactivating mutations in enzymes inherent to the cell. Further, the method includes fermenting the engineered non-plant cells in a batch reactor by incubating the engineered non-plant cells for a period of time of at least about 5 minutes to produce a solution comprising the TA product and cellular material. The method also includes separating the TA product from the cellular material using at least one separation unit to provide the product stream comprising the TA product.

In another aspect, the present invention provides a method for forming a product stream having a TA product. The method includes providing engineered non-plant cells and a feedstock including nutrients and water into a reactor. The method further includes fermenting the engineered non-plant cells in a reactor by incubating the engineered yeast cells for a period of time of at least about 5 minutes (e.g., 5 minutes or more) to produce a solution comprising cellular material and a TA product. Further, the method includes separating the TA product from the cellular material using at least one separation unit to provide a product stream comprising the TA product.

Another aspect of the invention provides an engineered non-plant cell that produces a Tropane Alkaloid (TA) product, the engineered non-plant cell having at least one modification selected from the group consisting of: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; and inactivating mutations in enzymes inherent to the cell. The engineered non-plant cell comprises at least one heterologous coding sequence encoding at least one enzyme selected from the group consisting of: arginine decarboxylase, agmatine urea hydrolase, agmatine enzyme, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase (pyrrolidine ketosynthase), tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, ritorine (littoraine) synthase, ritorine mutase, hyoscyamine dehydrogenase, hyoscyamine 6 β -hydroxylase/dioxygenase, and cocaine synthase. In some examples, the engineered non-plant cell comprises a plurality of heterologous coding sequences encoding an enzyme selected from the group consisting of: arginine decarboxylase, agmatine urea hydrolase, agmatine enzyme, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, ritoprene synthase, ritoprinogenase, hyoscyamine dehydrogenase, hyoscyamine 6 beta-hydroxylase/dioxygenase, and cocaine synthase. In some examples, a heterologous coding sequence can be operably linked. The operably linked heterologous coding sequence may be within the same pathway that produces the particular tropane alkaloid product. In some examples, the engineered non-plant cell comprises one or more improvements to intracellular compartmentalization selected from the group including, but not limited to: improved intracellular trafficking of enzymes, improved intracellular localization of enzymes, and improved intracellular transport of metabolites.

In another aspect of the invention, a therapeutic agent is provided. The therapeutic agent includes a tropane alkaloid product.

Drawings

The invention is best understood from the following detailed description when read with the accompanying drawing figures. It should be emphasized that, according to common practice, the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. The drawings include the following illustrations.

FIG. 1 shows an exemplary biosynthetic scheme for the conversion of L-arginine to a non-pharmaceutically acceptable TA. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine urea hydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont, spontaneous (non-enzymatic) step; PYKS, pyrrolidone synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP⁺A reductase; TR2, tropinone reductase 2; p450, cytochrome P450. Arginine, ornithine, spermine, spermidine and putrescine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end product indicated inside the box is an example of a non-medicinal TA.

Figure 2 shows an exemplary biosynthetic pathway by which amino acids can be converted into pharmaceutically useful TA molecules of interest and their precursor molecules. This example shows the conversion of L-arginine and L-phenylalanine to pharmaceutically acceptable TA. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine urea hydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont, spontaneous (non-enzymatic) step; PYKS, pyrrolidone synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP + reductase; TR1, tropinone reductase 1; ArAT, aromatic aminotransferase; PPR, phenylpyruvate reductase; UGT84a27, 3-phenyllactic acid UDP-glucosyltransferase; LS, ritodrine synthase; CYP80F1, ritopressin mutase; HDH, (S) -hyoscyamine dehydrogenase; H6H, (S) -hyoscyamine 6 β -hydroxylase/dioxygenase. Arginine, ornithine, spermine, spermidine, putrescine, phenylalanine, 3-phenylpyruvic acid and a trace amount of 3-phenyllactic acid are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end product indicated inside the box is an example of a pharmaceutically acceptable TA.

FIG. 3 shows an exemplary biosynthetic pathway by which amino acids can be converted to non-pharmaceutically acceptable TA and its precursor molecules. In this example, L-arginine and L-phenylalanine are converted to unnatural TA. ADC, arginine decarboxylase; ARG, arginase; AUH, agmatine urea hydrolase; ODC, ornithine decarboxylase; PAO, polyamine oxidase; PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; spont, spontaneous (non-enzymatic) step; PYKS, pyrrolidone synthase; CYP82M3, tropinone synthase; CPR, cytochrome P450-NADP⁺A reductase; TR1, tropinone reductase 1; PAL, phenylalanine ammonia lyase; 4CL, 4-coumarate-CoA ligase; CS, cocaine synthase. Arginine, ornithine, spermine, spermidine, putrescine and phenylalanine are naturally synthesized in yeast. All other metabolites shown are not naturally produced in yeast. The end product indicated inside the box is an example of non-native TA.

Figure 4 shows an exemplary biosynthetic pathway for production of putrescine from amino acids and other polyamine molecules. The figure shows how putrescine can be prepared from a central metabolite using endogenous yeast and heterologous biosynthetic pathways.

Figure 5 shows that yeast strains engineered to overexpress endogenous biosynthetic enzymes involved in arginine and polyamine metabolism can produce putrescine in liquid culture. Additional copies of the native gene are expressed from low copy plasmids in wild type yeast (cen. pk2). Prior to LC-MS/MS analysis, the transformed strains were cultured in a selective medium with 2% dextrose at 30 ℃ for 48 h. All data represent the mean of at least three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001. Unless otherwise indicated, statistical significance relative to the corresponding control (i.e., cen. pk2) is shown.

Figure 6 shows that yeast strains engineered to heterologously express biosynthetic enzymes from organisms other than yeast that are involved in arginine and polyamine metabolism can produce putrescine in liquid culture. In this example, yeast strains are engineered to express heterologous biosynthetic pathways from plants and bacteria. The heterologous enzyme is expressed from a low copy plasmid in wild-type yeast. Prior to LC-MS/MS analysis, the transformed strains were cultured in a selective medium with 2% dextrose at 30 ℃ for 48 h. All data represent the mean of at least three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001. Unless otherwise indicated, statistical significance relative to the corresponding control (i.e., cen. pk2) is shown.

Figure 7 shows that yeast strains engineered to heterologously express biosynthetic enzymes from organisms other than yeast that are involved in arginine and polyamine metabolism can produce TA precursors and intermediates agmatine, N-carbamoylputrescine and putrescine in liquid culture. This figure shows the functional validation of agmatine/putrescine biosynthetic pathway genes in yeast. Pk2 was transformed with three low copy plasmids to co-express between zero (negative control) and the three designated biosynthetic genes. A plasmid expressing Blue Fluorescent Protein (BFP) was used as a negative control for each of the three auxotrophic selection markers URA3, TRP1, and LEU 2. The transformed strains were cultured in selective medium with 2% dextrose at 30 ℃ for 48h before LC-MS/MS analysis of metabolite production. All data show titers measured by LC-MS/MS peak area relative to negative control (cen. pk2). Data represent the mean of three biological replicates and error bars show standard deviation.

FIG. 8 shows an endogenous regulatory pathway that tightly controls intracellular putrescine levels during normal yeast growth.

Figure 9 shows a heat map of putrescine production in yeast strains in which the endogenous polyamine biosynthesis regulatory mechanisms are disrupted. For overexpression of the native or heterologous putrescine pathway, the designated genes are expressed from low copy plasmids in wild type yeast (WT) or individual single disruption strains. Prior to LC-MS/MS analysis, the strains were cultured in selective (YNB-DO) medium with 2% dextrose for 72h at 30 ℃. All data represent the mean of at least three biological replicates. The figure shows that yeast strains with a single disruption and overexpression of the polyamine metabolism gene, endogenous or heterologous putrescine biosynthetic pathway, can produce putrescine in liquid culture.

Fig. 10 provides a summary of the engineering efforts to improve putrescine production in yeast. The '+' symbol indicates the expression of at least one gene from the pathway, while the '-' indicates no gene expression from the pathway. Prior to LC-MS/MS analysis, the strains were cultured in selective medium with 2% dextrose at 30 ℃ for 48 h. All data represent the mean of at least three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001. Unless otherwise indicated, statistical significance relative to the corresponding control (i.e., cen. pk2) is shown.

Figure 11 shows an LC-MS/MS chromatogram showing the stepwise conversion of putrescine to TA intermediate NMPy and byproduct 4MAB acid by intermediate NMP and 4MAB in engineered yeast according to an embodiment of the invention. The proposed mechanism of formation of 4MAB acid by-product by the activity of endogenous yeast enzymes (ALD) is shown. Extracted ion chromatogram MRM traces showing each metabolite along the pathway and the true standard (authetic standards) using the highest precursor/product ion transition for each metabolite are shown. The control represents strain CSY1235 expressing SPE1, AsADC and speB on a low copy plasmid (see example 1.5). Chromatogram traces represent three biological replicates. Enzyme symbol: PMT, putrescine N-methyltransferase; MPO, N-methylputrescine oxidase; ALD, aldehyde dehydrogenase.

Fig. 12 shows an LC-MS/MS chromatogram showing the relative yields of TA precursors (a) putrescine, (B) NMP, (C, E)4MAB and (D, F) NMPy in liquid cultures of engineered yeast expressing AbPMT1 and MPO enzyme, according to an embodiment of the invention. (A) MRM chromatogram of putrescine (m/z +89 → 72) of CSY1235 containing pCS4239 to overproduce putrescine. (B) MRM chromatogram of NMP (m/z +103 → 72) of CSY1235 containing pCS4239 and expressing AbPMT1 from a low copy plasmid. (C, D) corresponding MRM chromatograms of 4MAB (m/z +102 → 71) and NMPy (m/z +84 → 57) of CSY1235 containing pCS4239 and expressing AbPMT1 and NtMPO1 from low copy plasmids. (E, F) corresponding MRM chromatograms of 4MAB (m/z +102 → 71) and NMPy (m/z +84 → 57) of CSY1235 containing pCS4239 and expressing AbPMT1 and DmMPO1 Δ C-PTS1 from low copy plasmids. The Y-axis of the trace is the raw MRM ion count. After 48 hours of growth in selective medium with 2% dextrose at 30 ℃, all chromatograms were generated by LC-MS/MS analysis of the extracellular medium. Traces represent at least three biological replicates.

Figure 13 shows the effect of MEU1 disruption on SAM-dependent putrescine N-methylation by AbPMT 1. Wild type strains cen. pk2 or meu1 disrupted strain CSY1229 was co-transformed with low copy plasmids expressing SPE1, AsADC and speB and AbPMT 1. The data indicate the average NMP titer relative to cen. pk2 control, as quantified by LC-MS/MS peak area of 3 biological replicates after 48 hours of growth in selective medium with 2% dextrose at 30 ℃. Error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

Figure 14 shows in silico predictions of the subcellular localization of NMPy biosynthetic genes in plant and yeast/fungal cells using SherLoc2 web server. Values and paint represent probability scores (0 to 1) for localization to each of the following compartments: CYT, cytosol; NUC, cell nucleus; VAC, vacuole; CHL, chloroplast; MIT, mitochondria; POX, peroxisome.

Fig. 15 shows the effect of (a) co-localization of NtMPO1 with N-and C-terminal GFP-tagged and PEX3 peroxisome marker, and (B) N-and C-terminal GFP-tagging of NtMPO1 on the yield of TA precursors 4MAB and NMPy in liquid cultures of engineered yeast according to an embodiment of the invention. The figure shows experimental validation of subcellular localization of NtMPO 1. (A) Fluorescence microscopy of NtMPO 1N-terminal and C-terminal GFP fusions co-expressed with peroxisome marker mCherry-PEX3 in wild-type yeast (cen. pk2). White arrows indicate co-localization of GFP-tagged NtMPO1 with peroxisomes. Scale bar, 10 μm. (B) Effect of forced cytosolic localization of NtMPO1 on 4MAB or NMPy production. Wild type yeast (cen. pk2) was co-transformed with a low copy plasmid expressing wild type NtMPO1 or an N-or C-terminal GFP fusion and a low copy plasmid expressing SPE1, AsADC and speB and AbPMT 1. LC-MS/MS analysis was performed after 48 hours of growth in selective medium with 2% dextrose at 30 ℃. Data represent the mean of three biological replicates; error bars show standard deviation. The most likely subcellular compartments are indicated based on the microscopy data in (a).

Figure 16 provides fluorescence microscopy data depicting subcellular localization of AbPMT1 and NtMPO1 when expressed heterologously in yeast. Microscopy was performed on wild type yeast expressing either AbPMT1 or NtMPO1 with either an N-terminal or C-terminal GFP tag from low copy plasmids. Scale bar, 10 μm.

FIG. 17 shows a comparison of the sequence alignments of (A) NtMPO1 and putative MPO enzymes identified from plant transcriptome data AbMPO1 and DmMPO1 (from top to bottom: SEQ ID NOS: 27-29), (B) the yields of TA precursor 4MAB and NMPy in liquid cultures of engineered yeast strains expressing NtMPO1, AbMPO1 or DmMPO1, and (C) the predicted three-dimensional structures of NtMPO1, AbMPO1 and DmMPO1 as determined by homology modeling, according to embodiments of the present invention. (A) The NtMPO1 sequences were queried for alignment with the AbMPO1 and DmMPO1 candidates from the 1000Plants Project database. Blue indicates conservation of amino acid structure; red indicates no match. (B) Comparison of the relative activities of MPO orthologs. Putrescine overproducing strains (Putrescine overproducing strains) CSY1235 (see example 1.5) were co-transformed with low copy plasmids expressing SPE1, AsADC and speB, AbPMT1 and one of the three MPO variants. LC-MS/MS analysis was performed after 48 hours of growth at 30 ℃ in selective medium. Data represent the mean of three biological replicates; error bars show standard deviation. (C) A homology model for MPO enzyme (pink) was constructed based on the crystal structure (PDB: 1KSI, blue) of pea (Pisum sativum) copper-containing amino oxidase using a RaptorX web server. Top: NtMPO 1; center: AbMPO 1; bottom: DmMPO 1.

Fig. 18 shows 4MAB yield in liquid culture of engineered yeast strains overproducing putrescine and expressing AbPMT1 and N-and C-terminal truncations of NtMPO1 and DmMPO 1. This figure shows the effect of N-and C-terminal truncations of methyl putrescine oxidase on 4MAB yield in engineered yeast. The Wild Type (WT) enzyme and the indicated truncates were expressed from a low copy plasmid in the putrescine overproducing strain CSY1235 (see example 1.5). Prior to LC-MS/MS analysis, the strains were cultured in selective medium with 2% dextrose at 30 ℃ for 48 h. All data represent the mean of at least three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

Figure 19 shows the production of TA precursor 4MAB and NMPy and byproduct 4MAB acid in liquid cultures of engineered yeast strains with a single disruption of one of the four native aldehyde dehydrogenase genes. The figure shows the effect of disrupting a single aldehyde dehydrogenase on 4MAB acid accumulation. The putrescine overproducing strain CSY1235 (control) or a daughter strain with a nonsense mutation disruption of hfd1, ald4, ald5 or ald6 was used to express SPE1, AsADC and speB, AbPMT1 and Low copy plasmid transformation of (4). Bars indicate relative 4MAB acid titers as measured by LC-MS/MS peak area normalized to CSY1235 (no ALD damage) after 48 hours of growth in selective media at 30 ℃. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p<0.05；**P<0.01；***P<0.001。

FIG. 20 shows the yields of (A)4MAB acid byproduct and (B) TA precursors 4MAB and NMPy in liquid cultures of engineered yeast strains containing one or more disruptions of native aldehyde dehydrogenase. This figure shows the effect of aldehyde dehydrogenase gene disruption on the yield of (A)4MAB acid by-product and (B)4MAB and NMPy in engineered yeast. The '+' and '-' symbols indicate the presence or absence, respectively, of a functional enzyme. Prior to LC-MS/MS analysis, the strains were cultured in selective (YNB-DO) medium with 2% dextrose for 48h at 30 ℃. All data represent the mean of at least three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001. Statistical significance relative to the corresponding control (CSY1235) is shown unless otherwise indicated.

Fig. 21 shows a comparison of the yield of the TA precursor NMPy in liquid cultures of engineered yeast strains in the case of low copy plasmid-based or genomic expression of the putrescine overproducing genes AbPMT1 and DmMPO1 truncations, according to an embodiment of the invention. The figure provides a comparison of the yields of 4MAB and NMPy in the case of plasmid-based expression of the NMPy biosynthetic genes (CSY1241) and genomic expression (CSY 1243). Strain CSY1241 was overproduced with the expression putrescine genes (SPE1, AsADC, speB), AbPMT1 and Low copy plasmid transformation of (4). Strain CSY1243 expresses all the above genes from genomically integrated copies. After growth in selective (CSY1241) or non-selective (CSY1243) medium at 30 ℃ for 48h, NMPy levels were quantified by LC-MS/MS. Data represent the mean of at least two biological replicates, and error bars indicate standard deviation.

Figure 22 shows a biosynthetic pathway for the production of byproduct coumarine from NMPy and MPOB in accordance with an embodiment of the invention. Putative major and minor side reactions in yeast are indicated by bold and dashed arrows, respectively.

Figure 23 shows a comparison of the production of the TA precursor tropinone and tropine and the byproduct coumarine in liquid cultures of engineered yeast strains expressing low copy plasmid based AbPYKS, AbCYP82M3, DsTR1, and one of four different CPRs. The figure shows the yields of tropine and related intermediates in case AbPYKS, AbCYP82M3 and DsTR1 are expressed in engineered yeast. The designated gene is expressed from a low copy plasmid in CSY 1246; the prime and '-' symbols indicate the presence or absence of the enzyme. Prior to LC-MS/MS analysis, the strains were cultured in selective medium with 2% dextrose at 30 ℃ for 48 h. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

Figure 24 shows (a) LC-MS/MS chromatograms showing characteristic triplets of the TA precursor MPOB produced in liquid cultures of engineered yeast strains, and (B) the production of the TA precursors NMPy and MPOB in liquid cultures of yeast strains engineered to express AbPYKS, AbCYP82M3, and one of the four CPRs from plasmids. The figure shows the accumulation of NMPy and MPOB in the medium of engineered strains expressing AbPYKS. (A) Representative LC-MS/MS Multiple Reaction Monitoring (MRM) chromatograms used to detect MPOB in the extracellular medium of CSY1246, which expresses only AbPYKS from a low copy plasmid. The three characteristic MPOB isoform peaks are labeled with (I), (II) and (III). LC-MS/MS analysis was performed after 48h growth in selective medium at 30 ℃. (B) Relative abundance of NMPy and MPOB in the extracellular medium of CSY1246 expressing AbPYKS, AbCYP82M3 and one of the four CPRs from low copy plasmids (all 3 peaks) after 48h of growth in selective medium at 30 ℃. The prime and prime symbols indicate the presence or absence of a gene. Data represent the mean of three biological replicates; error bars indicate standard deviation.

FIG. 25 shows the effect of growth temperature on the production of TA precursor tropine and the byproduct coumarine in liquid cultures of engineered yeast. This shows the effect of growth temperature on spontaneous coumarine production in a tropina-producing yeast strain (CSY 1248). Relative selectivity represents the ratio of relative tropine titer to relative coumarine titer. Prior to LC-MS/MS analysis, the strains were cultured in non-selective medium with 2% dextrose at 30 ℃ or 25 ℃ for 48 h. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

FIG. 26 shows the effect of (A) ALD4 and ALD6 reconstitution on growth of a tropicalized engineered yeast strain on acetate supplemented or unsupplemented media, and (B) elimination of acetate auxotrophy on production of by-product 4MAB acid and cocaine in liquid cultures of the tropicalized engineered yeast strain, according to embodiments of the invention. The figure shows the effect of eliminating acetate auxotrophy in engineered torpedo-producing yeast strains. (A) Reconstitution of the effect of functional ALD4 or ALD6 genes on growth of NMPy-producing yeast strains (CSY1246) with and without acetate supplementation. ALD4 and ALD6 were expressed from low copy plasmids. 'WT' indicates CSY1246 with control (BFP) plasmid. The adjacent columns show ten fold dilutions. (B) Production of 4MAB acid and the byproduct coumarine by metabolism of reconstituted acetate in engineered yeast. The '+' and '-' symbols indicate the presence or absence of a feed metabolite (acetate) or ALD4 and ALD6 genes expressed from low copy plasmids. Prior to LC-MS/MS analysis, the strains were cultured in selective (YNB-DO) medium with 2% dextrose for 48h at 30 ℃. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

Fig. 27 shows representative LC-MS/MS chromatograms of (a) acetate auxotrophy effect on accumulation of TA precursor between NMPy and tropinone in liquid cultures of yeast strains engineered to produce tropine, and (B) TA precursor MPOB produced in liquid cultures of yeast strains engineered to produce tropine with and without acetate auxotrophy, according to embodiments of the invention. The figure shows the effect of reconstituting ALD6 activity on metabolite flux in engineered yeast towards tropine by NMPy. (A) Yield of intermediate between NMPy and tropinone in engineered strains with and without functional Ald6 p. Intermediate abundance was measured by LC-MS/MS MRM in extracellular medium of either an integrated strain of the tropine-producing strain (CSY1248) grown at 25 ℃ for 48h in non-selective medium supplemented with 0.1% w/v potassium acetate (grey) or the tropine-producing strain with reconstituted ALD6 (CSY1249) grown at 25 ℃ in non-selective medium not supplemented with acetate (pink). Data represent the mean of three biological replicates; error bars indicate standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001. (B) Representative MRM chromatograms of MPOB production from CSY1248 (grey) and CSY1249 (red) cultured as described in (a).

FIG. 28 shows the progress of improvement in the production of TA precursor tropine and the byproduct coumarine in liquid culture of engineered yeast strains. The figure provides a summary of strains engineered to increase tropine production in yeast. The prime symbol indicates that no gene is present; 'p' and 'i' indicate gene expression from low copy plasmids or genomic integrations, respectively. Prior to LC-MS/MS analysis, the strains were cultured in selective or non-selective medium with 2% dextrose at 30 ℃ or 25 ℃ for 48 h. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

Fig. 29 shows the effect of expressing additional copies of the heterologous biosynthetic enzymes PMT, MPO, PYKS, and CYP82M3 on the yield of each TA precursor between putrescine and tropine in liquid cultures of engineered yeast, according to an embodiment of the invention. This figure identifies metabolic bottlenecks in the optimized tropine-producing strain (CSY 1249). Using a control plasmid expressing BFP ("no overexpression") or expression of AbPMT1,Low copy plasmid transformation of additional copies of AbPYKS or AbCYP82M3 strain CSY 1249. After 48h of growth in selective medium at 25 ℃, the level of intermediates in the extracellular medium was quantified by LC-MS/MS. Data indicate the mean of three biological replicates and error bars show standard deviation.

Figure 30 shows the effect of additional copies of the bottleneck enzymes PMT and PYKS on tropine yield in engineered yeast. The figure shows that the metabolic bottleneck is alleviated by genomic integration of additional copies of PMT and PYKS enzymes. Prior to LC-MS/MS analysis of the growth medium, the tolerogenic strains CSY1249 and CSY1251 were cultured in non-selective medium at 25 ℃ for 48 h. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p < 0.05; p < 0.01; p < 0.001.

FIG. 31 shows the production of the TA precursor acyl donor compound PLA in liquid culture of engineered yeast strains expressing heterologous lactate dehydrogenase and phenylpyruvate reductase. The figure shows a protein engineered to convert L-phenylalanine to 3-phenyllactic acidLC-MS/MS analysis of the yeast strains of (1). Yeast strains were engineered to have a low copy CEN/ARS plasmid containing the LEU2 selectable marker, the TDH3 promoter, and the coding sequences for: BFP as a negative control; LDH variants from bacillus coagulans (b.coagulans) (BcLLDH), lactobacillus casei (l.casei) (LcLLDH), lactobacillus plantarum (l.plantarum) (LpLLDH); or a PPR variant from belladonna (a. belladonna) (AbPPR), lactobacillus plantarum (LpPPR), Escherichia coli (hcxB) or rhodotorula fluorescens (w. fluoroscens) (WfPPR). Yeast from freshly transformed colonies were grown in 300. mu.L of selective medium (-Leu) in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 25 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. The data show that the ion chromatogram is based on extraction (ammonium adduct, EIC m/z) ⁺184) relative 3-phenyllactic acid titer normalized against trace levels present in the negative control. Data represent the mean of three biological replicates and error bars show standard deviation. Two-tailed t test of students: p<0.05；**P<0.01；***P<0.001。

Figure 32 shows an LC-MS/MS chromatogram showing the production of the TA precursor acyl donor compound cinnamic acid in liquid culture of an engineered yeast strain expressing phenylalanine ammonia lyase. The figure shows an LC-MS/MS analysis of yeast strains engineered to convert L-phenylalanine to cinnamic acid. Yeast strains were engineered to have a low copy CEN/ARS plasmid containing a TRP1 selectable marker, TEF1 promoter, and the coding sequence for (i) BFP or (ii) arabidopsis thaliana (a. thaliana) phenylalanine ammonia lyase (AtPAL 1). Yeast from freshly transformed colonies were grown in 300. mu.L of selective medium (-Trp) in 96-well deep-well microtiter plates. After 48 hours of growth in a shaking incubator at 30 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. The chromatogram traces show the production of cinnamic acid by these strains based on the most abundant Multiple Reaction Monitoring (MRM) transition (m/z +149 → 131) of cinnamic acid. Each trace represents three samples.

FIG. 33 shows the substrate specificity of a UDP-glucosyltransferase 84A27(UGT84A27) ortholog from a TA producing solanaceae plant expressed in engineered yeast. The figure shows a comparison of the activity of UGT84a27 ortholog against three different phenylpropane (phenylpropanoid) compounds expressed in engineered yeast. (A) Phenylpropane tested as glucose (Glu) receptor for UGT84a27 in engineered yeast. Top, (D) -3-phenyllactic acid (PLA); intermediate, trans Cinnamic Acid (CA); bottom, trans-Ferulic Acid (FA). (B) Heat map of the percentage of fed phenylpropane to glucoside conversion achieved by yeast engineered to express UGT84a 27. UGT84a27 ortholog or BFP negative control was expressed from a low copy plasmid in CSY 1251. Prior to LC-MS/MS analysis, transformed cells were cultured for 72h in selective medium supplemented with 500. mu.M PLA, CA or FA. Data represent the mean ± standard deviation of n-3 biologically independent samples.

Fig. 34 shows an example of chromatographic and mass spectrometric analysis of UGT84a27 activity. The figure shows a representative LC-MS/MS trace showing conversion of PLA, CA and FA to homologous glucosides by AbUGT in CSY1251 incubated for 120h as in figure 33B to achieve more complete glycosylation. For PLA, the acid (top trace in each plot) and glucoside (bottom trace in each plot) are passed through different NH ₄ ⁺The parent masses of the adducts (parent vessels) and the different retention times are distinguished. For CA and FA, rapid fragmentation (fragmentation) requires detection of glucosides based on the lower retention peak produced by their phenylpropane fragments.

Figure 35 shows structure-guided active site engineering of AbUGT to alter substrate specificity. The figure shows structural analysis of the AbUGT 3D structure to identify potential mutations that increase activity on PLA. (A) A homology model for AbUGT84A27 was constructed based on the crystal structure of the Arabidopsis thaliana UDP-glucosyltransferase UGT74F2 with bound UDP (PDB: 5V 2K). Based on docking simulations, PLA (orange) is shown with a preferred binding position with UDP-glucose (pink). (B) Magnified view of the AbUGT active site with docked D-PLA and UDP-glucose. Shows potential mutations identified to improve PLA selectivity (F130Y, L205F, I292Q); the dashed lines indicate putative polar/hydrogen bonding interactions.

Figure 36 shows the substrate specificity of the active site mutant of AbUGT84a 27. This figure shows a heat map of the percentage of fed phenylpropane to glucoside conversion achieved by yeast engineered to express the abutt mutant. The AbUGT wild type, active site mutant or BFP negative control was expressed from a low copy plasmid in CSY 1251. Prior to LC-MS/MS analysis, transformed cells were cultured for 72h in selective medium supplemented with 500. mu.M PLA, CA or FA. Data represent the mean ± standard deviation of n-3 biologically independent samples.

FIG. 37 shows an LC-MS/MS chromatogram which verifies the stepwise biosynthesis of PLA glucoside in yeast engineered to produce tropine. The figure shows Multiple Reaction Monitoring (MRM) and Extracted Ion Chromatogram (EIC) traces of media from yeast strains engineered to stepwise reconstitute PLA glucoside. The strains were grown in non-selective medium for 72h prior to LC-MS/MS analysis of the culture supernatants. Chromatogram traces represent three biological replicates.

FIG. 38 shows a schematic of the biosynthetic pathway for the dual metabolic fate of glucose in yeast. This figure shows the effect of citrate on glucoside production by inhibiting glycolysis. Abbreviations: HXK, hexokinase; GPI, glucose-6-phosphate isomerase; PFK, phosphofructokinase; PGM, phosphoglucomutase; UGP, UDP-glucose pyrophosphorylase.

FIG. 39 shows the effect of citrate supplementation on heteroglucoside production in engineered yeast. This figure shows the effect of supplementation with 2% citrate on the conversion of phenylalanic acid to glucoside by yeast engineered to express AbUGT. Strain CSY1288 was cultured in non-selective medium with or without 2% citrate and without additional supplementation (to assess glucosylation of endogenously produced PLA) or with 500 μ M trans-Cinnamic Acid (CA) or trans-Ferulic Acid (FA). Cultures were grown for 72h prior to LC-MS/MS analysis. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001.

FIG. 40 shows relative PLA glucoside production in yeast strains engineered to overexpress UDP-glucose biosynthetic enzymes. This figure shows the effect of over-expressing natural enzymes involved in the biosynthesis of the glucoside precursor UDP-glucose on the yield of PLA glucoside in engineered yeast. The enzyme or negative control (BFP) was expressed from a low copy plasmid in strain CSY 1288. The strains were cultured in selective medium for 72h prior to LC-MS/MS analysis of metabolites in the culture supernatant. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001. Statistical significance was shown relative to the corresponding control.

Figure 41 shows relative PLA glucoside production in CSY1288 with disruption of endogenous glucosidase. This figure shows the effect of disruption of each of the three native glucosidase genes on PLA glucoside accumulation in engineered yeast. The strains were cultured in non-selective medium for 72h prior to LC-MS/MS analysis of the culture supernatants. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001. Statistical significance was shown relative to the corresponding control.

Figure 42 shows an LC-MS/MS chromatogram showing the production of the TA precursor hyoscyaldehyde from ritopram in a liquid culture of engineered yeast cells expressing AbCYP80F 1. This figure shows an LC-MS/MS analysis of yeast strains engineered to convert (R) -ritodrine to hyoscyaldehyde. The yeast strain was engineered to have a low copy CEN/ARS plasmid containing the LEU2 selectable marker, the TDH3 promoter, and the coding sequence for the atropine mutase CYP80F1 from belladonna (AbCYP80F 1). The strain also has a second low copy plasmid containing a TRP1 selectable marker, a TDH3 promoter, and coding sequences for: (i) BFP as negative control, (ii) saccharomyces cerevisiae CPR (NCP1), or (iii) arabidopsis CPR (atrr 1). Yeast from freshly transformed colonies were grown in 300. mu.L selective medium (-Leu- -Trp) supplemented with 1mM ritoprene in 96-well deep-well microtiter plates. After 48 hours of growth in a shaking incubator at 30 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. Chromatogram traces show the production of scopolamine by these strains based on the most abundant MRM transition (m/z +288 → 124). Arrows indicate the putative hyoscyaldehyde peak. Each trace represents three samples.

Figure 43 shows the production of pharmaceutical TA scopolamine from pharmaceutical TA scopolamine in liquid culture of engineered yeast cells expressing an ortholog of scopolamine 6 β -hydroxylase/dioxygenase (H6H). This figure shows the conversion of (S) -scopolamine to (S) -scopolamine by an engineered yeast strain expressing the H6H ortholog. Yeast strains were engineered to have low copy CEN/ARS plasmids containing LEU2 selection marker, TDH3 promoter and BFP as negative control or coding sequences from the H6H variant of datura stramonium (d.straamonium) (DsH6H), scopolia acutangula (a.acutangulus) (AaH6H), woody stramonium (b.arborea) (BaH6H) or datura flower (d.metel) (DmH 6H). Yeast from freshly transformed colonies were grown in 300 μ L of selective medium (-Leu) supplemented with 1mM hyoscyamine in 96-well deep-well microtiter plates. After 48 hours of growth in a shaking incubator at 30 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. The data represent the average of three biological replicates and are normalized to the amount of scopolamine contaminant in the scopolamine feed. Error bars represent standard deviation. Relative scopolamine titers were quantified based on the peak area of the m/z +304 → 138MRM transition.

Figure 44 shows the effect of cofactor availability and supplementation in the media on the conversion of scopolamine to scopolamine in liquid cultures of engineered yeast cells expressing DsH 6H. This figure shows the effect of cofactor supplementation on the conversion of (S) -scopolamine to (S) -scopolamine in engineered yeast. Yeast strains were engineered to have a low copy CEN/ARS plasmid containing the LEU2 selectable marker, the TDH3 promoter, and the coding sequences for: (i) (ii) BFP as a negative control, or (iii) hyoscyamine 6 β -hydroxylase/dioxygenase from datura stramonium (DsH 6H). Yeast from freshly transformed colonies were grown in 300. mu.L of selective medium (-Leu) supplemented with the indicated substrates and/or cofactors in 96-well deep-well microtiter plates. After 48 hours of growth in a shaking incubator at 30 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. Relative (S) -scopolamine titers were quantified based on the integrated peak areas of the m/z +304 → 138MRM transition and normalized against a strain expressing DsH6H and having all cofactors and substrates supplemented. Data represent the mean of three biological replicates and error bars indicate standard deviation. Hyo, (S) -hyoscyamine; 2-OG, 2-oxoglutaric acid; L-AA, L-ascorbic acid.

Figure 45 shows hierarchical clustering heatmaps of hyoscyamine dehydrogenase gene candidates identified from the belladonna transcriptome by analysis of tissue co-expression data. This figure shows clustering of tissue-specific expression profiles of transcripts in the belladonna transcriptome, which might encode enzymes with hyoscyamine dehydrogenase activity. Transcript expression for each candidate was scaled in rows using a normal distribution. Dendrograms indicate hierarchical clustering of candidates obtained by tissue-specific expression profiling. Known TA pathway genes are identified by name; putative HDH candidates are indicated with locus ID. Black triangles indicate candidates screened for activity; double black triangles indicate candidates with experimentally validated HDH activity.

Figure 46 shows production of TA scopolamine from ritopram in liquid cultures of engineered yeast cells expressing a scopolamine dehydrogenase (HDH) candidate. This figure shows an experimental screening for activity of HDH candidates identified from the belladonna transcriptome in engineered yeast. The yeast strains were engineered to express belladonna ritorilin mutase (AbCYP80F1) and scopolamine 6 β -hydroxylase/dioxygenase (DsH6H) from constitutive promoters integrated into the expression cassettes in the genome, and to express each of the 13 HDH candidates from a low copy CEN/ARS plasmid containing a TRP1 selection marker and the TDH3 promoter. Yeast from freshly transformed colonies were grown in 300. mu.L of selective medium (-Trp) supplemented with 1mM ritodrine in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 30 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. Relative hyoscyaldehyde titers were based on m/z ⁺288→124 integrated peak areas for MRM transitions were quantified and normalized against engineered strains expressing BFP, but not HDH candidates. (S) -scopolamine Titers based on m/z⁺304 → 138 integrated peak area of MRM transition and standard curve of true scopolamine standard. Data represent the mean of three biological replicates and error bars indicate standard deviation.

FIG. 47 shows the three-dimensional structure of hyoscyamine dehydrogenase from belladonna. This figure shows a cartoon representation of the structure of AbHDH, a homology model constructed based on the crystal structure of Populus tremuloides sinapyl alcohol dehydrogenase (PtSAD; PDB: 1YQD) as a template. NADPH and Zn²⁺Shown in the active site. The inset shows a magnified view of the AbHDH active site with NADPH and docked scopolamine. The dashed lines indicate interactions important for catalysis.

Figure 48 shows the phylogenetic tree of the three identified HDH orthologs (AbHDH, DiHDH, DsHDH) and the closest protein hits in the UniProt/SwissProt database. This figure shows the clustering of three identified HDH enzyme orthologs with closely related protein sequences based on a BLAST search of the UniProt/SwissProt database. The sequence shown includes the first 50 BLASTp hits based on the E value, and 10 additional hits selected from the next 100 orderings. Phylogenetic relationships were derived by bootstrap ortho-ligation (bootstrap neighbor-join) using n-1000 experiments in ClustalX2 and the resulting trees were visualized using the FigTree software. Abbreviations: ADH, alcohol dehydrogenase; CADH, cinnamyl alcohol dehydrogenase; MTDH, mannitol dehydrogenase; DPAS, dehydroprecordylcardine acetate synthase (dehydroprecordylcardine acetate synthase); 8HGDH, 8-hydroxygeraniol dehydrogenase; GDH, geraniol dehydrogenase; GS, a jubosizine (geissochizine) synthase; REDX, unspecified redox protein.

Figure 49 shows the production of TA scopolamine for use as a drug from ritodrine in liquid culture of engineered yeast cells expressing a hyoscyamine dehydrogenase ortholog. This figure shows a comparison of activity between the identified HDH enzyme orthologs expressed in engineered yeast. YeastThe strains were engineered to express belladonna ritorilin mutase (AbCYP80F1) and scopolamine 6 β -hydroxylase/dioxygenase (DsH6H) from constitutive promoters integrated into the expression cassette in the genome, to express each of 3 HDH orthologs (AbHDH, DiHDH, DsHDH) from a low copy CEN/ARS plasmid containing the TRP1 selection marker and the TDH3 promoter, and to express an additional copy of DsH6H from a low copy CEN/ARS plasmid containing the LEU2 selection marker and the TDH3 promoter. Yeast from freshly transformed colonies were grown in 300. mu.L selective medium (-Leu-Trp) supplemented with 1mM ritoprene in 96-well deep-well microtiter plates. After 72 hours of growth in a shaking incubator at 30 ℃ and 460rpm, the yeast was pelleted and the media supernatant was analyzed by LC-MS/MS. Relative hyoscyaldehyde titers were based on m/z⁺288 → 124 integrated peak area quantification of MRM transition and normalization against engineered strains expressing AbHDH and BFP instead of DsH 6H. (S) -scopolamine Titers based on m/z ⁺304 → 138 integrated peak area of MRM transition and standard curve of true scopolamine standard. Data represent the mean of three biological replicates and error bars indicate standard deviation.

Figure 50 shows experimental validation of the conversion of ritodrine feed to scopolamine by yeasts engineered to express CYP80F1, HDH and H6H. This figure shows the Multiple Reaction Monitoring (MRM) LC-MS/MS trace of media from yeast strains engineered to convert ritodrine to scopolamine. The strains were cultured for 72h in non-selective medium supplemented with 1mM ritodrine before LC-MS/MS analysis of the metabolites in the culture supernatant. The dark trace in the lower right panel (CSY1294, scopolamine) represents a 125nM (38. mu.g/L) scopolamine standard. Chromatogram traces represent three biological replicates.

FIG. 51 shows the classical plant ER to vacuole trafficking and maturation pathway of SCPL acyltransferase (SCPL-AT). This figure shows a schematic representation of a typical ER to vacuolar protein transport pathway followed by SCPL-AT in plants, where belladonna ritoprene synthase (AbLS) is shown as an example. The circled numbers indicate major steps in SCPL-AT expression and activity, including maturation in (1) the ER lumen and (2) the golgi, (3) transport to the vacuole, and vacuole (4) inward substrate transport and (5) outward product transport.

FIG. 52 shows the co-localization of wild-type ritodrine synthase from belladonna expressed in engineered yeast. This figure shows an epifluorescence microscopy of yeast engineered to express an AbLS with an N-terminal GFP-tag (GFP-AbLS) and stained with vacuolar stain FM 4-64. CSY1294 expressing GFP-AbLS from a low copy plasmid was examined microscopically. Scale bar, 5 μm.

FIG. 53 shows a strategy for forced localization of ritodrine synthase to different yeast subcellular compartments by signal sequence replacement. This figure shows a protein engineering approach to improve the subcellular localization of AbLS to address potential limitations on substrate availability in different compartments. (A) Schematic representation of yeast subcellular compartments for localization of AbLS by signal sequence exchange. The signal sequence source protein for each compartment is indicated. (B) The termini and residues selected for substitution of the AbLS signal sequence. Residues comprising each signal sequence domain were selected based on structural annotation in the UniProt/SwissProt database.

Figure 54 shows a western blot of wild-type AbLS expressed in tobacco and treated with deglycosylase. This figure shows the identification of the type of glycosylation modification of AbLS expressed in plants. AbLS with a C-terminal HA-tag was transiently expressed in leaves of nicotiana benthamiana (n. The crude leaf extract was either untreated (lane 1: '-'), or treated with peptide N-glycosidase F (PNGase F; lane 2: '. N') or O-glycosidase (lane 3:. '. O') to remove N-or O-linked glycosylation, respectively. The crude extract was separated by electrophoresis on a NuPAGE 4-12% Bis-Tris gel and then transferred to nitrocellulose membrane for immunodetection using chimeric rabbit IgG kappa anti-HA HRP conjugated antibody. All electrophoresis and blotting steps were performed under disulfide reduction conditions (see on-line Methods). Lane 'L', Bio-Rad Precision Plus Dual Color protein ladder.

Figure 55 shows western blots of AbLS glycosylation site mutants expressed in yeast and tobacco. This figure shows a comparison of the N-glycosylation pattern present for AbLS expressed in yeast and tobacco. Wild-type AbLS with a C-terminal HA-tag, a single glycosylation site-point mutant (N → Q) or a quadruple mutant is transiently expressed in nicotiana benthamiana ('Nb') (a) by agrobacteria or in CSY1294 ('yeast') (B) by a low copy plasmid. The preparation of crude tobacco and yeast extracts was carried out under denaturing disulfide reduction conditions (see in-line method). The crude extract was separated by electrophoresis on a NuPAGE 4-12% Bis-Tris gel and then transferred to nitrocellulose membrane for immunodetection using chimeric rabbit IgG kappa anti-HA HRP conjugated antibody. All electrophoresis and blotting steps were performed under disulfide reduction conditions (see in-line methods). For (a) and (B), corresponding yeast and tobacco expression controls were included for comparison. Lane 'L', Bio-Rad Precision Plus Dual Color protein ladder.

FIG. 56 shows the phylogenetic identification of putative endoproteolytic propeptide removal in ritodrine synthase. This figure shows the sequence alignment of AbLS with the characterized serine carboxypeptidase and SCPL acyltransferase known to have (atctc, AsSCPL1, TaCBP2) or lack (atcmt, yPRC1) an internal propeptide linker removed by proteolytic means (bold, grey). The putative N-terminal signal peptide is shown in bold (black); disulfide bonds are represented as connecting lines. AtSCT, Arabidopsis thaliana sinapoyl glucose choline sinapoyl transferase; AtSMT, Arabidopsis thaliana sinapoyl glucose, malic acid sinapoyl transferase; AbLS, belladonna ritoprim synthase; AsSCPL1, Avena sativa (Avena striatosa) avenanthrase synthase; TaCBP2, Triticum aestivum (Triticum aestivum) carboxypeptidase 2; yPRC1, yeast carboxypeptidase Y. From top to bottom: 30-35 of SEQ ID NO.

FIG. 57 shows structural identification of putative endoproteolytic propeptide removal in ritodrine synthase. This figure shows a comparison of the three-dimensional structures of two SCPL-ATs, one of which is known to contain an internal propeptide sequence that is proteolytically removed. Left side: the crystal structure of TaCBP2 in the form of (top) cartoon and (bottom) surface representations (PDB: 1WHT) shows disulfide bonds and internal propeptide removal sites. Right side: homology models of AbLS based on the crystal structure of TaCBP2 in the form of (top) cartoon and (bottom) surface representations, showing an N-terminal signal peptide, disulfide bonds, and a putative internal propeptide that appears to block active site entry.

Figure 58 shows analysis of proteolytic cleavage patterns of AbLS cleavage control and putative propeptide exchange variants in yeast. This figure shows western blot analysis of protein fragment sizes generated from AbLS split control and propeptide variants expressed in engineered yeast. The C-terminal HA-tagged AbLS variant was expressed from a low copy plasmid in CSY1294 (lanes 1-6); HA-tagged wild-type AbLS expressed in nicotiana benthamiana (Nb) was shown as another control (lane 7). Gel electrophoresis and blotting were performed under disulfide-reducing conditions, and detection was performed using anti-HA antibodies (see online methods). Lane symbols: l, protein molecular weight ladder; WT, wild-type AbLS; SPL, AbLS split at the putative propeptide with a signal peptide on both fragments; SPL-T, AbLS split at the putative propeptide, which has no signal peptide on either fragment; GS, wild-type propeptide exchange to AbLS variant of flexible Gly-Ser linker; SCT, exchange of wild type propeptide to AbLS variant of atctc propeptide sequence; CUT, wild-type propeptide exchanged for an AbLS variant at a synthetic polyarginine site recognized and cleaved by the Kex2p protease.

Figure 59 shows the production of de novo scopolamine and scopolamine in yeast strains engineered to express AbLS N-terminal fusions. This figure shows a comparison of the production of de novo scopolamine and scopolamine in yeast strains expressing AbLS with different soluble protein domains fused to the N-terminus. Wild type (control) or AbLS fusions were expressed from low copy plasmids in CSY 1294. The transformed strains were cultured in selective medium for 96h before LC-MS/MS analysis of the metabolites in the culture supernatant. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001.

Figure 60 shows fluorescence microscopy of tobacco alkaloid transporter expressed in CSY1296 to alleviate vacuolar TA transport restriction. This figure shows fluorescence microscopy images of engineered yeast expressing tobacco alkaloid transporter fused at its C-terminus to GFP, enabling identification of its subcellular localization. (A) The C-terminal GFP fusion of NtJAT1 and (B) NtMATE2 was expressed from a low copy plasmid in CSY 1296. Scale bar, 5 μm.

Figure 61 shows the production of tropine, scopolamine and scopolamine in CSY1296 engineered to express a heterologous alkaloid transporter. This figure shows the utility of different plant alkaloid transporters in alleviating intracellular substrate transport limitations in yeast engineered for TA production. Common tobacco jasmonate inducible alkaloid transporter 1(NtJAT1), multidrug and toxin efflux (MATE) transporter 1 or 2, or negative control (BFP) is expressed from a low copy plasmid in CSY 1296. The transformed strains were cultured in selective medium for 96h before LC-MS/MS analysis of the metabolites in the culture supernatant. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001.

FIG. 62 shows LC-MS/MS chromatograms in (A) product ion mode and (B) multiple reaction monitoring mode, showing de novo production of non-native TA cinnamoyl tropine in engineered yeast. This figure shows LC-MS/MS analysis of engineered yeast strains that produce non-native TA cinnamoyl tropine. (A) Tandem MS/MS spectra of extracellular media of: (i) a tropine-producing strain CSY 1251; (ii) CSY1251 expressing phenylalanine ammonia lyase (AtPAL1), 4-coumarate-CoA ligase 5(At4CL5) and cocaine synthase (EcCS), denoted CSY 1282; or (iii) a true cinnamoyl tropine standard with a parent mass m/z + ═ 272. The blue diamonds represent the parent compound peaks. (B) The activity of EcCS acyltransferase on cinnamic acid and alpha-tropine is verified by substrate feeding. Strains were transformed with combinations of plasmids expressing AtPAL1 (low copy plasmid pCS4252) and/or At4CL5 and EcCS (high copy plasmid pCS4207) and then cultured in media with different supplemental substrates as follows: (i) cen. pk2+ At4CL5+ EcCS +0.1mM trans-cinnamic acid; (ii) CEN. PK2+ At4CL5+ EcCS +0.5mM α -tropine; (iii) pk2+ AtPAL1+ At4CL5+ EcCS; (iv) pk2+ AtPAL1+ At4CL5+ EcCS +0.5mM α -tropine; (v) CSY1251+ At4CL5+ EcCS; (vi) CSY1251+ At4CL5+ EcCS +0.2mM trans-cinnamic acid; (vii) CSY1251+ AtPAL1+ At4CL5+ EcCS; (viii)25nM cinnamoyl tropine standard. For (A) and (B), the yeast strains were cultured in selective medium (YNB-DO + 2% dextrose + 5% glycerol) for 72h at 25 ℃ prior to LC-MS/MS analysis.

FIG. 63 shows the effect of (A) different carbon sources fed alone or (B) with dextrose on the production of tropine and related TA precursors in liquid cultures of engineered yeast. This figure shows the optimization of carbon sources to increase the yield of tropine in engineered yeast. An overnight culture of the tropicalis strain CSY1249 (see example 3.3.4) was grown in non-selective enrichment medium (YPD). The overnight cultures were pelleted and resuspended in a non-selective limiting medium (YNB-SC) with all amino acids and either (A) 2% of each carbon source or (B) 2% of dextrose and 2% of each additional carbon source, including dextrose. Cultures were grown at 25 ℃ for 48h before analysis of the growth medium by LC-MS/MS. The data show the relative titers of each metabolite normalized against (a) 2% dextrose or (B) 2% + 2% dextrose. Data represent the mean of three biological replicates and error bars indicate standard deviation.

FIG. 64 shows a metabolic bottleneck analysis of scopolamine producing strain CSY 1296. This figure shows the effect of expressing additional copies of the flux limiting enzyme on the yield of TA and TA precursors in engineered yeast. Additional copies of each biosynthetic enzyme between tropine and scopolamine were expressed in strain CSY1296 from the following low copy plasmids: (A) WfPPR, pCS 4436; (B) AbUGT, pCS 4440; (C) DsRed-AbLS, pCS 4526; (D) AbCYP80F1, pCS 4438; (E) DsHDH, pCS 4478; (F) DsH6H, pCS 4439; or BFP controls (pCS4208, pCS4212 or pCS4213) corresponding to the same auxotrophic marker as the respective biosynthetic gene plasmid. The transformed strains were cultured in an appropriate selective medium at 25 ℃ for 96 hours before quantification of the metabolites in the growth medium by LC-MS/MS. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001.

Figure 65 shows the effect of alleviating flux and transport limitations on scopolamine and scopolamine production in engineered yeast. This figure shows a comparison of the production of de novo scopolamine and scopolamine in yeast strains CSY1296 and CSY1297, the latter having additional genomic copies of the flux restriction enzymes (WfPPR and DsH6H) and a tobacco vacuolar alkaloid inward transporter (NtJAT 1). The strains were cultured in non-selective medium for 96h before LC-MS/MS analysis of the metabolites in the culture supernatant. Data represent the mean (open circles) of 3 biologically independent samples and error bars show standard deviation. Two-tailed t test of students: p <0.05, P <0.01, P < 0.001.

Definition of

Before describing exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used in the specification.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2 nd edition, John Wiley AND Sons, New York (1994), AND Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide the skilled artisan with a general meaning for many OF the terms used herein. Nevertheless, for clarity and ease of reference, certain terms are defined below.

It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "primer" refers to one or more primers, i.e., a single primer and multiple primers. It is also to be noted that the claims may be drafted to exclude any optional element. Accordingly, the statements are intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only," etc., or use of a "negative" limitation in connection with the recitation of claim elements.

As used herein, the terms "determining," "measuring," "evaluating," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

The term "polypeptide" as used herein refers to a polymeric form of amino acids of any length, including peptides in the range of 2-50 amino acids in length and polypeptides greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone in which the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone. The polypeptide may be of any convenient length, for example 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. A "peptide" may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the length of the peptide is between 5 and 30 amino acids.

As used herein, the term "isolated" refers to a portion of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free of other components associated with the portion prior to purification.

As used herein, the term "encoded by … …" refers to a nucleic acid sequence that encodes a polypeptide sequence, wherein the polypeptide sequence or portion thereof contains an amino acid sequence of 3 or more amino acids, such as 5 or more, 8 or more, 10 or more, 15 or more, or 20 or more amino acids from a polypeptide encoded by the nucleic acid sequence. The term also encompasses polypeptide sequences that are immunologically identifiable using the polypeptides encoded by the sequences.

A "vector" is capable of transferring a gene sequence to a target cell. As used herein, the terms "vector construct", "expression vector" and "gene transfer vector" are used interchangeably to mean any nucleic acid construct capable of directing the expression of a gene of interest, and which can transfer a gene sequence to a target cell, either by genomic integration of all or a portion of the vector, or transient or genetic maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning and expression vehicles as well as integrating vectors.

An "expression cassette" includes any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest operably linked to a promoter of the expression cassette. Such cassettes are constructed as "vectors", "vector constructs", "expression vectors" or "gene transfer vectors" for the transfer of the expression cassette into a target cell. Thus, the term includes cloning and expression vehicles as well as viral vectors.

A "plurality" comprises at least 2 members. In certain instances, a plurality may have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 106 or more, 107 or more, 108 or more, or 109 or more members. In any embodiment, a plurality may have 2-20 members.

The term "tropane alkaloid product" is intended to mean any molecule whose backbone contains an 8-azabicyclo [3.2.1] octane core group comprising a cycloheptane ring and a nitrogen bridge connecting carbon atoms 1 and 5, wherein the 8-azabicyclo [3.2.1] octyl group is covalently bonded to the acyl group through an ester bond at position 3, and/or wherein the 8-azabicyclo [3.2.1] octyl group is functionalized at position 3 with a hydroxyl group and is functionalized at positions 2, 4, 5, 6 and/or 7 with one or more hydroxyl groups. Tropane alkaloid products include, but are not limited to, ritodrine, hyoscyamine, atropine, anisodamine, scopolamine, cocaine, and any other similar tropine/pseudotropine + acyl natural or non-natural tropane alkaloids (e.g., calamine).

The term "precursor of a tropane alkaloid product" is intended to mean any molecule that can be biosynthesized by an organism from a carbon source and a nitrogen source and that can be converted to a tropane alkaloid product in one or more (e.g., one or two) biosynthetic steps; wherein the carbon source is a carbohydrate, a non-carbohydrate sugar, a sugar alcohol, a lipid, a fatty acid, or a substrate that is converted by a metabolic pathway to one or more of the foregoing carbon sources; and wherein the nitrogen source is ammonia, urea, nitrate, nitrite, any amino acid other than glutamic acid, arginine, ornithine and citrulline, a peptide, a protein, or any substrate that is converted by a metabolic pathway to one or more of the above nitrogen sources.

The term "derivative of a tropane alkaloid product" is intended to mean any molecule that is not naturally produced by an unmodified organism, wherein the backbone of said molecule comprises the tropane alkaloid product and is distinguished from said tropane alkaloid product by the attachment of functional groups, without modification of the backbone itself. As used herein, attachment of functional groups includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation, and halogenation.

Numerical ranges include the numbers defining the range.

The methods described herein comprise a number of steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. Thus, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, a step may be performed after completion of an incubation or waiting period (e.g., a few minutes to an overnight waiting period) following a previous step.

Other definitions of terms may appear throughout the specification.

Detailed Description

Host cells engineered to produce Tropane Alkaloids (TAs) of interest, such as scopolamine and scopolamine, are provided. The host cell may have one or more engineered modifications selected from: a feedback inhibition mitigating mutation in an enzyme gene; transcriptional regulatory modifications of biosynthetic enzyme genes; an inactivating mutation in the enzyme; and heterologous coding sequences. Also provided are methods of using the host cells to produce the TA of interest, as well as compositions, e.g., kits, systems, etc., that can be used in the methods of the invention.

Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Certain ranges are presented herein as numerical values preceded by the term "about". The term "about" is used herein to provide literal support for the exact number following it, as well as numbers that are near or approximate to the number following the term. In determining whether a number is near or approximate to a specifically recited number, a near or approximate non-recited number may be a number that provides substantial equivalence of the specifically recited number in the context in which it appears.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and were set forth in its entirety herein to disclose and describe the methods and/or materials in connection with which the publications were cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method proceeds in the order of recited events or in any other order that is logically possible.

In further describing the invention, the TA precursors, TA and modifications of TA of interest, including derivatives of TA, are first described in more detail, followed by a description of the host cells used to produce them. Next, an overview of the methods of interest in which host cells can be used is given. Kits useful for practicing the methods of the invention are also described.

Tropane Alkaloid (TA) precursors

As described above, host cells are provided that produce tropane alkaloid precursors (TA precursors). The TA precursor can be any intermediate or precursor compound in a synthetic pathway (e.g., as described herein) that results in the production of a TA of interest (e.g., as described herein). In some cases, a TA precursor has a structure that can be characterized as TA or a derivative thereof. In certain instances, a TA precursor has a structure that can be characterized as a fragment of TA. In some cases, the TA precursor is early TA. As used herein, "early TA" refers to an early intermediate in the synthesis of the TA of interest in a cell, where the early TA is produced by the host cell from host cell starting materials or simple starting compounds. In some cases, early TA is a TA intermediate produced by the subject host cell solely from host cell feedstocks (e.g., carbon and nutrient sources) without the need to add starting compounds to the cell. The term early TA may refer to the precursor of the TA end product of interest, whether or not the early TA itself may be characterized as a tropane alkaloid.

In some cases, the TA precursor is an early TA, such as a pronotropin tropane alkaloid or a pronitolins tropane alkaloid. Thus, host cells are provided that produce both a pronotropic tropane alkaloid (pronotropic TA) and a pronotropic tropane alkaloid (proniline TA). Tropine is a major branch point intermediate of interest in downstream TA synthesis through cell engineering efforts to produce end products such as pharmaceutical TA products derived from ritorelin (figure 2). The subject host cells can produce TA precursors from simple and inexpensive starting materials that can be used to produce tropine, ritodrine, and downstream TA end products.

As used herein, the terms "pre-esterified tropane alkaloid", "pre-esterified TA", and "pre-esterified TA precursor" are used interchangeably and refer to a biosynthetic precursor of ritodrine, cinnamoyl tropine, or other products of esterification of an acyl donor and acyl acceptor, whether or not the structure of the esterified precursor itself is characterized as a tropane alkaloid. The term pre-esterified TA is intended to include biosynthetic precursors, intermediates and metabolites thereof of any convenient member of the host cell biosynthetic pathway that can produce an esterification product such as ritulin. In some cases, the pre-esterified TA comprises a tropane alkaloid fragment, such as a tropine fragment, a phenylpropane fragment, or a precursor or derivative thereof. In some cases, the pre-esterified TA has a structure that can be characterized as a tropane alkaloid or derivative thereof.

TA precursors of interest include, but are not limited to, tropine and phenyllactic acid (PLA), as well as tropine and PLA precursors such as arginine, ornithine, agmatine, N-carbamoylputrescine (NCP), putrescine, N-methylputrescine (NMP), 4-methylaminobutyraldehyde, N-methylpyrrolidinium (NMPy), 4- (1-methyl-2-pyrrolidinyl) -3-oxobutanoic acid (MPOB), tropinone, phenylalanine, prephenate, and phenylpyruvic acid (PPA). In some embodiments, the one or more TA precursors are trope and PLA. In certain instances, the one or more TA precursors are trope and a phenylpropanoic acid other than PLA, such as cinnamic acid. Figures 1, 2 and 3 show the biosynthesis of non-medicinal, medicinal and non-natural TAs from various TA and non-TA precursor molecules, respectively.

The synthetic pathway for the TA precursor may be produced in the host cell and may begin with any convenient starting compound or material. FIGS. 1-4 show interesting synthetic pathways for TA precursors starting from amino acids. The starting material may be non-naturally occurring, or the starting material may be naturally occurring in the host cell. Any convenient compounds and materials may be used as starting materials, based on the synthetic pathways present in the host cell. The source of the starting material may be from the host cell itself, e.g. arginine or phenylalanine, or the starting material may be added or supplemented to the host cell from an external source. Thus, in some instances, a starting compound refers to a compound in a cellular synthetic pathway that is added to a host cell from an external source that is not a growth feedstock or part of the cell growth medium. Starting compounds of interest include, but are not limited to, N-methylputrescine, 4-methylaminobutyraldehyde, tropinone, tropine, PLA, cinnamic acid, and any of the compounds shown in FIGS. 1-4. For example, if the host cell is grown in liquid media, the cell culture media can be supplemented with starting materials that are transported into the cell and converted by the cell into the desired product. Starting materials of interest include, but are not limited to, inexpensive raw materials and simple precursor molecules. In some cases, the host cell utilizes a feedstock comprising a simple carbon source as a starting material, which the host cell utilizes to produce compounds of the cellular synthetic pathway. The host cell growth feedstock may comprise one or more components such as a carbon source such as cellulose, starch, free sugars and a nitrogen source such as ammonium salts or inexpensive amino acids. In some cases, the growth feedstock useful as a starting material can be derived from sustainable sources, such as biomass grown on marginal land, including switchgrass and algae, or biomass waste products from other industrial or agricultural activities.

Tropane Alkaloids (TA)

As described above, host cells are provided that produce a Tropane Alkaloid (TA) of interest. In some embodiments, the engineered strains of the present invention will provide a platform for the production of tropane alkaloids of interest and their modifications across several classes, including but not limited to pharmaceutical TAs, such as those derived from tropes and PLAs; non-pharmaceutically acceptable TAs, such as those derived from tropinone, pseudotropine, or norpseudotropine; and non-native TA, such as those derived from esterification of TA precursors (e.g., acyl donor and acyl acceptor compounds) rather than tropine and PLA. Each of these classes is intended to include biosynthetic precursors, intermediates and metabolites thereof of any convenient member of the host cell biosynthetic pathway that can produce a member of that class. Non-limiting examples of compounds are given below for each of these classes. In some embodiments, the structure of a given example may or may not itself be characterized as a tropane alkaloid. The chemical entities of the present invention are intended to include all possible isomers, including single enantiomers, racemic mixtures, optically pure forms, mixtures of diastereomers, and intermediate mixtures.

The pharmaceutically acceptable TA may include, but is not limited to, ritodrine, hyoscyamine, atropine, anisodamine, scopolamine, and derivatives thereof, which are naturally produced by plants.

Non-medicinal TAs may include, but are not limited to, calyculin, cocaine and derivatives thereof naturally produced by plants.

Non-natural TA may include, but is not limited to, cinnamoyl tropine, cinnamoyl-3 β -tropine, coumaroyl-3 β -tropine, benzoyl-3 β -tropine, caffeoyl-3 β -tropine, feruloyl tropine, and feruloyl-3 β -tropine.

Modifications of TA including derivatives

As described above, host cells are provided that produce modified derivatives of a Tropane Alkaloid (TA) of interest. In some embodiments, the engineered strains of the invention will provide a platform for derivatizing the TA of interest, including derivatizing a TA precursor, a pharmaceutically acceptable TA, a non-pharmaceutically acceptable TA, and a non-native TA of the engineered host cell produced by the engineered host cell or fed into a growth medium.

As used herein, the terms "derivatize," "functionalize," "modify by derivatizing," and "modify by functionalization" refer to the modification of a TA or TA precursor by the attachment of a functional group, without modifying the TA backbone itself. As used herein, attachment of functional groups includes, but is not limited to, hydroxylation, alkylation and N-alkylation, acetylation and N-acetylation, acylation and N-acylation, and halogenation.

In some embodiments of the invention, derivatization of the TA of interest may be accomplished enzymatically by feeding a pre-functionalized TA precursor (e.g., a halogenated or alkylated amino acid) to a host cell engineered to uptake the fed TA precursor and then convert it to the TA of interest. In other embodiments of the invention, derivatization of the TA of interest may be accomplished enzymatically by engineering the host cell to express an enzyme having the desired activity of attaching a functional group to the target TA, in addition to the enzymes and cellular modifications required to produce an unmodified TA. In other embodiments of the invention, derivatization of the TA of interest may be accomplished enzymatically by treating the unmodified TA produced by the engineered host cell with a purified enzyme capable of attaching the desired functional group or with a crude lysate of the host cell engineered to express the enzyme having the desired derivatization activity. In other embodiments of the invention, derivatization of the TA of interest can be achieved non-enzymatically by treating the unmodified TA produced by the engineered host cell with a chemical agent having the desired functional group.

Modified derivatives of TA include, but are not limited to, p-hydroxy atropine, p-hydroxy scopolamine, p-fluoroscopolamine, p-chloroscopolamine, p-bromoscopolamine, N-methyl scopolamine, N-butyl scopolamine, N-acetyl scopolamine, and N-acetyl scopolamine.

Host cell

As noted above, one aspect of the invention is a host cell that produces one or more TA of interest. Any convenient cell may be used in the subject host cells and methods. In some cases, the host cell is a non-plant cell. In some cases, the host cell can be characterized as a microbial cell. In certain instances, the host cell is an insect cell, a mammalian cell, a bacterial cell, or a fungal cell. Any convenient type of host cell can be used to produce the subject TA-producing cells, see, e.g., US2008/0176754, US2014/0273109, and WO2014/143744, now published as U.S. patent No. 8,975,063); the disclosure of which is incorporated by reference in its entirety. Host cells of interest include, but are not limited to, bacterial cells such as Bacillus subtilis, Escherichia coli, Streptomyces (Streptomyces), Anabaena (Anabaena), Arthrobacter (Arthrobacter), Acetobacter (Acetobacter), Bacillus (Bacillus), Bifidobacterium (Bifidobacterium), Brevibacterium (Brachybacterium), Brevibacterium (Brevibacterium), Carnobacterium (Carnobacterium), Clostridium (Clostridium), Corynebacterium (Corynebacterium), Enterobacter (Enterobacter), Escherichia (Escherichia), Gluconobacter (Gluconobacter), Hafnia (Hafnia), Halomonas, Klebsiella, Leuconobacter (Klebsiella), Leuconobacter (Methylobacter), Lactobacillus (Lactobacillus), and Lactobacillus (Lactobacillus), Lactobacillus (Lactobacillus), and Lactobacillus (Lactobacillus) strains, and the like, Methylcellular genus (Methylcella), Methylcoccus genus (Methylcoccus), Microbacterium genus (Micrococcus), Micrococcus genus (Micrococcus), Microcystis genus (Microcystis), Moorella genus (Moorella), Oenococcus genus (Oenococcus), Pediococcus genus (Pediococcus), Prochlorococcus genus (Prochlorococcus), Propionibacterium genus (Propionibacterium), Proteus genus (Proteus), Pseudoalteromonas genus (Pseudomonas), Pseudomonas genus (Pseudomonas), psychrophilum genus (Psychlorobacterium), Rhodococcus genus (Rhodobacter), Rhodopseudomonas genus (Rhodopseudomonas), Serratia genus (Serratia), Staphylococcus genus (Photococcus genus), Streptococcus genus (Streptococcus genus), Streptomyces genus (Synechococcus), Synechococcus genus (Synechococcus), Salmonella genus (Salmonella) and Spirosoma cells such as Spirosoma typhus, Spirosoma cells (Spirosoma) and Spirosoma cells such as Spirosoma typhus genus (Spirosoma) and Spirosoma cells such as Spirosoma sp, and yeast cells such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Pichia pastoris (Pichia pastoris), Yarrowia lipolytica (Yarrowia lipolytica), Candida albicans (Candida albicans), Aspergillus sp., Rhizopus sp., Penicillium sp., and Trichoderma reesei (Trichoderma reesei) cells. In some embodiments, the host cell is a yeast cell or an e. In some cases, the host cell is a yeast cell. In some cases, the host cell is from a yeast strain engineered to produce the TA of interest. Any of the host cells described in US2008/0176754, US2014/0273109, and WO2014/143744, which have now been published as US patent No. 8,975,063, may be suitable for use in the subject cells and methods. In certain embodiments, the yeast cell may belong to the species saccharomyces cerevisiae (s. In certain embodiments, the yeast cell may belong to the species schizosaccharomyces pombe. In certain embodiments, the yeast cell can belong to the pichia species. Yeast is of great interest as a host cell because cytochrome P450 proteins involved in some biosynthetic pathways of interest are able to fold correctly into the endoplasmic reticulum membrane, thereby retaining their activity.

Yeast strains of interest that can be used in the present invention include, but are not limited to, CEN.PK (genotype: MATa/α ura3-52/ura3-52trp1-289/trp1-289 leu2-3_112/leu2-3_112his3 Δ 1/his3 Δ 1MAL2-8C/MAL2-8C SUC2/SUC2), S288C, W303, D273-10 962180, A364A, Σ 1278B, AB, SK1 and FL 100. In some cases, the yeast strain is any one of the following: S288C (MAT α; SUC2 mal mel gal2 CUP1 flo1 flo 1-1 hap1), BY4741(MAT α; his 1 Δ 1; leu 1 Δ 0; MET1 Δ 0; ura 1 Δ 0), BY4742(MAT α; his 1 Δ 1; leu 1 Δ 0; LYS 1 Δ 0; ura 1 Δ 0), BY 43(MATa/MAT α; his 1 Δ 1/his 1 Δ 1; leu 1 Δ 0/leu 1 Δ 0; MET1 Δ 0/MET 1; LYS 1/LYS 1 Δ 0; ura 1 Δ 0/ura 1 Δ 0) and WAT1 or a derivative of W (MATR), W303-B strain (MATa; MATa-cyE 1-NADPH 1; NADPH 1-NADPH 1-NADPH 1; NADPH 1-NADPH 1, NADPH 1-1, NADPH-1, and NADPH-1, respectively. In another embodiment, the yeast cell is W303 α (MAT α; his3-11, 15trp1-1 leu2-3 ura3-1 ade 2-1). The identity and genotype of other yeast strains of interest can be found in EUROSCARF (web. uni-frankfurt. de/fb 15/mikro/eurocarf/col _ index. html).

In some cases, the host cell is a fungal cell. In certain embodiments, the fungal cell may belong to the Aspergillus species and the strains include Aspergillus niger (Aspergillus niger) (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (Aspergillus oryzae) (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624, ATCC 20542), and Aspergillus nidulans (FGSC a 4).

In certain embodiments, the heterologous coding sequence may be codon optimized for expression in aspergillus species and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from the group consisting of a phosphoglycerate kinase Promoter (PGK), an MbfA promoter, a cytochrome c oxidase subunit promoter (CoxA), an SrpB promoter, a TvdA promoter, a malate dehydrogenase promoter (MdhA), a β -mannosidase promoter (ManB). In certain embodiments, the terminator may be selected from a glucoamylase terminator (GlaA) or TrpC terminator. In certain embodiments, the expression cassette consisting of the promoter, heterologous coding sequence, and terminator may be expressed from a plasmid or integrated into the genome of the host. In certain embodiments, selection of cells to maintain the plasmid or integration cassette can be performed by antibiotic selection such as hygromycin or a nitrogen source, using, for example, acetamide as the sole nitrogen source. In certain embodiments, the DNA construct may be introduced into the host cell using established transformation methods such as protoplast transformation, lithium acetate, or electroporation. In certain embodiments, cells can be cultured, with or without selection, in liquid ME or solid MEA (3% malt extract, 0.5% peptone, and ± 1.5% agar) or in Vogel minimal medium.

In some cases, the host cell is a bacterial cell. The bacterial cells may be selected from any bacterial species. Examples of species from which the bacterial cells may be derived include anabaena, arthrobacter, acetobacter, bacillus, bifidobacterium, brevibacterium, carnobacter, clostridium, corynebacterium, enterobacter, escherichia, gluconacetobacter, hafnia, halophilomonas, klebsiella, cocklebur, lactobacillus, leuconostoc, megacoccus, methylomonas, methylobacter, methylcellus, microbacterium, micrococcus, moore, oenopoccus, oenococcus, pediococcus, protochlorococcum, propionibacterium, proteobacterium, pseudoalteromonas, pseudomonas, psychrophilum, rhodobacter, rhodococcus, rhodopseudomonas, serratia, rhodobacter, and rhodobacter, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, and Zymomonas. Examples of bacterial species that may be used with the methods of the present disclosure include Arthrobacter nicotianae (Arthrobacter nicotianae), Acetobacter xylinum (Acetobacter aceti), Arthrobacter albus (Arthrobacter arilotenis), Bacillus cereus (Bacillus cereus), Bacillus coagulans (Bacillus coagulosus), Bacillus licheniformis (Bacillus licheniformis), Bacillus pumilus (Bacillus pumilus), Bacillus sphaericus (Bacillus sphaericus), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus subtilis (Bacillus subtilis), Bacillus adolescentis (Bifidobacterium adolescentis), Bacillus brevis (Bacillus typhimurium), Bacillus expansus (Bacillus subtilis), Bacillus euroticus), Bacillus subtilis (Bacillus adolescentis), Bacillus subtilis (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus amyloliquefaciens), Bacillus amyloliquefaciens (Corynebacterium glutamicum), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus amyloliquefaciens, Bacillus acidophylum, and Bacillus amyloliquefaciens) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) can be used in, Halophilous bacteria (Halomonas elongata), Rhizophilus (Kocuria rhizophila), Lactobacillus acidovorans (Lactobacillus acidophilus), Lactobacillus jensenii (Lactobacillus jensenii), Lactococcus lactis (Lactobacillus lactis), Lactobacillus sorbus (Lactobacillus yamanasensis), Leuconostoc citreum (Leuconostoc citreum), Pediococcus casei (Microbacterium caseii), Microbacterium lobutanus (Microbacterium foliorum), Micrococcus reevesii (Micrococcus lylae), Alcoholisobus (Ocococcus oensis), Pediococcus acidilactici (Pediococcus acidilactici), Propionibacterium propionicum (Propionibacterium propionicum), Pseudomonas comatus (Pseudomonas fluorescens), Streptococcus thermophilus (Streptococcus faecalis), Streptococcus faecalis (Streptococcus thermophilus), Streptococcus faecalis (Streptococcus faecalis), Streptococcus thermophilus (Streptococcus faecalis), Streptococcus faecalis (Streptococcus), Streptococcus faecalis (Streptococcus), Streptococcus faecalis (Streptococcus), Streptococcus faecalis (Streptococcus faecalis), Streptococcus faecalis (Bacillus), Streptococcus faecalis (Bacillus), Streptococcus faecalis (Bacillus), Streptococcus faecalis (Bacillus), Streptococcus faecalis (Bacillus), Streptococcus (Bacillus faecalis), Streptococcus faecalis (Bacillus, Streptococcus), Streptococcus (Bacillus, Streptococcus), Streptococcus (Bacillus faecalis (Bacillus, Streptococcus), Streptococcus (Bacillus, Streptococcus), Streptococcus (Bacillus faecalis (Bacillus, Streptococcus), Bacillus faecalis (Bacillus, Streptococcus), Streptococcus (Bacillus, Streptococcus), Bacillus faecalis (Bacillus, Streptococcus), Bacillus, Streptococcus (Bacillus faecalis (Bacillus, Streptococcus), Bacillus, Streptococcus (Bacillus, Streptococcus), Bacillus, Streptococcus (Bacillus faecalis (Bacillus, Streptococcus), Bacillus, Streptococcus (Bacillus, Streptococcus), Bacillus, Streptococcus (Bacillus, Streptococcus), Bacillus, Streptococcus (Bacillus, Streptococcus, Bacillus, Streptococcus, Bacillus, Streptococcus, corynebacterium glutamicum (Corynebacterium glutamicum), Bifidobacterium breve/longum (Bifidobacterium bifidum/breve/longum), Streptomyces lividans (Streptomyces lividans), Streptomyces coelicolor (Lactobacillus coelicolor), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus sakei (Lactobacillus sakei), Lactobacillus casei (Lactobacillus casei), Pseudomonas citrobacter (Pseudomonas citrobacter), Pseudomonas putida (Pseudomonas putida), Young/Acetoxynol/Butanol/Clostridium bailii/Clostridium butyricum (Clostridium ljungdahlii/Aceticum/Acetobacter/Acetobutylicum), and Tyr/Thermoacetobacter xylinum (Thermomyces thermophilus/Thermomyces thermophilus).

In certain embodiments, the bacterial cell may belong to an escherichia coli strain. In certain embodiments, the E.coli strain may be selected from BL21, DH5 α, XL1-Blue, HB101, BL21, and K12. In certain embodiments, the heterologous coding sequence may be codon optimized for expression in e.coli and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from the group consisting of the T7 promoter, tac promoter, trc promoter, tetracycline inducible promoter (tet), lac operator promoter (lac), lacO1 promoter. In certain embodiments, the expression cassette consisting of the promoter, heterologous coding sequence, and terminator may be expressed from a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pUC19 or pBAD. In certain embodiments, selection of cells that maintain the plasmid or integration cassette can be performed by antibiotic selection, such as kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamicin, erythromycin, or ampicillin. In certain embodiments, the DNA construct may be introduced into the host cell using established transformation methods such as conjugation, heat shock chemical transformation, or electroporation. In certain embodiments, the cells may be cultured in liquid Luria-Bertani (LB) medium at about 37 ℃ with or without antibiotics.

In certain embodiments, the bacterial cell may be a strain of bacillus subtilis. In certain embodiments, the strain of Bacillus subtilis may be selected from 1779, GP25, RO-NN-1, 168, BSn5, BEST195, 1A382, and 62178. In certain embodiments, the heterologous coding sequence may be codon optimized for expression in a bacillus species and expressed from an appropriate promoter. In certain embodiments, the promoter may be selected from the grac promoter, the p43 promoter, or the trnQ promoter. In certain embodiments, the expression cassette consisting of the promoter, heterologous coding sequence, and terminator may be expressed from a plasmid or integrated into the genome. In certain embodiments, the plasmid is selected from pHP13 pE194, pC194, pHT01, or pHT 43. In certain embodiments, an integration vector such as pDG364 or pDG1730 may be used to integrate the expression cassette into the genome. In certain embodiments, selection of cells that maintain the plasmid or integration cassette can be performed by antibiotic selection, such as erythromycin, kanamycin, tetracycline, and spectinomycin. In certain embodiments, the DNA construct may be introduced into the host cell using established transformation methods such as natural competence, heat shock, or chemical transformation. In certain embodiments, the cells may be cultured in liquid Luria-Bertani (LB) medium at 37 ℃ or in M9 medium supplemented with glucose and tryptophan.

Genetic modification of host cells

The host cell can be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of the TA of interest. In some cases, a modification refers to a genetic modification, such as a mutation, addition, or deletion of a gene or a fragment thereof, or transcriptional regulation of a gene or a fragment thereof. In some cases, one or more (such as two or more, three or more, or four or more) modifications are selected from: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; inactivating mutations in enzymes indigenous to the cell; a heterologous coding sequence encoding an enzyme; and a heterologous coding sequence encoding a protein that improves subcellular trafficking and/or localization of the enzyme or metabolite. A cell that includes one or more modifications may be referred to as a modified cell.

The modified cell may overproduce one or more of the precursor TA, or modified TA molecule. Overproduction refers to cells that have improved or increased production of the TA molecule of interest relative to a control cell (e.g., an unmodified cell). Improved or increased production refers to production of an amount of the TA of interest when the control is not producing TA precursors, and an increase of about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, such as 2-fold or more, such as 5-fold or more, including 10-fold or more, in the case of a control with some TA production of interest.

In some cases, the host cell is capable of producing an increased amount of putrescine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of putrescine is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of N-methylpyrrolidinium relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of N-methylpyrrolidinium is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of a tropine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, an increased amount of a tropine is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of phenylpyruvic acid relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of phenylpyruvic acid is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of phenyllactic acid relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of phenyllactic acid is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of ritodrine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of ritodrine is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of scopolamine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of scopolamine is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some cases, the host cell is capable of producing an increased amount of scopolamine relative to a control host cell lacking one or more modifications (e.g., as described herein). In certain instances, the increased amount of scopolamine is an increase of about 10% or more relative to a control host cell, such as an increase of about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, 2-fold or more, 5-fold or more, or even 10-fold or more relative to a control host cell.

In some embodiments, the host cell is capable of producing 10% or greater yield of the tropine from a starting compound, such as arginine, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield of the tropine from the starting compound.

In some embodiments, the host cell is capable of producing 10% or greater yield of phenyllactic acid from a starting compound, such as phenylalanine, such as 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even 90% or greater yield of phenyllactic acid from a starting compound.

In some embodiments, the host cell is capable of producing a 10% or higher yield of hyoscyamine from a starting compound, such as arginine or phenylalanine, such as a 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, or even a 90% or higher yield of hyoscyamine from the starting compound.

In some embodiments, the host cell is capable of producing a 10% or greater yield of scopolamine from a starting compound, such as arginine or phenylalanine, such as a 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, or even a 90% or greater yield of scopolamine from the starting compound.

In some embodiments, the host cell overproduces one or more TA molecules of interest selected from the group consisting of: arginine, ornithine, agmatine, putrescine, N-methylputrescine, 4-methylaminobutyraldehyde, N-methylpyrrolidinium, 4- (1-methyl-2-pyrrolidinyl) -3-oxobutanoic acid, tropinone, tropine, phenylalanine, prephenate, phenylpyruvic acid, phenyllactic acid, glucose-1-O-phenyllactate, ritoline, hyoscyaldehyde, hyoscyamine, anisodamine, and scopolamine.

Any convenient combination of one or more modifications may be included in a subject host cell. In some cases, two or more (such as two or more, three or more, or four or more) different types of modifications are included. In certain instances, two or more (such as three or more, four or more, five or more, or even more) different modifications of the same modification type are included in a subject cell.

In some embodiments of the host cell, when the cell comprises one or more heterologous coding sequences encoding one or more enzymes, it comprises at least one additional modification selected from the group consisting of: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; and inactivating mutations in enzymes inherent to the cell. In certain embodiments of the host cell, when the cell comprises one or more feedback inhibition mitigating mutations in one or more biosynthetic enzyme genes native to the cell, it comprises at least one additional modification selected from the group consisting of: transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; inactivating mutations in enzymes indigenous to the cell; and a heterologous coding sequence encoding an enzyme. In some embodiments of the host cell, when the cell comprises one or more transcriptional regulatory modifications of one or more biosynthetic enzyme genes native to the cell, it comprises at least one additional modification selected from the group consisting of: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; inactivating mutations in enzymes indigenous to the cell; a heterologous coding sequence encoding an enzyme; and a heterologous coding sequence encoding a protein that improves subcellular trafficking and/or localization of the enzyme or metabolite. In certain instances of the host cell, when the cell comprises one or more inactivating mutations in one or more enzymes native to the cell, it comprises at least one additional modification selected from the group consisting of: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; a heterologous coding sequence encoding an enzyme; and a heterologous coding sequence encoding a protein that improves subcellular trafficking and/or localization of the enzyme or metabolite.

In certain embodiments of the host cell, the cell comprises one or more feedback inhibition mitigating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more transcriptional regulatory modifications of one or more biosynthetic enzyme genes native to the cell. In certain embodiments of the host cell, the cell comprises one or more feedback inhibition mitigating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more inactivating mutations in enzymes native to the cell. In certain embodiments of the host cell, the cell comprises one or more feedback inhibition mitigating mutations in one or more biosynthetic enzyme genes native to the cell; and one or more heterologous coding sequences. In some embodiments, the host cell comprises one or more modifications (e.g., as described herein) comprising one or more genes of interest described in table 1.

Feedback inhibition mitigating mutations

In some cases, the host cell is a cell that includes one or more feedback inhibition mitigating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell (e.g., present in an unmodified cell). As used herein, the term "feedback inhibition mitigating mutation" refers to a mutation that mitigates the feedback inhibition control mechanism of a host cell. Feedback inhibition is a control mechanism of a cell in which enzymes in the synthetic pathway of a regulated compound are inhibited when the compound accumulates to a certain level, thereby balancing the amount of the compound in the cell. In some cases, the one or more feedback inhibition mitigating mutations are in an enzyme depicted in the biosynthetic pathway of fig. 1-4 or the schematic of fig. 8. Mutations that mitigate feedback inhibition reduce the inhibition of the regulated enzyme in the cell of interest relative to a control cell and provide increased levels of the regulated compound or its downstream biosynthetic products. In some cases, alleviating the inhibition of a regulated enzyme refers to the IC of inhibition ₅₀Increase by a factor of 2 or more, such as 3 or more, 5 or more, 10 or more, 30 or more, 100 or more, 300 or more, 1000 or more, or even more. An increased level refers to a level that is 110% or more of the level of the modulated compound or its downstream product in a control cell, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 18% or more of the level of the modulated compound or its downstream product in a host cell0% or more, 190% or more, or 200% or more, such as at least 3 times or more, at least 5 times or more, at least 10 times or more, or even more.

Various feedback inhibition control mechanisms and biosynthetic enzymes inherent to host cells that are intended to regulate the level of TA precursor can be targeted for remission in the host cell. The host cell may include one or more feedback inhibition mitigating mutations in one or more biosynthetic enzyme genes native to the cell. The mutation may be in any convenient biosynthetic enzyme gene inherent to the host cell in which the biosynthetic enzyme is under regulatory control. In some embodiments, the one or more biosynthetic enzyme genes encode one or more enzymes selected from the group consisting of: ornithine Decarboxylase (ODC), ornithine decarboxylase resistant (antityme) and putrescine N-methyltransferase. In some embodiments, the one or more biosynthetic enzyme genes encode ornithine decarboxylase. In some cases, the one or more biosynthetic enzyme genes encode an ornithine decarboxylase enzyme. In some embodiments, the one or more biosynthetic enzyme genes encode putrescine N-methyltransferase. In certain instances, one or more feedback inhibition mitigating mutations are present in a biosynthetic enzyme gene selected from SPE1, OAZ1, and PMT. In certain instances, one or more feedback inhibition mitigating mutations are present in the biosynthetic enzyme gene that is SPE 1. In certain instances, one or more feedback inhibition mitigating mutations are present in the biosynthetic enzyme gene that is OAZ 1. In some cases, one or more feedback inhibition mitigating mutations are present in the biosynthetic enzyme gene that is PMT. In some embodiments, the host cell comprises one or more feedback inhibition mitigating mutations in one or more biosynthetic enzyme genes (such as one of those described in table 1).

Any convenient number and type of mutations may be utilized to alleviate the feedback inhibition control mechanism. As used herein, the term "mutation" refers to a deletion, insertion or substitution of one or more amino acid residues or one or more nucleotide residues relative to a reference sequence or motif. Mutations can be incorporated as directed mutations to the native gene at the original locus. In some cases, the mutation may be incorporated as an additional copy of the gene (introduced as genetic integration at a separate locus), or as an additional copy on an episomal vector, such as a 2 μ or centromeric plasmid. In some cases, the feedback-inhibited copy of the enzyme is under the transcriptional regulation of the native cell. In some cases, feedback-inhibiting copies of the enzyme are introduced by placing them under the control of a synthetic promoter, with engineered constitutive or dynamic regulation of protein expression.

In certain embodiments, a host cell of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition mitigating mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 feedback inhibition mitigating mutations, in one or more biosynthetic enzyme genes native to the host cell.

Transcriptional regulatory modifications

The host cell may include one or more transcriptional regulatory modifications (such as two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell. Any convenient biosynthetic enzyme gene of the cell can be targeted for transcriptional regulation. Transcriptional regulation refers to the regulation of expression, e.g., increase or decrease, enhancement or repression, of a gene of interest in a modified cell relative to a control cell (e.g., an unmodified cell). In some cases, transcriptional regulation of a gene of interest includes increasing or enhancing expression. By increasing or enhancing expression is meant an increase in the expression level of the gene of interest by 2-fold or more, such as 5-fold or more, and sometimes by 25, 50, or 100-fold or more, and in certain embodiments by 300-fold or more, as compared to a control (i.e., expression in the same cell without modification), for example, by using any convenient gene expression assay. Alternatively, in the case where the expression of the gene of interest in the cell is undetectable, the expression level of the gene of interest is considered to be increased if the expression is increased to a readily detectable level. In some cases, transcriptional regulation of a gene of interest includes reducing or repressing expression. By reduced or repressed expression is meant a 2-fold or greater reduction, such as 5-fold or greater, and sometimes a 25, 50, or 100-fold or greater reduction, and in certain embodiments a 300-fold or greater reduction in the expression level of a gene of interest as compared to a control. In some cases, expression is reduced to undetectable levels. Improvements in host cell processes of interest that may be applicable to the subject host cells are described in U.S. publication No. 20140273109(14/211,611) to Smolke et al, the disclosure of which is incorporated herein by reference in its entirety.

Any convenient biosynthetic enzyme gene can be transcriptionally regulated and includes, but is not limited to, those biosynthetic enzymes described in figures 1-3, such as ARG2, CAR1, SPE1, FMS1, PHA2, ARO8, ARO9, and UGP 1. In some cases, the one or more biosynthetic enzyme genes are selected from ARG2, CAR1, SPE1, and FMS 1. In some cases, the one or more biosynthetic enzyme genes is ARG 2. In certain instances, the one or more biosynthetic enzyme genes is CAR 1. In some embodiments, the one or more biosynthetic enzyme genes is SPE 1. In some embodiments, the one or more biosynthetic enzyme genes is FMS 1. In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes (such as one of those described in table 1). In some embodiments, the host cell comprises one or more transcriptional regulatory modifications to one or more genes, such as one of those genes depicted in the biosynthetic pathway of one of fig. 1-4 or the schematic of fig. 8.

In some embodiments, the transcriptional regulatory modification comprises replacing the native promoter of one or more biosynthetic enzyme genes with a strong promoter or expressing one or more additional copies of one or more genes under the control of a strong promoter. The promoter driving expression of the gene of interest may be a constitutive promoter or an inducible promoter, provided that the promoter may be active in the host cell. The gene of interest may be expressed from its native promoter, or a non-native promoter may be used. Although not required, such promoters should have moderate to high strength in the host in which they are used. Promoters may be regulated or constitutive. In some embodiments, promoters are used that are not subject to glucose repression or are only slightly repressed by the presence of glucose in the culture medium. There are many suitable promoters, examples of which include the promoter of the glycolytic gene, such as the promoter of the Bacillus subtilis tsr gene (encoding fructose diphosphate aldolase) or the GAPDH promoter from Saccharomyces cerevisiae (encoding glyceraldehyde-phosphate dehydrogenase) (Bitter G.A., meth. enzymol.152: 673684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter of baker's Yeast (baker's year) (Ruohonen L. et al, J.Biotechnol.39: 193203 (1995)), a phosphate starvation-inducible promoter such as the Yeast PHO5 promoter (Hinnen, A. et al, in Yeast Genetic Engineering, Barr, P.J. et al, edited by Butterworth (1989), the alkaline phosphatase promoter from Bacillus licheniformis (B.licheniformis) (Lee.J.W.K. et al, J.Gen.Microbiol.137: 11271133 (1991)), GPD1, and the Yeast promoters of interest include, but are not limited to, inducible promoters such as Gal1-10, Gal1, GalS, repressible promoters Met25, tetO, and constitutive promoters such as the glycerol aldehyde 3-dehydrogenase (MRF- α -dehydrogenase), the cytochrome oxidase promoter (ADH-alpha-reductase), the ADH-inducible promoter (ADH-P-7), the ADH-inducible promoter (ADH-P-5), the promoter of Bacillus licheniformis, ADH-L-enzyme, ADH-2, ADH-A-dehydrogenase, and the like, Phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), and the like. In some cases, the strong promoter is GPD 1. In some cases, the strong promoter is TEF 1. Autonomously replicating yeast expression vectors containing hormone-inducible promoters such as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, Glucocorticoid Response Element (GRE) and thyroid hormone response element (TRE), see, for example, those promoters described in U.S. patent No. 7,045,290. Vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH can be used. In addition, any Promoter/enhancer combination (based on Eukaryotic Promoter Data Base, EPDB)) can also be used to drive expression of the gene of interest. It will be appreciated that any convenient promoter specific for the host cell (e.g.E.coli) may be selected. In some cases, promoter selection can be used to optimize transcription, and thus enzyme levels, to maximize yield while minimizing energy resources.

Inactivating mutations

The host cell may include one or more inactivating mutations (such as two or more, three or more, four or more, five or more, or even more) of the enzymes of the cell. The inclusion of one or more inactivating mutations can improve the throughput of the synthetic pathway of the host cell to increase the level of the TA of interest or the desired enzyme or precursor that produces the TA. In some cases, the one or more inactivating mutations are directed to an enzyme native to the cell. Figure 8 shows the natural regulatory mechanisms acting on the polyamine production pathway in yeast, and figure 9 shows the effect of disruption of these natural regulatory systems on putrescine production. As used herein, "inactivating mutation" refers to one or more mutations in a gene or regulatory DNA sequence of a cell, wherein the one or more mutations inactivate a biological activity of a protein expressed by the gene of interest. In some cases, the gene is native to the cell. In some cases, the gene encodes an enzyme that is inactive and is part of, or associated with, the synthetic pathway of the TA of interest produced by the host cell. In some cases, the inactivating mutation is located in a regulatory DNA sequence that controls the gene of interest. In some cases, the inactivating mutation is directed to a promoter of the gene. Any convenient mutation (e.g., as described herein) may be used to inactivate a gene or regulatory DNA sequence of interest. "inactivated" or "inactivating a.. means that the biological activity of a protein expressed by a mutant gene is reduced by 10% or more, such as 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, relative to a control protein expressed by a non-mutant control gene. In some cases, the protein is an enzyme and the inactivating mutation reduces the activity of the enzyme.

In some embodiments, the cell comprises an inactivating mutation in an enzyme native to the cell. Any convenient enzyme may be targeted for inactivation. Enzymes of interest include, but are not limited to, those depicted in FIGS. 1-4, 8, 11, 22, and 41, whose role in the biosynthetic pathway of the host cell tends to reduce the level of TA of interest. In some cases, the enzyme has methylthioadenosine phosphorylase activity. In certain embodiments, the enzyme comprising an inactivating mutation is MEU1 (see, e.g., fig. 8, 9, and 13). In some cases, the enzyme has ornithine decarboxylase anti-enzyme activity. In certain embodiments, the enzyme comprising an inactivating mutation is OAZ 1. In some cases, the enzyme has spermidine synthase activity. In certain embodiments, the enzyme comprising an inactivating mutation is SPE 3. In some cases, the enzyme has spermine synthase activity. In some embodiments, the enzyme comprising an inactivating mutation is SPE 4. In some cases, the enzyme is a membrane transporter having polyamine outward transport activity (export activity). In certain embodiments, the enzyme or protein comprising an inactivating mutation is TPO 5. In some cases, the enzyme has a cinnamic acid decarboxylase activity. In certain embodiments, the enzyme comprising an inactivating mutation is PAD 1. In some cases, the enzyme has alcohol dehydrogenase activity. In some embodiments, the enzyme comprising an inactivating mutation is selected from the group consisting of ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA 1. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH 2. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH 3. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH 4. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH 5. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH 6. In certain embodiments, the enzyme comprising one or more inactivating mutations is ADH 7. In some cases, the enzyme has aldehyde oxidoreductase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from HFD1, ALD2, ALD3, ALD4, ALD5, and ALD 6. In certain embodiments, the enzyme comprising one or more inactivating mutations is HFD 1. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD 2. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD 3. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD 4. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD 5. In certain embodiments, the enzyme comprising one or more inactivating mutations is ALD 6. In some cases, the enzyme has glucosidase activity. In certain embodiments, the enzyme comprising an inactivating mutation is selected from EXG1, SPR1 and EGH 1. In certain embodiments, the enzyme comprising one or more inactivating mutations is EXG 1. In certain embodiments, the enzyme comprising one or more inactivating mutations is SPR 1. In certain embodiments, the enzyme comprising one or more inactivating mutations is EGH 1. In some embodiments, the host cell comprises one or more inactivating mutations of one or more genes described in table 1.

Method for TA acyltransferase reactions using functional expression of acyltransferases in non-plant hosts

Some of the methods, processes, and systems provided herein describe the concerted reaction of one or more TA precursors comprising an acyl donor group with one or more TA precursors comprising an acyl acceptor group in a non-plant cell to produce one or more TAs (hereinafter referred to as TA transacylation reactions). Some of these methods, processes, and systems may include engineered host cells. In some examples, the TA transacylation reaction is a key step in the conversion of substrates to various alkaloids. In some examples, the TA transacylation reaction comprises a condensation reaction.

In some examples, TA transacylation may involve at least one condensation reaction. In some cases, at least one of the condensation reactions is carried out in the presence of an enzyme. In some cases, at least one of the condensation reactions is catalyzed by an enzyme. In some cases, at least one enzyme may be used to catalyze the condensation reaction.

In some of the methods, processes, and systems described herein, the condensation reaction can be carried out in the presence of an enzyme. In some examples, the enzyme may be an acyltransferase. Acyltransferases may use TA with alcohol or carboxylate functionality as a substrate. Acyltransferases may use as substrates TA which contains a carboxylate group activated by a 1-O-. beta.glycosidic bond to a sugar (hereinafter referred to as glycoside). Acyltransferases can convert the TA alcohol and carboxylate/glycoside functionalities into the corresponding ester derivatives. Non-limiting examples of enzymes suitable for condensation of a TA precursor in the present disclosure include serine carboxypeptidase-like acyltransferase (SCPL-AT). For example, ritodrine synthase (EC 2.3.1.-) can condense tropine and other TA precursors containing alcohol functionality with 1-O-. beta. -phenyllactoyl-glucose and other TA glycoside precursors to ritodrine and other corresponding ester products. In some examples, a protein comprising the SCPL-AT domain of any of the foregoing examples can undergo condensation. In some examples, SCPL-AT can catalyze a condensation reaction within a host cell (such as an engineered host cell), as described herein. In other examples, SCPL-AT can catalyze a condensation reaction within a subcellular compartment inside a host cell (such as an engineered host cell), as described herein.

In some embodiments of the invention, the amino acid sequence of an acyltransferase (such as an SCPL-AT enzyme) for use in performing a TA acyltransferase reaction is subjected to one or more modifications that alter post-translational processing, trafficking, folding, oligomerization, and/or subcellular localization of the enzyme. Since some acyltransferases, including the SCPL-AT enzyme, have never been demonstrated to exhibit catalytic activity in living non-plant cells, such modifications may prove useful or may be necessary for activity in non-plant host cells. Examples of such modifications include, but are not limited to: adding, removing or replacing an N-terminal signal peptide sequence; adding, removing or replacing an internal propeptide sequence; adding or removing asparagine-linked N-glycosylation sites; (ii) addition or removal of serine-linked O-glycosylation sites; and fusion of the protein domain to the N-and/or C-terminus of the acyltransferase domain.

In one embodiment of the invention, the SCPL-AT enzyme domain is modified AT its N-terminus by fusion of a soluble protein domain. This soluble domain masks any internal signal sequences in the acyltransferase domain, thereby improving the trafficking and/or subcellular localization of the fused SCPL-AT domain. In some examples, the N-terminally fused domain induces trafficking of the SCPL-AT domain to subcellular compartments including, but not limited to, the ER membrane, ER lumen, cis-golgi, trans-golgi, lysosomes, vacuolar membrane, and vacuolar lumen. The N-terminally fused soluble domain may also change the oligomerization state of the SCPL-AT domain from its native state (monomer) to any state including, but not limited to, homodimers, heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, homohexamers, heterotrimers, homooctamers, heterotoctamers or higher degrees of oligomerization.

In one example, the N-terminally fused soluble protein domain is a fluorescent protein selected from the group including, but not limited to: fluorescent proteins from species of the genus Aequorea (Aequoria) and fluorescent proteins from species of the genus Corallocta (Discosoma). In one example, the N-terminally fused soluble protein domain is red fluorescent protein (DsRed) from a species of the genus lentinus. In other examples, the N-terminally fused soluble protein domain is another enzyme in the TA biosynthetic pathway, including but not limited to ornithine decarboxylase, putrescine N-methyltransferase, pyrrolidone synthase, tropinone reductase, phenylpyruvate reductase, phenyllactate UDP-glucosyltransferase 84A, and hyoscyamine dehydrogenase.

Examples of amino acid sequences of soluble protein domains that can be fused to the N-terminus of the SCPL-AT domain (which can then be used to perform TA acyl transfer reactions in non-plant cells) are provided in table 3. The amino acid sequence of the SCPL-AT enzyme comprising a fused N-terminal domain and used for TA acyltransferase reactions in non-plant cells may have 50% or more identity to a given amino acid sequence as listed in table 3. For example, the amino acid sequence of such an acyltransferase may comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence as provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence comprises at least 80% -99% identity at the amino acid level to the particular amino acid sequence. In some instances, an "identical" amino acid sequence comprises at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequences may be identical, but the DNA sequence is altered, such as to optimize codon usage of a host organism, for example.

An engineered non-plant host cell may be provided that produces an acyltransferase that catalyzes a TA acyltransferase reaction, wherein the acyltransferase comprises an amino acid sequence N-terminally fused to an amino acid sequence of a soluble protein domain selected from the group consisting of those sequences in table 3. The acyltransferase produced in the engineered host cell may be recovered and purified to form a biocatalyst. One or more enzymes recovered from an engineered host cell that produces an acyltransferase may be used in a process for performing a TA acyltransferase reaction. The process can include contacting a TA precursor having an alcohol and/or carboxylate/glycoside functional group with an acyltransferase in an amount sufficient to convert the alcohol and/or carboxylate/glycoside groups to the corresponding ester groups. In an example, a TA precursor having alcohol and/or carboxylate/glycoside functionality can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the TA precursor is converted to the corresponding ester. In further examples, a TA having an alcohol and/or carboxylate/glycoside functionality can be contacted with a sufficient amount of one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the TA precursor is converted to the corresponding ester.

One or more enzymes useful for performing a TA transacylation reaction may contact a TA precursor in vitro. Additionally or alternatively, one or more enzymes useful for performing a TA transacylation reaction may contact a TA precursor in vivo. In addition, one or more enzymes useful for performing a TA transacylation reaction can be provided to cells having a TA precursor therein, or the enzyme can be produced in engineered non-plant host cells.

In some examples, the methods provide engineered non-plant host cells that produce alkaloid products, wherein TA transacylation reactions can constitute a key step in alkaloid product production. In some examples, the alkaloid produced is a pharmaceutically acceptable TA. In other embodiments, the produced alkaloids are derived from a pharmaceutically acceptable TA, including, for example, non-native TA. In other embodiments, the alkaloid product is selected from the group consisting of a pharmaceutically acceptable TA, a non-pharmaceutically acceptable TA, and a non-native TA.

In some examples, the substrate is a TA precursor selected from the group consisting of: tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid and glycosides of the listed compounds.

In some examples, the methods provide engineered non-plant host cells that produce alkaloid products from tropine and 1-O- β -phenyllactyl glucose. The condensation of tropine and 1-O- β -phenyllactylglucose to ritoprene may constitute a key step in the production of various alkaloid products from precursors. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). Various alkaloid products can include, but are not limited to, pharmaceutically acceptable TA, non-pharmaceutically acceptable TA and non-native TA.

Any suitable carbon source may be used as a precursor for the TA transacylation reaction. Suitable precursors can include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some examples, unpurified mixtures from renewable feedstocks (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysate) may be used. In other embodiments, the carbon precursor may be a mono-carbon compound (e.g., methanol, carbon dioxide) or a di-carbon compound (e.g., ethanol). In other embodiments, other carbon-containing compounds may be used, such as methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine). In some examples, TA or a TA precursor having alcohol and/or carboxylate/glycoside functionality can be added directly to the engineered host cells of the invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, phenyllactic acid, cinnamic acid, ferulic acid, coumaric acid, and glycosides of the listed compounds.

In some embodiments, the substrate used to perform the vacuolar TA transacylation reaction may comprise one or more alcohol and/or carboxylate/glycoside functionalities, wherein only one of the functionalities is condensed with the corresponding ester.

TA alcohol-aldehyde interconversion

Some of the methods, processes, and systems provided herein describe the conversion of a TA having an aldehyde functional group to a TA having an alcohol (hydroxyl) functional group, and the conversion of a TA having an alcohol functional group to a TA having an aldehyde functional group (hereinafter referred to as TA alcohol-aldehyde interconversion). Some of these methods, processes, and systems may include engineered host cells. In some examples, TA alcohol-aldehyde interconversion is a key step in the conversion of substrates to various alkaloids. In some examples, the conversion of the TA aldehyde group to a TA alcohol group comprises a reduction reaction. In some cases, the reduction of the substrate TA aldehyde to an alcohol can be carried out by reducing the aldehyde substrate to the corresponding tetrahedral oxyanion intermediate, and then protonating this intermediate to a hydroxyl group, as provided in figure 2 and as generally represented in scheme 1. As provided in scheme 1, R¹May be H, CH₃Or higher alkyl; r²And R³May be H, OH or OCH₃；R⁴May be H; and R is⁵Can be H, OH, C₁-C₄Alkyl radical, C₁-C₄Alkoxy radical, C₁-C₄Acyl, F, Cl or Br.

Scheme 1

In some examples, the TA alcohol-aldehyde interconversion can involve at least one oxidation reaction or at least one reduction reaction. In some cases, at least one of the oxidation or reduction reactions is performed in the presence of an enzyme. In some cases, at least one of the oxidation or reduction reactions is catalyzed by an enzyme. In some cases, both the oxidation and reduction reactions are performed in the presence of at least one enzyme. In some cases, at least one enzyme may be used to catalyze the oxidation and reduction reactions. The oxidation and reduction reactions may be catalyzed by the same enzyme.

In some of the methods, processes, and systems described herein, the oxidation or reduction reaction can be performed in the presence of an enzyme. In some examples, the enzyme may be a dehydrogenase. Dehydrogenases may use TA having alcohol or aldehyde functionality as a substrate. The dehydrogenase can convert the TA alcohol or aldehyde functionality to the corresponding aldehyde or alcohol derivative. The dehydrogenase may be referred to as a Hyoscyamine Dehydrogenase (HDH). Non-limiting examples of enzymes suitable for oxidation and/or reduction of TA in the present disclosure include cytochrome P450 oxidase, 2-oxoglutarate-dependent oxidase, flavoprotein oxidase, short-chain dehydrogenase-reductase (SDR), medium-chain dehydrogenase-reductase (MDR), Cinnamyl Alcohol Dehydrogenase (CAD), and aldehyde-ketone reductase (AKR). For example, tropinone reductase 1(EC 1.1.1.206) can oxidize tropinone and other TA precursors with ketone functionality to tropine (3 α -tropine) and other corresponding alcohol products. In some examples, a protein comprising a dehydrogenase domain of any of the foregoing examples can be oxidized or reduced. In some examples, the dehydrogenase can catalyze oxidation and/or reduction reactions within a host cell (such as an engineered host cell), as described herein.

Examples of amino acid sequences of dehydrogenases useful for performing a TA alcohol-aldehyde interconversion are provided in Table 2. The amino acid sequence of a dehydrogenase used for TA alcohol-aldehyde interconversion can have 50% or more identity to a given amino acid sequence as set forth in table 2. For example, the amino acid sequence of such a dehydrogenase can comprise an amino acid sequence having at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence as provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence comprises at least 80% -99% identity at the amino acid level to the particular amino acid sequence. In some instances, an "identical" amino acid sequence comprises at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequences may be identical, but the DNA sequence is altered, such as to optimize codon usage of a host organism, for example.

An engineered host cell can be provided that produces a dehydrogenase that catalyzes a TA alcohol-aldehyde interconversion, wherein the dehydrogenase comprises an amino acid sequence selected from the group consisting of those sequences in table 2. The dehydrogenase produced within the engineered host cell may be recovered and purified to form a biocatalyst. One or more enzymes recovered from the engineered host cell that produces dehydrogenase can be used in a process for performing TA alcohol-aldehyde interconversion. The process can include contacting a TA having alcohol and/or aldehyde functional groups with a dehydrogenase in an amount sufficient to convert the alcohol and/or aldehyde groups of the TA to the corresponding aldehyde and/or alcohol groups. In an example, a TA having alcohol and/or aldehyde functional groups can be contacted with a sufficient amount of one or more enzymes such that at least 5% of the TA is converted to its corresponding aldehyde and/or alcohol group. In further examples, a TA having an alcohol and/or aldehyde functional group can be contacted with a sufficient amount of one or more enzymes such that at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, or 100% of the TA is converted to its corresponding aldehyde and/or alcohol functional group.

One or more enzymes useful for performing a TA alcohol-aldehyde interconversion can contact TA in vitro. Additionally or alternatively, one or more enzymes useful for performing a TA alcohol-aldehyde interconversion can contact TA in vivo. In addition, one or more enzymes useful for performing TA alcohol-aldehyde interconversion can be provided to cells having TA therein, or the enzymes can be produced in engineered host cells.

In some examples, the methods provide engineered host cells that produce alkaloid products, wherein TA alcohol-aldehyde interconversion can constitute a key step in the production of the alkaloid products. In some examples, the alkaloid produced is a pharmaceutically acceptable TA. In other embodiments, the produced alkaloids are derived from a pharmaceutically acceptable TA, including, for example, non-native TA. In another embodiment, the TA having alcohol and/or aldehyde functionality is an intermediate to the product of the engineered host cell. In other embodiments, the alkaloid product is selected from the group consisting of a pharmaceutically acceptable TA, a non-pharmaceutically acceptable TA, and a non-native TA.

In some examples, the substrate is TA or a precursor of TA selected from the group consisting of: ritodrine, hyoscyaldehyde, hyoscyamine, anisodamine and scopolamine.

In some examples, the methods provide an engineered host cell that produces an alkaloid product from scopolamine. The reduction of hyoscyaldehyde to hyoscyamine may constitute a key step in the production of various alkaloid products from precursors. In some examples, the precursor is an L-amino acid or a sugar (e.g., glucose). Various alkaloid products can include, but are not limited to, pharmaceutically acceptable TA, non-pharmaceutically acceptable TA and non-native TA.

Any suitable carbon source may be used as a precursor for the TA alcohol-aldehyde interconversion. Suitable precursors can include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some examples, unpurified mixtures from renewable feedstocks (e.g., corn steep liquor, beet molasses, barley malt, biomass hydrolysate) may be used. In other embodiments, the carbon precursor may be a mono-carbon compound (e.g., methanol, carbon dioxide) or a di-carbon compound (e.g., ethanol). In other embodiments, other carbon-containing compounds may be used, such as methylamine, glucosamine, and amino acids (e.g., L-arginine and L-phenylalanine). In some examples, TA or a TA precursor having alcohol and/or aldehyde functionality can be added directly to the engineered host cells of the invention, including, for example, tropine, pseudotropine, ecgonine, methylecgonine, ritorine, hyoscyaldehyde, hyoscyamine, anisodamine, and scopolamine.

In some embodiments, the substrate used to perform the TA alcohol-aldehyde interconversion can comprise one or more alcohol and/or aldehyde functional groups, wherein only one of the functional groups is oxidized or reduced to the corresponding aldehyde or alcohol group.

Methods for increasing intracellular and extracellular metabolite transport

Some of the methods, processes, and systems provided herein describe the use of proteins (hereinafter "transporters") to translocate metabolites across lipid membranes (hereinafter "transmembrane transport"). Some of these methods, processes, and systems may include engineered host cells. In some instances, transmembrane transport is a critical step in the conversion of substrates to various alkaloids.

In certain embodiments, the host cell comprises one or more heterologous coding sequences for one or more transporters or active fragments thereof that localize to a lipid membrane and translocate TA or a TA precursor across the same lipid membrane. In some examples, the lipid membrane is a vacuolar membrane. In other examples, the lipid membrane is the ER membrane. In some examples, the lipid membrane is a peroxidase membrane. In other examples, the lipid membrane is a cytoplasmic membrane.

In some examples, the TA and TA precursors transported in this manner include, but are not limited to, putrescine, N-methyl putrescine, 4-methylaminobutyraldehyde, N-methylpyrrolidinium, tropinone, tropine, phenyllactic acid, 1-O- β -phenyllactylglucose, ritorine, hyoscyamine, anisodamine, and scopolamine. Accumulation of such TAs or TA precursors in a particular subcellular compartment can prevent entry of operably linked biosynthetic enzymes in different compartments; thus, the use of a transporter that translocates TA or a TA precursor from one compartment to another may alleviate such transport limitations. In certain instances, expression of heterologous coding sequences for one or more transporters in a host cell can increase production of TA or a TA precursor.

In some embodiments, the transporter protein or active fragment thereof is a multidrug and toxin efflux (MATE) transporter protein. Any convenient MATE transporter that transports one or more of the above-described TAs or TA precursors can be used in the subject host cells. Transporters of interest include, but are not limited to, enzymes such as nicotiana tabacum jasmonate inducible alkaloid transporter 1(NtJAT1), nicotiana tabacum MATE1, nicotiana tabacum MATE2, or any other protein as described in tables 1 and 4.

In certain embodiments, the transporter protein or active fragment thereof is a nitrate/peptide family (NPF) transporter protein. Any convenient NPF transporter that transports one or more of the above-described TAs or TA precursors can be used in the subject host cells. In other embodiments, the transporter or active fragment thereof is an ATP-binding cassette (ABC) transporter. Any convenient NPF transporter that transports one or more of the above-described TAs or TA precursors can be used in the subject host cells. In some embodiments, the transporter protein or active fragment thereof is a Pleiotropic Drug Resistant (PDR) transporter protein. Any convenient PDR transporter that transports one or more of the above-described TAs or TA precursors can be used in the subject host cells.

In certain embodiments, the host cell comprises a heterologous coding sequence for a transporter protein or an active fragment thereof. In some embodiments of the invention, the amino acid sequence of the transport protein is subjected to one or more modifications that alter subcellular localization, direction of substrate translocation, and/or topological orientation of the enzyme. Examples of such modifications include, but are not limited to: adding, removing or replacing N-terminal, C-terminal or internal signal sequences; adding, removing, replacing or rearranging a transmembrane helix; and fusion of protein domains to the N-and/or C-terminus of a transporter.

Examples of amino acid sequences of transporters that can be used to alleviate substrate transport limitations and/or increase accumulation of TA or TA precursors in particular cellular compartments are provided in table 4. The amino acid sequence of a transporter protein used in this manner in a non-plant cell may have 50% or more identity to a given amino acid sequence as set forth in table 4. For example, the amino acid sequence of such a transporter can comprise an amino acid sequence that is at least 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the amino acid sequence as provided herein. Furthermore, in certain embodiments, an "identical" amino acid sequence comprises at least 80% -99% identity at the amino acid level to the particular amino acid sequence. In some instances, an "identical" amino acid sequence comprises at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequences may be identical, but the DNA sequence is altered, such as to optimize codon usage of a host organism, for example.

An engineered non-plant host cell may be provided that produces a transporter that translocates one or more TAs or TA precursors from one cellular compartment to another, wherein the transporter comprises an amino acid sequence selected from the group consisting of those sequences in table 4. In some examples, the methods provide engineered non-plant host cells that produce alkaloid products, wherein TA transmembrane transport may constitute a critical step in alkaloid product production. In some examples, the alkaloid produced is a pharmaceutically acceptable TA. In other embodiments, the produced alkaloids are derived from a pharmaceutically acceptable TA, including, for example, non-native TA. In other embodiments, the alkaloid product is selected from the group consisting of a pharmaceutically acceptable TA, a non-pharmaceutically acceptable TA, and a non-native TA.

Heterologous coding sequences

In some cases, a host cell is a cell containing one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more, or even more) that encode one or more activities that enable the host cell to produce a desired TA of interest, e.g., as described herein. As used herein, the term "heterologous coding sequence" is used to denote any polynucleotide that encodes or ultimately encodes a peptide or protein or equivalent amino acid sequence thereof (e.g., an enzyme) that is not normally present in the host organism and that is expressible in the host cell under appropriate conditions. Thus, "heterologous coding sequence" includes multiple copies of the coding sequence that are normally present in a host cell, such that the cell expresses additional copies of the coding sequence that are normally not present in the cell. The heterologous coding sequence can be RNA or any type thereof (e.g., mRNA), DNA or any type thereof (e.g., cDNA), or a hybrid of RNA/DNA. Coding sequences of interest include, but are not limited to, full-length transcription units that include features such as coding sequences, introns, promoter regions, 3' -UTRs, and enhancer regions.

In an example, the engineered host cell comprises a plurality of heterologous coding sequences each encoding an enzyme. In some examples, the multiple enzymes encoded by the multiple heterologous coding sequences may be different from each other. In some examples, some of the plurality of enzymes encoded by the plurality of heterologous coding sequences may be different from each other, and some of the plurality of enzymes encoded by the plurality of heterologous coding sequences may be duplicate copies.

In some examples, a heterologous coding sequence can be operably linked. The operably linked heterologous coding sequence may be within the same pathway that produces the particular tropane alkaloid product. In some examples, the operably linked heterologous coding sequence may be directly contiguous along the pathway that produces a particular tropane alkaloid product. In some examples, an operably linked heterologous coding sequence can have one or more native enzymes between the one or more enzymes encoded by the plurality of heterologous coding sequences. In some examples, the heterologous coding sequence can have one or more heterologous enzymes between the one or more enzymes encoded by the plurality of heterologous coding sequences. In some examples, the heterologous coding sequence can have one or more non-native enzymes between the one or more enzymes encoded by the plurality of heterologous coding sequences.

In some embodiments, the host cell comprises putrescine N-methyltransferase (PMT) activity. Any convenient PMT enzyme may be used in the subject host cells. PMT enzymes of interest include, but are not limited to, enzymes such as EC 2.1.1.53, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for PMT or an active fragment thereof.

In some cases, the host cell includes one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMP to 4 MAB. In certain instances, the one or more enzymes are selected from plant Methyl Putrescine Oxidase (MPO) and eukaryotic MPO (e.g., EC 1.4.3.22).

In certain embodiments, the cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert NMPy to MPOB. In certain instances, the one or more enzymes are a type III polyketide synthase (e.g., EC 2.3.1. -). The one or more heterologous coding sequences can be derived from any convenient species (e.g., as described herein). In some cases, one or more heterologous coding sequences may be derived from a species described in table 1. In some cases, one or more heterologous coding sequences are present in a gene or enzyme selected from those described in table 1.

In certain embodiments, the host cell comprises tropinone synthase activity. Any convenient tropinone synthase (e.g., CYP82M3) may be used in the subject host cells. The tropinone synthases of interest include, but are not limited to, enzymes such as EC 1.14.14-, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for a tropinone synthase or an active fragment thereof.

In certain embodiments, the host cell comprises tropinone reductase activity. Any convenient tropinone reductase may be used in the subject host cells. The tropinone reductases of interest include, but are not limited to, enzymes such as EC 1.1.1.206, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for tropinone reductase or an active fragment thereof.

In some cases, the host cell comprises phenylpyruvate reductase (PPR) activity. Any convenient PPR enzyme may be used in the subject host cells. Some PPR enzymes of interest include, but are not limited to, enzymes such as EC 1.1.1.237, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for PPR or an active fragment thereof.

In certain embodiments, the host cell comprises phenyllactyltransferase activity. Any convenient phenyllactate glycosyltransferase can be used in the subject host cells. Glycosyltransferases include, but are not limited to, enzymes such as 2.4.1-that transfer a glucose moiety from UDP-glucose to phenyllactate via a glycosidic ester bond, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for a phenyllactate glycosyltransferase or an active fragment thereof.

In certain embodiments, the cell comprises one or more heterologous coding sequences for one or more enzymes or active fragments thereof that convert tropine and 1-O- β -phenyllactyl glucose to ritodrine. In some embodiments, the host cell comprises ritodrine synthase activity. Any convenient ritodrine synthase or enzyme comprising an active fragment of ritodrine synthase can be used in the subject host cells. Ritulin synthases of interest include, but are not limited to, enzymes such as EC 2.3.1-, as described in table 1, and enzymes comprising a ritulin synthase fused at its N-terminus to a soluble protein domain described in table 3. In certain embodiments, the host cell comprises a heterologous coding sequence for ritodrine synthase or an active fragment thereof.

In some cases, the host cell comprises rituximab activity. Any convenient rituximab may be used in the subject host cells. Rituximab of interest include, but are not limited to, enzymes such as EC 1.14.19-, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for rituximab, or an active fragment thereof.

In some embodiments, the host cell comprises a Hyoscyamine Dehydrogenase (HDH) activity. Any convenient HDH enzyme may be used in the subject host cells. Some HDH enzymes of interest include, but are not limited to, those sequences described in table 2. In certain embodiments, the host cell comprises a heterologous coding sequence for HDH or an active fragment thereof.

In certain embodiments, the host cell comprises hyoscyamine 6 β -hydroxylase/dioxygenase (H6H) activity. Any convenient H6H enzyme may be used in the subject host cells. Some H6H enzymes of interest include, but are not limited to, enzymes such as EC 1.14.11.11, as described in table 1. In certain embodiments, the host cell comprises a heterologous coding sequence for H6H or an active fragment thereof.

In certain examples, the engineered host cell comprises a plurality of heterologous coding sequences each encoding a transmembrane metabolite transporter. In some examples, the multiple transporters encoded by multiple heterologous coding sequences may differ from one another. In some examples, some of the plurality of transporters encoded by the plurality of heterologous coding sequences may be different from one another, and some of the plurality of transporters encoded by the plurality of heterologous coding sequences may be duplicate copies.

As used herein, the term "heterologous coding sequence" also includes coding portions of peptides or enzymes, i.e., cDNA or mRNA sequences of peptides or enzymes, as well as coding portions of full-length transcription units, i.e., genes including introns and exons, as well as "codon-optimized" sequences, truncated sequences, or other forms of altered sequences that encode enzymes or encode their equivalent amino acid sequences, provided that the equivalent amino acid sequences produce a functional protein. Such equivalent amino acid sequences may have a deletion of one or more amino acids, wherein the deletion is N-terminal, C-terminal, or internal. Truncated forms are contemplated, so long as they have the catalytic capabilities indicated herein. Fusions of two or more enzymes are also contemplated to facilitate transfer of the metabolite in the pathway, provided that catalytic activity is maintained. Also included are fusions of one or more enzymes or catalytic protein domains with one or more non-catalytic protein domains in such a way that the non-catalytic protein domains facilitate the solubilization, folding, maturation and/or activity of the fused catalytic domains.

Operable fragments, mutants or truncated forms can be identified by modeling and/or screening. This is made possible by: the addition or deletion of, for example, the N-terminal, C-terminal or internal region of the protein is carried out in a stepwise manner, and then the activity of the resulting derivative with respect to the desired reaction is analyzed as compared with the original sequence. If the derivative in question operates with this capacity, it is considered to constitute the equivalent derivative of the enzyme in its entirety.

Aspects of the invention also relate to heterologous coding sequences that encode amino acid sequences equivalent to the native amino acid sequences of the various enzymes. An amino acid sequence that is "equivalent" is defined as an amino acid sequence that: which is not identical to the specified amino acid sequence, but contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.), which do not substantially affect the biological activity of the protein, when used for the desired purpose, as compared to a similar activity of the specified amino acid sequence. In the case of a decarboxylase, biological activity refers to its catalytic activity. Equivalent sequences are also intended to include those that have been engineered and/or evolved to have properties that differ from the original amino acid sequence. Variable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. In certain embodiments, an "equivalent" amino acid sequence comprises at least 80% -99% identity at the amino acid level to a particular amino acid, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more, in some cases at least 95%, 96%, 97%, 98% and 99% identity at the amino acid level. In some cases, the amino acid sequences may be identical, but the DNA sequence is altered, such as to optimize codon usage of a host organism, for example.

The host cell may also be modified to have one or more genetic alterations to accommodate the heterologous coding sequence. Alterations of the native host genome include, but are not limited to, modifying the genome to reduce or ablate the expression of specific proteins that may interfere with the desired pathway. The presence of such native proteins can rapidly convert one of the intermediates or end products of the pathway to a metabolite or other compound that is not available in the desired pathway. Thus, if the activity of the native enzyme is reduced or absent altogether, the intermediate produced will be more readily available for incorporation into the desired product.

In some cases, where ablation of protein expression may be of interest, the alterations are in proteins involved in pleiotropic drug responses, including but not limited to ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, and related transcription factors. These proteins are involved in the outward transport of the TA molecule and TA precursor into the culture medium, and thus the absence of a control compound controls the outward transport of the compound into the culture medium, making them more useful for incorporation into the desired product. In some embodiments, the host cell gene deletion of interest includes genes associated with unfolded protein response and Endoplasmic Reticulum (ER) proliferation. Such gene deletions may result in increased TA production. Expression of cytochrome P450 can induce an unfolded protein response and can lead to ER proliferation. Deletion of genes associated with these stress responses may control or reduce the overall burden on the host cell and improve pathway performance. Genetic alteration may also include modification of the promoter of the endogenous gene to increase expression and/or introduction of additional copies of the endogenous gene. Examples of this include the construction/use of strains that overexpress the endogenous yeast NADPH-P450 reductase Ncp1P to increase the activity of the heterologous P450 enzyme. Furthermore, endogenous enzymes directly involved in the synthesis of intermediate metabolites, such as Spe1p, Fms1p, Car1p, Arg2p, Aro8p, Aro9p, Pha2p, Ugp1p, and Leu2p, may also be overexpressed.

Heterologous coding sequences of interest include, but are not limited to, sequences encoding enzymes, wild-type or equivalent sequences, which are generally responsible for the production of TA and precursors in plants. In some cases, the enzyme encoded by the heterologous sequence may be any enzyme in the TA pathway, and may be from any convenient source. The choice and quantity of enzymes encoded by a heterologous coding sequence for a particular synthetic pathway can be selected based on the desired product. In certain embodiments, a host cell of the invention may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 heterologous coding sequences.

In some cases, the polypeptide sequence encoded by the heterologous coding sequence is as reported in GENBANK. Enzymes of interest include, but are not limited to, those described herein and those shown in table 1. The host cell may include any combination of the listed enzymes from any source. The accession numbers in table 1 refer to GenBank unless otherwise indicated. Some accession numbers refer to the yeast genome database (SGD), which is available on the world wide web at yeastgenome.

In some embodiments, a host cell (e.g., a yeast strain) is engineered for the selective production of a TA of interest by targeting one or more enzymes to a compartment in the cell. In some cases, the enzyme may be located in a host cell such that a compound produced by the enzyme rearranges spontaneously or is converted to a desired metabolite by another enzyme before reaching a localized enzyme (localized enzyme) that can convert the compound to the undesired metabolite. The spatial distance between the two enzymes may be selected to prevent one of the enzymes from acting directly on the compound to produce undesirable metabolites and to limit the production of undesirable end products (e.g., undesirable opioid byproducts). In some other cases, an enzyme may be localized in a host cell such that the subcellular compartment in which it is localized provides better pH, cofactor concentration, redox potential, substrate concentration, and/or other biochemical parameters for its activity than the compartment in which the enzyme naturally occurs. In certain instances, the enzyme may be localized to a particular compartment within the host cell, such that the intracellular transport pathway that transports the enzyme to that compartment provides the necessary post-translational modifications to render the enzyme active. Such post-translational modifications include, but are not limited to, acetylation, acetylglycosylation, amidation, carboxylation, methylation, glutathionylation, hydroxylation, glycosylation, phosphorylation, sulfonation, disulfide bond formation, cleavage of signal sequences, and multienzyme complex formation. In certain embodiments, any of the enzymes described herein, alone or with a second enzyme, may be located in any convenient compartment in the host cell, including but not limited to, the organelles, endoplasmic reticulum, golgi apparatus, vacuoles, nuclei, plasma membranes, mitochondria, peroxisomes, periplasm, the lumen of any of the above organelles, or the membrane surrounding or associated with any of the above organelles. Where one or more enzymes are located in a membrane associated with any of the above organelles, the enzymes can be oriented such that the catalytic domain of the enzyme faces the cytosol, the lumen of the organelle, and/or any other intracellular space. In some embodiments, the host cell comprises one or more enzymes comprising a localization tag. Any convenient label may be used. In some cases, the localization tag is a peptide sequence attached to the N-terminus and/or C-terminus of the enzyme.

The tag may be attached to the enzyme using any convenient method. In some cases, the localization tag is derived from an endogenous yeast protein. Such tags may provide access to a variety of yeast organelles, including but not limited to Endoplasmic Reticulum (ER), Golgi Apparatus (GA), Mitochondria (MT), Plasma Membrane (PM), Peroxisomes (POX), and vacuole (V). In certain embodiments, the label is an ER routing label (e.g., ER 1). In certain embodiments, the label is a vacuolar label (e.g., V1). In certain embodiments, the label is a plasma membrane label (e.g., P1). In certain embodiments, the tag is a peroxisome targeting sequence (e.g., PTS 1). In some cases, the tag includes or is derived from a transmembrane domain within a protein of the tail anchor class. In some embodiments, the localization tag localizes the enzyme outside of the organelle. In certain embodiments, the localization tag localizes the enzyme inside the organelle. In some embodiments, the localization tag localizes the enzyme such that one or more portions of the enzyme are found both inside and outside the organelle.

In some embodiments of the invention, the host cell is modified by expression of one or more coding sequences encoding one or more enzymes comprising a localization tag as described above. In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that the one or more enzymes are expressed in the cytosol. Examples of such enzymes include, but are not limited to, arginine decarboxylase, putrescine N-methyltransferase, pyrrolidone synthase, tropinone reductase, phenylpyruvate reductase, UDP-glucosyltransferase, and 2-oxoglutarate-dependent dioxygenase, such as hyoscyamine 6 β -hydroxylase/dioxygenase. In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that the one or more enzymes are expressed in the ER membrane. Examples of such enzymes include, but are not limited to, cytochrome P450 s, such as tropinone synthase (CYP82M3) and ritopressin mutase (CYP80F1), and NADP ⁺-a cytochrome P450 reductase. In certain embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that the one or more enzymes are expressed in the mitochondria. Examples of such enzymes include, but are not limited to, N-acetylglutamate synthase. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that the one or more enzymes are expressed in the peroxisome. Examples of such enzymes include, but are not limited to, amine oxidases, such as N-methylputrescine oxidase. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that the one or more enzymes are expressed in the vacuolar lumen. Examples of such enzymes include, but are not limited to, serine carboxypeptidase-like acyltransferases, such as ritoprene synthase, and engineered variants thereof. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes or proteins are expressed in the vacuolar membraneSo as to achieve the purpose. Examples of such proteins include, but are not limited to, multidrug and toxin efflux transporters, nitrate/peptide family transporters, and ATP-binding cassette transporters. In other embodiments, the host cell is modified by expression of one or more heterologous coding sequences such that one or more enzymes or proteins are expressed in the plasma membrane. Examples of such proteins include, but are not limited to, ATP-binding cassette transporters, multi-effect drug resistant transporters, and multi-drug resistant transporters.

In some cases, expression of each type of enzyme is increased by additional gene copies (i.e., multiple copies), which increases intermediate accumulation and/or production of the TA of interest. Embodiments of the invention include increasing the production of TA of interest in a host cell by simultaneously expressing multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient method may be used, including multiple copies of heterologous coding sequences for the enzyme in the host cell.

In some embodiments, the host cell comprises multiple copies, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies, of the heterologous coding sequence for the enzyme. In certain embodiments, the host cell comprises multiple copies, such as multiple copies of two or more, three or more, four or more, etc., of heterologous coding sequences for one or more enzymes. In some cases, multiple copies of a heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell. For example, a host cell may comprise multiple copies of a heterologous coding sequence, wherein each copy is derived from a different source organism. Thus, each copy may include some variation in the exonic sequence based on inter-species differences in the enzyme of interest encoded by the heterologous coding sequence.

In some embodiments of the host cell, the heterologous coding sequence is from a source organism selected from the group consisting of: coli, Bacillus coagulans, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus species, Saccharomyces cerevisiae, jellyfish species, Cordiculus species, Arabidopsis thaliana, oats, tomato (Solanum lycopersicum), potato (Solanum tuberosum), common tobacco (Nicotiana tabacum), Nicotiana benthamiana (Nicotiana benthamiana), Atropa belladonna, Hyoscyamus henryi (Hyoscyamus niger), Hyoscyamus nigra (Hyoscyamus musculus), Datura stramonium (Datura stramunium), Datura nitida (Datura metura), Datura Innoxia (Datura Innoxia), Duboisia myrtle (Duboisia myopooides), Bellam belladonna (Androsaria), Scopolia japonica (Androsaria), Anisodus tanguticus, Anisodus acutus, Anisotropicus, Sapinus acutangulus, Brussia obovata (Brugura), Gymnolia elata (Solidago), Aconitum (Solidago grandiflora), Codonia sp), Codonia indica (Rosemaria spp), Codonia sp) Hyoscyamus species (Hyoscyamus spp), Datura species (Datura spp), Duboisia species (Duboisia spp), Scopolia species (Anisodus spp), Datura species (Brugmansia spp), Erythroxylom spp or Cardamine species (Cochlearia spp). In some cases, the heterologous coding sequence is from a source organism selected from belladonna, henbane and datura. In some embodiments, the host cell comprises a heterologous coding sequence from one or more of the source organisms described in table 1.

The engineered host cell culture medium can be sampled and monitored for the production of the TA of interest. Any convenient method can be used to observe and measure the TA of interest. Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein), wherein a sample of interest is analyzed by comparison to a known amount of a standard compound. Identity can be confirmed, for example, by m/z and MS/MS fragmentation patterns, and quantification or measurement of compounds can be achieved by LC trace peak and/or EIC MS peak analysis of known retention times, with reference to the corresponding LC-MS analysis of known amounts of compound standards.

Method

Procedure step

As noted above, aspects of the invention include methods of making a Tropane Alkaloid (TA) of interest. Thus, aspects of the invention include culturing a host cell under conditions in which one or more host cell modifications (e.g., as described herein) functionally express such that the cell converts a starting compound of interest to a product of interest TA or a precursor thereof (e.g., a pre-esterified TA). Also provided are methods comprising culturing a host cell under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and converting a starting compound of interest into a product of interest, TA. In some cases, the method is a method of making a Tropane Alkaloid (TA), comprising culturing a host cell (e.g., as described herein); adding a starter compound to the cell culture; and recovering the TA from the cell culture. In some embodiments of the methods, the starting compound, the TA product, and the host cell are described by one of the entries in table 1.

The fermentation medium may comprise a suitable carbon substrate. Carbon sources suitable for performing the methods of the present disclosure can include a wide variety of carbon-containing substrates. Suitable substrates may include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or combinations thereof. In some cases, unpurified mixtures from renewable feedstocks (e.g., corn steep liquor, beet molasses, barley malt) may be used. In some cases, the carbon substrate can be a one-carbon substrate (e.g., methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). In other cases, other carbon-containing compounds may be used, such as methylamine, glucosamine, and amino acids.

Any convenient method of culturing host cells can be used to produce TA precursors and downstream TAs of interest. The specific protocol employed may vary, for example, depending on the host cell, heterologous coding sequence, desired TA precursor, and downstream TA of interest, and the like. The cell may be present in any convenient environment, such as an environment in which the cell is capable of expressing one or more functional heterologous enzymes. In vitro, as used herein, refers only to outside of a living cell, regardless of the location of the cell. As used herein, the term in vivo refers to inside a living cell, regardless of the location of the cell. In some embodiments, the cells are cultured under conditions conducive to expression of the enzyme and with appropriate substrates available to allow production of the TA precursor and downstream TA of interest in vivo. In some embodiments, the functional enzyme is extracted from the host to produce TA under in vitro conditions. In some cases, the host cell is returned to the multicellular host organism. The host cell is in any growth phase including, but not limited to, stationary phase, logarithmic growth phase, and the like. Furthermore, the cultures themselves may be continuous cultures or they may be batch cultures.

The cells may be grown in a suitable fermentation medium at a temperature between 20-40 ℃. The cells can be grown under agitation at any convenient speed (e.g., 200 rpm). The cells may be grown at a suitable pH. A pH range suitable for fermentation may be between pH 5-9. The fermentation may be carried out under aerobic, anaerobic or microaerophilic conditions. Any suitable growth medium may be used. Suitable growth media may include, but are not limited to, common commercial preparation media such as Synthetic Definition (SD) minimal media or Yeast Extract Peptone Dextrose (YEPD) enrichment media. Any other enriched, defined or synthetic growth medium suitable for microorganisms may be used.

The cells can be cultured in containers of essentially any size and shape. Examples of vessels suitable for performing the methods of the present disclosure may include, but are not limited to, multi-well shake plates, test tubes, flasks (baffled and unbaffled), and bioreactors. The volume of the medium can range from 10 microliters to greater than 10,000 liters.

May include adding to the growth medium an agent known to regulate metabolism in a manner desired for alkaloid production. In one non-limiting example, cyclic adenosine 2 '3' -monophosphate can be added to the growth medium to modulate catabolite repression.

Any convenient cell culture conditions for a particular cell type may be utilized. In certain embodiments, host cells comprising one or more modifications are cultured with standard cell culture media and supplements under standard or readily optimized conditions. For example, when selective pressure for plasmid maintenance is not required, a standard growth medium may contain 20g/L yeast extract, 10g/L peptone, and 20g/L dextrose (YPD). Host cells containing the plasmids were grown in Synthetic Complete (SC) medium containing 1.7g/L yeast nitrogen base, 5g/L ammonium sulfate and 20g/L dextrose, supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources that may be used for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. In the laboratory, cells are grown at any convenient temperature (e.g., 30 ℃) under agitation at any convenient rate (e.g., 200rpm) in a container, such as a test tube or flask having a volume in the range of 1-1000mL or more.

For example, as part of an industrial process, the culture volume can be expanded to grow in a larger fermentation vessel. The industrial fermentation process may be carried out under closed batch, fed-batch or continuous chemostat conditions or any suitable fermentation mode. In some cases, the cells may be immobilized on a substrate as a monolithic cell catalyst and subjected to fermentation conditions to produce the alkaloid.

Batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and does not change during the fermentation. One or more desired organisms are inoculated into the culture medium at the beginning of the fermentation. In some cases, batch fermentations were run with changes made to the system to control factors such as pH and oxygen concentration (but not carbon). In this type of fermentation system, the biomass and metabolite composition of the system is constantly changing during the fermentation process. Cells typically undergo a lag phase, then enter a log phase (high growth rate), then enter a resting phase (growth rate is reduced or stopped), and finally enter a death phase (if left untreated).

Fed-batch fermentation is similar to batch fermentation except that the substrate is added to the system at intervals during the fermentation process. Fed-batch systems are used to reduce the effects of catabolite repression on host cell metabolism, and in other cases where limited amounts of substrate are desired in the growth medium.

Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor and an equal amount of fermentation medium is continuously withdrawn from the vessel for processing. Continuous fermentation systems are typically operated to maintain steady state growth conditions such that cell loss due to medium withdrawal must be balanced by the growth rate in the fermentation. Continuous fermentation is typically operated under conditions where the cells are at a constant high cell density. Continuous fermentation allows for the modulation of one or more factors that affect target product concentration and/or cell growth.

The liquid medium may include, but is not limited to, an enriched or synthetically defined medium having the additive components described above. The media components may be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Several media components can be prepared and sterilized separately and then compounded in a fermentation vessel. The medium may be buffered to help maintain a constant pH throughout the fermentation.

Process parameters including temperature, dissolved oxygen, pH, agitation, aeration rate and cell density can be monitored or controlled during fermentation. For example, the temperature of the fermentation process can be monitored by a temperature probe immersed in the medium. The culture temperature can be controlled at a set point by adjusting the jacket temperature. Water can be cooled in an external cooler and then flowed into the bioreactor control column and circulated to the jacket at the temperature required to maintain the set point temperature in the vessel.

In addition, gas flow parameters can be monitored during fermentation. For example, gas can be flowed into the media through a sparger (sparger). Gases suitable for use in the methods of the present disclosure may include compressed air, oxygen, and nitrogen. The gas flow may be at a fixed rate or regulated to maintain a dissolved oxygen set point.

The pH of the medium can also be monitored. In an example, the pH can be monitored by a pH probe immersed in the medium inside the container. If pH control is effective, the pH can be adjusted by acid and base pumps that add each solution to the medium at the desired rate. The acid solution used to control the pH may be sulfuric acid or hydrochloric acid. The base solution used to control the pH may be sodium hydroxide, potassium hydroxide or ammonium hydroxide.

In addition, dissolved oxygen in the culture medium can be monitored by a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is effective, the oxygen content can be adjusted by increasing or decreasing the stirring speed. The dissolved oxygen content can also be adjusted by increasing or decreasing the gas flow rate. The gas may be compressed air, oxygen or nitrogen.

The stirring speed can also be monitored during the fermentation. In an example, the agitator motor may drive the agitator. The agitator speed can be set at a consistent rpm throughout the fermentation process or can be dynamically adjusted to maintain a set dissolved oxygen content.

In addition, turbidity can be monitored during fermentation. In an example, cell density can be measured using a turbidity probe. Alternatively, cell density can be measured by taking a sample from the bioreactor and analyzing it in a spectrophotometer. Furthermore, samples can be taken from the bioreactor at time intervals by means of a sterile sampling device. The sample may be analyzed for alkaloids produced by the host cell. Samples can also be analyzed for other metabolites and consumption of sugars, media components, or cell density.

In another example, feedstock parameters can be monitored during the fermentation process. In particular, feedstocks including sugars and other carbon sources, nutrients, and cofactors may be added to the fermentation using external pumps. Other components may also be added during fermentation, including but not limited to defoamers, salts, chelating agents, surfactants, and organic liquids.

Any convenient codon optimization technique for optimizing expression of a heterologous polynucleotide in a host cell may be adapted for use in the subject host cells and methods, see, e.g., Gustafsson, c.et al (2004) Trends Biotechnol,22, 346-.

The subject methods can further comprise adding a starting compound to the cell culture. Any convenient method of addition may be applied to the subject method. The cell culture may be supplemented with a sufficient amount of the starting material of interest (e.g., as described herein), e.g., a mM to μ M amount, such as between about 1-5mM of the starting compound. It will be appreciated that the amount of starting material added, the time and rate of addition, the form of the material added, and the like, can vary depending on a number of factors. The starting materials may be added neat or pre-dissolved in a suitable solvent (e.g., cell culture medium, water, or organic solvent). The starting material may be added in concentrated form (e.g., 10x over the desired concentration) to minimize dilution of the cell culture medium after addition. The starting materials may be added in one or more batches or by continuous addition over an extended period of time (e.g., hours or days).

Process for separating a product from a fermentation medium

The subject methods can also include recovering the TA of interest from the cell culture. Any convenient separation and isolation methods (e.g., chromatography or precipitation) can be applied to the subject methods to recover the TA of interest from the cell culture. Filtration methods can be used to separate the soluble fraction from the insoluble fraction of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) can be used to separate the TA of interest from other soluble components of the cell culture. In some cases, extraction methods (e.g., liquid extraction, pH-based purification, etc.) can be used to separate the TA of interest from other components of the cell culture.

The produced alkaloids can be isolated from the fermentation medium using methods known in the art. Multiple recovery steps may be performed immediately after fermentation (or in some cases, during fermentation) to initially recover the desired product. Through these steps, alkaloids (e.g., TAs) can be separated from cells, cell debris and waste, and other nutrients, sugars and organic molecules can remain in the waste medium. This process can be used to obtain a TA rich product.

In one example, a product stream having a Tropane Alkaloid (TA) product is formed by providing engineered yeast cells and a feedstock comprising nutrients and water into a batch reactor. The engineered yeast cell may have at least one modification selected from the group consisting of: a feedback inhibition mitigating mutation in a cell's native biosynthetic enzyme gene; transcriptional regulatory modifications of cell-resident biosynthetic enzyme genes; and inactivating mutations in enzymes inherent to the cell. The engineered yeast cells can be fermented while the engineered yeast cells are in a batch reactor. Specifically, the engineered yeast cells can be fermented by incubating the engineered yeast cells for a period of time of at least about 5 minutes to produce a solution comprising the TA product and cellular material. Once the engineered yeast cells have been fermented, the TA product can be separated from the cellular material using at least one separation unit to provide a product stream comprising the TA product. In particular, the product stream may include the TA product as well as additional components, such as clarified yeast medium. In addition, the TA product can comprise one or more TAs of interest, such as one or more TA compounds.

Different methods can be used to remove cells from a bioreactor medium including the TA of interest. In an example, cells can be removed by sedimentation over time. This settling process can be accelerated by cooling or by the addition of a clarifying agent (such as silica). The spent media can then be siphoned from the top of the reactor, or the cells can be decanted from the bottom of the reactor. Alternatively, cells may be removed by filtration using a filter, membrane or other porous material. The cells may also be removed by centrifugation, for example by continuous flow centrifugation or by using a continuous extractor.

If some of the valuable TA of interest is present inside the cell, the cell can be permeabilized or lysed and cell debris can be removed by any of the methods described above. Agents for permeabilizing a cell can include, but are not limited to, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods of lysing cells may include the addition of surfactants such as sodium dodecyl sulfate, or mechanical disruption by bead milling or sonication.

By adding an organic liquid that is immiscible with the aqueous medium, the TA of interest can be extracted from the clarified spent medium by liquid-liquid extraction. Examples of suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methyl isobutyl ketone, methyl oleate, toluene, oleyl alcohol, ethyl butyrate. The organic liquid may be added to as little as 10% or as much as 100% of the volume of the aqueous medium.

In some cases, the organic liquid may be added at the beginning of the fermentation or at any time during the fermentation. This extractive fermentation process can increase the yield of TA of interest from the host cell by continually removing TA precursors or TA into the organic phase.

Agitation can cause the organic phase to form an emulsion with the aqueous medium. Methods to facilitate separation of the two phases into distinct layers may include, but are not limited to, addition of demulsifiers or nucleating agents, or adjustment of pH. The emulsion may also be centrifuged to separate the two phases, for example by continuous conical plate centrifugation.

Alternatively, the organic phase can be separated from the aqueous medium for physical removal after extraction. For example, the solvent may be encapsulated in a film.

In an example, the TA of interest can be extracted from the fermentation medium using an adsorption process. Specifically, the resin may be added by adding a resin such asXAD4 or other agent that removes TA by adsorption extracts the TA of interest from the clarified spent medium. An organic solvent can then be used to release the TA of interest from the resin. Examples of suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone.

Filtration can also be used to extract the TA of interest from the fermentation medium. At high pH, the TA of interest can form a crystal-like precipitate in the bioreactor. Such precipitates can be removed directly by filtration using a filter, membrane or other porous material. The precipitate may also be collected by centrifugation and/or decantation.

The extraction process described above can be performed in situ (in the bioreactor) or ex situ (e.g., in an external loop through which the culture medium exits the bioreactor and contacts the extractant, and is then recycled back to the vessel). Alternatively, the extraction process may be performed after termination of the fermentation using a clarified medium removed from the bioreactor vessel.

Process for purifying a product from an alkaloid rich solution

Subsequent purification steps may involve treatment of the post-fermentation TA precursor or TA rich product using methods known in the art to recover each product species of interest in high purity.

In one example, the TA precursor or TA extracted in the organic phase can be transferred to an aqueous solution. In some cases, the organic solvent may be evaporated by heating and/or vacuum, and the resulting powder may be dissolved in an aqueous solution of suitable pH. In another example, the TA precursor or TA can be extracted from the organic phase by adding an aqueous solution of suitable pH that facilitates extraction of the TA precursor or TA into the aqueous phase. The aqueous phase may then be removed by decantation, centrifugation, or another method.

The solution containing the TA precursor or TA may be further treated to remove the metal, for example, by treatment with a suitable chelating agent. The solution containing the TA precursor or TA may be further treated by precipitation to remove other impurities such as proteins and DNA. In one example, a solution containing a TA precursor or TA is treated with a suitable precipitating agent such as ethanol, methanol, acetone, or isopropanol. In an alternative example, DNA and proteins may be removed by dialysis or by other size exclusion methods that separate smaller alkaloids from contaminating biological macromolecules.

In further examples, solutions containing TA precursors, TA or modified TA can be extracted to high purity by continuous cross-flow filtration using methods known in the art.

If the solution contains a TA precursor or mixture of TA, it can be subjected to an acid-base treatment using methods known in the art to obtain the individual TA species of interest. In this process, the pH of the aqueous solution is adjusted to precipitate the TA precursor alone or TA at the respective pKa.

For high purity, small scale preparations, the TA precursor or TA can be purified by liquid chromatography in a single step.

Yeast-derived alkaloid API versus plant-derived API

Clarified yeast medium (CYCM) may contain a variety of impurities. The clarified yeast medium can be dehydrated by vacuum and/or heat to produce an alkaloid enriched powder. This product is similar to the concentrate of solanum leaves (CNL) which the Active Pharmaceutical Ingredient (API) manufacturer uses to extract the tropane alkaloids to be subjected to further chemical treatment and purification. For the purposes of the present invention, CNL is a representative example of any type of purified plant extract from which one or more desired alkaloid products can ultimately be further purified. Table 5 highlights impurities in both products, which may be unique to either CYCM or CNL, or may be present in both. By analyzing products of unknown origin for a subset of these impurities, one skilled in the art can determine whether the products are derived from yeast or plant production hosts.

API-grade pharmaceutical ingredients are highly purified molecules. Thus, plant or yeast derived impurities (such as those listed in tables 2 and 3) that may indicate API may not be present at the API stage of the product. Indeed, many API products derived from yeast strains of the invention may be largely indistinguishable from traditional plant-derived APIs. However, in some cases, conventional alkaloid compounds can be chemically modified using chemical synthesis methods, which can be manifested as chemical impurities in plant-based products in need of such chemical modifications. For example, chemical derivatization often results in a set of impurities associated with the chemical synthesis process. In certain instances, these modifications can be made biologically in the yeast production platform, thereby avoiding some of the impurities associated with chemical derivatization from being present in the yeast-derived product. In particular, these impurities from chemically derived products may be present in API products produced using chemical synthetic processes, but may not be present in API products produced using yeast derived products. Alternatively, if the yeast-derived product is mixed with the chemically-derived product, the resulting impurities may be present, but in an amount that is lower than would be expected in an API comprising only or predominantly the chemically-derived product. In this example, by analyzing the API product for a subset of these impurities, one skilled in the art can determine whether the product is derived from a yeast production host or a traditional chemical derivatization pathway.

Non-limiting examples of impurities that may be present in the chemically derivatized tropane alkaloid API, but not in the biosynthetic API, include hydrogen halides, such as hydrogen chloride, hydrogen iodide, and hydrogen bromide, formed by chemical N-alkylation (such as N-methylation and N-butylation) of hyoscyamine and scopolamine.

However, in the case where both the yeast-derived compound and the plant-derived compound are chemically modified by chemical synthesis methods, the same impurities associated with the chemical synthesis process can be expected to be present in the product. In this case, the starting material (e.g., CYCM or CNL) may be analyzed as described above.

Methods of engineering host cells

Also included are methods of engineering host cells for the production of a TA of interest or a precursor thereof. Insertion of the DNA into the host cell may be accomplished using any convenient method. The methods are used to insert a heterologous coding sequence into a host cell such that the host cell functionally expresses an enzyme and converts a starting compound of interest into a product of interest, TA.

Any convenient promoter may be used in the subject host cells and methods. The promoter driving expression of the heterologous coding sequence may be a constitutive promoter or an inducible promoter, provided that the promoter is active in the host cell. Heterologous coding sequences may be expressed from their native promoter, or non-native promoters may be used. Such promoters may have low to high strength in the host in which they are used. Promoters may be regulated or constitutive. In certain embodiments, a promoter that is not repressed by glucose or is only slightly repressed by the presence of glucose in the culture medium is used. Promoters of interest include, but are not limited to, promoters of glycolytic genes, such as the promoter of the Bacillus subtilis tsr gene (the promoter region encoding the fructose bisphosphate aldolase gene) or from the Saccharomyces cerevisiae gene encoding glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, phosphate starvation-induced promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from Bacillus licheniformis (B. licheniformis), yeast-induced promoters such as Gal1-10, Gal1, GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as the glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), the alcohol dehydrogenase promoter (ADH), the translation elongation factor-1-alpha promoter (TEF), the cytochrome c-oxidase promoter (CYC1), MRP7 promoter, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), etc. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used and include, but are not limited to, Glucocorticoid Response Element (GRE) and thyroid hormone response element (TRE). These and other examples are described in U.S. patent No. 7,045,290, which is incorporated by reference, including the references cited therein. Additional vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. In addition, any promoter/enhancer combination (EPDB can also be used to drive gene expression according to eukaryotic promoter databases any convenient appropriate promoter can be selected for the host cell, e.g., E.coli.

Any convenient vector may be used in the subject host cells and methods. Vectors of interest include vectors for yeast and other cells. The types of yeast vectors can be divided into 4 major categories: integrating vectors (YIp), autonomously replicating high copy number vectors (YEp or 2 μ plasmid), autonomously replicating low copy number vectors (YCp or centromeric plasmid), and vectors for cloning large fragments (YAC). Vector DNA is introduced into prokaryotic or eukaryotic cells by any convenient transformation or transfection technique.

Practicality of use

The host cells and methods of the invention, for example, as described above, can be used in a variety of applications. Applications of interest include, but are not limited to: research applications and therapeutic applications. The method of the present invention can be used in a number of different applications, including any convenient application that is of interest in the generation of TA.

The subject host cells and methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those applications of interest in the preparation of pharmaceutical products including TA. The host cells described herein produce tropane alkaloid precursors (TA precursors) and the TA of interest. Tropinone and tropine are the main branch point intermediates of interest in TA synthesis, including engineering efforts to produce end products such as pharmaceutical TA products. The subject host cells can be used to produce TA precursors from simple and inexpensive starting materials that can be used to produce the TA of interest, including tropinone, tropine, and TA end products. Thus, the subject host cells can be used to supply therapeutically active TA or precursors thereof.

In some cases, host cells and methods can be used to produce commercial scale quantities of TA or its precursors, where the chemical synthesis yields of these compounds are low and not viable means for large scale production. In some cases, the host cells and methods are used in a fermentation facility that will include a bioreactor (fermentor) of, for example, 5,000-200,000 liter capacity, allowing for rapid production of the TA of interest or its precursor for the therapeutic product. Such applications may include the production of the TA of interest from fermentable carbon sources such as cellulose, starch and free sugars on an industrial scale.

The subject host cells and methods find use in a variety of research applications. The subject host cells and methods can be used to analyze the effect of various enzymes on the biosynthetic pathways of various TA or precursors thereof of interest. In addition, host cells can be engineered to produce TA or precursors thereof that can be used to test for biological activity of interest in terms of therapeutic function that has not yet been demonstrated. In some cases, engineering a host cell to include multiple heterologous coding sequences encoding multiple enzymes elucidates a high yield biosynthetic pathway toward a TA or precursor thereof of interest. In some cases, research applications include the generation of precursors of therapeutic molecules of interest, which can then be further chemically modified or derivatized into desired products, or used to screen for increased therapeutic activity of interest. In some cases, host cell strains are used to screen for enzymatic activities of interest in such pathways, which may result in the discovery of the enzymes through transformation of TA metabolites produced in these strains.

The subject host cells and methods can be used as a platform for the production of plant specific metabolites. The subject host cells and methods can be used as a platform for drug library development and plant enzyme discovery. For example, the subject host cells and methods can be used to develop natural product-based drug libraries by obtaining yeast strains that produce scaffold molecules of interest (such as scopolamine and scopolamine) and further functionalizing the compound structures by combinatorial biosynthetic or chemical means. By generating drug libraries in this manner, any potential drug hits have been associated with production hosts suitable for large-scale culture and production. As another example, the subject host cells and methods can be used for plant enzyme discovery. The subject host cells provide a clean background of defined metabolites to express plant Expressed Sequence Tag (EST) libraries to identify novel enzymatic activities. The subject host cells and methods provide methods of expression and culture conditions for functional expression and increased activity of plant enzymes in yeast.

Kit and system

Aspects of the invention also include kits and systems, wherein the kits and systems can include one or more components used in the methods of the invention, e.g., host cells, starting compounds, heterologous coding sequences, vectors, culture media, and the like, as described herein. In some embodiments, a subject kit comprises a host cell (e.g., as described herein) and one or more components selected from the group consisting of: starting compounds, heterologous coding sequences and/or vectors comprising the heterologous coding sequences, vectors, growth materials, components suitable for use in expression systems (e.g., cells, cloning vectors, Multiple Cloning Sites (MCS), bidirectional promoters, Internal Ribosome Entry Sites (IRES), etc.), and culture media.

Any of the components described herein can be provided in a kit, e.g., including one or more modified host cells, starting compounds, culture media, and the like. A variety of components suitable for making and using heterologous coding sequences, cloning vectors, and expression systems can be used in the subject kits. The kit may also include tubes, buffers, and the like, as well as instructions for use. The various reagent components of the kit may be present in separate containers, or some or all of them may be pre-compounded into a reagent mixture in a single container, as desired.

Also provided are systems for producing a TA of interest, wherein the systems can include engineered host cells, starting compounds, media, fermenters, and fermentation devices comprising one or more modifications (e.g., as described herein), e.g., devices suitable for maintaining growth conditions of the host cells, sampling and monitoring devices and components, and the like. A variety of components suitable for large scale fermentation of yeast cells can be used in the subject systems.

In some cases, the system includes components for large scale fermentation of engineered host cells, and monitoring and purification of TA compounds produced by the fermented host cells. In certain embodiments, one or more starting compounds (e.g., as described herein) are added to the system under conditions whereby the engineered host cells in the fermentor are produced one or more desired TA products or precursors thereof. In some cases, the host cell produces the TA of interest (e.g., as described herein). In some instances, the TA product of interest is a pharmaceutically acceptable TA product such as hyoscyamine, N-methyl hyoscyamine, anisodamine, scopolamine, N-methyl scopolamine, and N-butyl scopolamine.

In some cases, the system includes a means for monitoring and or analyzing one or more TA compounds or precursors thereof produced by the subject host cell. For example, an LC-MS analysis system as described herein, a chromatography system, or any convenient system in which a sample can be analyzed and compared to a standard, for example as described herein. The fermentation medium can be monitored by sampling and analysis at any convenient time prior to and during fermentation. When the conversion of the starting compound to the TA product or precursor of interest is complete, the fermentation can be stopped and purification of the TA product can be performed. Thus, in some cases, the subject systems include purification components suitable for purifying a TA product or precursor of interest from a host cell culture medium in which it is produced. Purification components may include any convenient means that can be used to purify the TA product or precursor of the fermentation, including but not limited to silica chromatography (silica chromatography), reverse phase chromatography, ion exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction, and pH extraction methods. In some cases, the subject systems provide for the production and isolation of a TA fermentation product of interest after one or more starting compounds are input to the system.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Exemplary method

The following section provides examples of methods and procedures that can be used to construct, culture, and test microbial strains (such as yeast strains) for the production of TA precursors and TA, and to perform fermentations of such strains to produce TA precursors and TA. Also included are examples of methods, procedures and materials that can be used to generate DNA sequences required for modification of a microbial host and to introduce desired DNA sequences into a microbial host.

Chemical compounds and standards. TA precursors and TA chemical standards for verifying and quantifying the identity of metabolites produced by engineered host cells are available from commercial suppliers. For example, putrescine dihydrochloride, N-methylputrescine, guline, tropinone, and tropine are available from Santa Cruz Biotechnology (Dallas, TX). 4- (methylamino) butanoic acid hydrochloride was purchased from Sigma (St. Louis, Mo.). Gamma-methylaminobutyraldehyde (4MAB) diacetal and ritalin are commercially available from Torto Research Chemicals (Torto, ON). Chemical standards of NMPy can be synthesized by: deprotection of one volume of diacetal with five volumes of 2M HCl at 60 ℃ for 30 minutes was performed as previously described (see Feth, F., Wray, V., and Wagner, K.G. determination of methyl pultrescine oxidase by high performance chromatography. phytochemistry 24, 1653-propanoic 1655(1985)), overnight at room temperature, and the resulting concentrate was then washed twice with three volumes of diethyl ether to remove residual organic impurities.

And (5) constructing a plasmid. Oligonucleotides for generating new DNA sequences by Polymerase Chain Reaction (PCR) and for DNA sequencing can be obtained from DNA synthesis companies, such as IDT DNA, Twist Bioscience, or Stanford Protein and Nucleic Acid Facility (Stanford, CA). Native yeast genes can be amplified from s.cerevisiae genomic DNA by colony PCR (see Kwiatkowski, t.j., Zoghbi, h.y., Ledbetter, s.a., Ellison, k.a., and chinaut, a.c. rapid identification of yeast specific microorganisms by matrix placement and plasmid PCR. nucleic Acids res.18,7191 (1990)). The gene sequence of the heterologous enzyme can be codon optimized using suitable codon optimization software, such as GeneArt GeneOptimizer software (Thermo Fisher Scientific), to improve expression in Saccharomyces cerevisiae. The heterologous gene sequence can then be synthesized by commercial DNA synthesis into a linear double-stranded DNA fragment. Two types of plasmids can be used for gene expression in yeast: direct Expression (DE) plasmids for testing biosynthetic genes of interest and Yeast Integration (YI) maintenance plasmids that provide a template for genomic integration of selected promoter-gene-terminator cassettes.

The DE plasmid contains the gene of interest flanked by a constitutive promoter and terminator, a low copy CEN6/ARS4 yeast origin of replication, and an auxotrophic selectable marker. The DE plasmid can be constructed in the following manner: the gene of interest is PCR amplified to attach 5 'and 3' restriction sites using primer overhangs, the PCR product or the synthetic gene fragment is digested with the appropriate restriction enzyme pairs (e.g., SpeI, BamHI, EcoRI, PstI or XhoI), and then ligated into a similarly digested vector with the appropriate yeast promoter, terminator and replication sequences, such as plasmid pAG414GPD-ccdB, pAG415GPD-ccdB or pAG416GPD-ccdB using T4 DNA ligase (see Alberti, S., Gitler, A.D. and Lindquist, S.A. suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24,913-9 (2007)).

The YI plasmid contains the gene of interest flanked by a constitutive promoter and terminator, but lacks a yeast origin of replication or an auxotrophic selection marker. The YI plasmid can be constructed by linearizing an empty-retaining vector with appropriate promoter and terminator using 'around-the-horn' PCR using primers designed to bind to the 3 'and 5' ends of the promoter and terminator, respectively. The gene of interest can also be PCR amplified to attach 5 'and 3' overhangs with homology of 35-40bp to the ends of the linearized vector backbone. Genes can then be assembled into YI vectors using Gibson assembly. The DE plasmid expressing the GFP fusion of the biosynthetic enzymes can be prepared by: PCR-amplified DNA fragments encoding GFP, target enzyme and YI vector backbone, respectively, were first assembled using Gibson assembly, and the fusion construct from the YI plasmid was subsequently subcloned into the DE vector using restriction enzymes and ligation cloning as described.

PCR amplification can be performed using any high fidelity recombinant DNA polymerase available from commercial suppliers, and linear DNA can be purified using a suitable DNA column purification kit. Assembled plasmids can be propagated in any chemically competent E.coli strain using heat shock transformation and selection in Luria-Bertani (LB) broth with carbenicillin (100. mu.g/mL), kanamycin (50. mu.g/mL), or another antibiotic selection or LB agar plates. Plasmid DNA can be isolated by alkaline lysis from overnight E.coli cultures grown in selective LB medium at 37 ℃ and 250rpm, using a plasmid purification column according to the manufacturer's protocol. The plasmid sequence should be verified by Sanger sequencing.

And (5) constructing a yeast strain. Any suitable laboratory yeast strain may be used as the host organism. The yeast strains described in the examples of the experimental part were derived from the parent strain CEN. PK2-1D (see Entian, K.D. andP.25Yeast Genetic Strain and Plasmid collections, methods Microbiol.36, 629-666 (2007), referred to as CEN.PK2. The strain can be in yeast-peptone medium supplemented with 2% w/v dextrose (YPD medium), Yeast Nitrogen Base (YNB) defined medium supplemented with synthetic complete amino acid mixture (YNB-SC) and 2% w/v dextrose, Or non-selectively on agar plates of the above-mentioned medium. Strains transformed with plasmids carrying auxotrophic selection markers (URA3, TRP1, HIS3 and/or LEU2) can grow selectively in YNB medium supplemented with 2% w/v dextrose and appropriate deletion (dropout) solutions (YNB-DO) or on YNB-DO agar plates. Yeast strains deficient in acetate metabolism can be grown on the above media (i.e., YPAD or YNBA) supplemented with 0.1% w/v potassium acetate.

Yeast genome modification can be performed using the CRISPRM method (see Ryan, O.W. et al Selection of chromosomal DNA libraries using a multiplex CRISPR system. Elife 3, 1-15 (2014)). The CRISPRm plasmid expresses Streptococcus pyogenes (Streptococcus pyogenes) Cas9 and a single guide RNA (sgrna) targeting a locus of interest in the yeast genome, and can be constructed by assembly PCR and Gibson assembly of DNA fragments encoding Sp Cas9, tRNA promoter and HDV ribozyme (guide RNA sequence of 20-nt), and tracrRNA and terminator. For gene insertion, PCR amplification can be used to construct an integrated fragment of the gene of interest that contains one or more unique promoters and terminators flanked by Gibson assemblies that are cloned into a maintenance vector. The integrated fragments were PCR amplified using a suitable high fidelity DNA polymerase at the integration site using adjacent fragments and/or regions of microhomology of 40bp flanking the yeast genome. For gene disruption, the integration fragment contains 6-8 stop codons in all three reading frames, flanked by 40bp microhomology to the disruption site, in the first half of the open reading frame. For a complete gene deletion, the integration fragment contains an auxotrophic marker gene flanked by 40bp microhomology to the deletion site. Each integrated fragment was co-transformed with the CRISPRm plasmid targeting the desired genomic site. Positive integrants can be identified by yeast colony PCR, Sanger sequencing, and/or functional screening by liquid chromatography and tandem mass spectrometry (LC-MS/MS).

And (5) yeast transformation. The yeast strain can be transformed using any suitable method, including heat shock, electroporation, and chemical transformation. For example, the Yeast strains described in the examples of the experimental part were chemically transformed using the Frozen-EZ Yeast Transformation II kit (Zymo Research). Individual yeast colonies were inoculated into YP (A) D medium and grown overnight at 30 ℃ and 250 rpm. The saturated cultures were back-diluted to between 1:10 and 1:50 in YP (A) D medium and grown for an additional 5-7 hours to reach exponential phase. The culture was pelleted by centrifugation at 500 Xg for 4 min and then washed twice by resuspending the pellet in 50mM Tris-HCl buffer, pH 8.5. The washed pellet was resuspended in 20. mu.L of EZ2 solution for each transformation and then mixed with 100. mu.L of 600ng total DNA and 200. mu.L of EZ3 solution. The yeast suspension was incubated at 30 ℃ for 1 hour with gentle rotation. For plasmid transformation, transformed yeast were spread directly onto YNB (A) -DO agar plates. For Cas 9-mediated chromosomal modification, the yeast suspension was instead mixed with 1mL YP (a) D medium, pelleted by centrifugation at 500 × g for 4 min, and then resuspended in 250 μ L fresh YP (a) D medium. The suspension was then incubated at 30 ℃ for two more hours with gentle rotation to generate the G418 resistance protein, which was then plated on YP (A) D plates containing 400mg/L G418 (geneticin) sulfate. The plates were then incubated at 30 ℃ for 48-60 hours to allow colony formation.

Spot dilution assay. The strain was inoculated into YNB (A) -DO medium and allowed to grow overnight at 30 ℃ and 250 rpm. Saturated overnight cultures were pelleted by centrifugation at 500 Xg for 4 min and resuspended in sterile Tris-HCl buffer (pH 8.0) to OD basis₆₀₀Is 10⁷Concentration of individual cells/mL. Then, 10-fold serial dilutions of each strain were made in Tris-HCl buffer and 10. mu.L of each dilution was spotted onto pre-heated YNB (A) -DO plates. The plates were incubated at 30 ℃ and imaged after 48 hours.

Growth conditions for metabolite assays. Small scale metabolite production assays can be performed in YNB (A) -SC or YNB (A) -DO medium. Yeast colonies can be inoculated into 500. mu.L of 300-L medium and grown in 2mL deep well 96-well plates covered with gas permeable membranes for 48-72 hours in a shaker at 30 ℃, 460rpm and 80% relative humidity.

Metabolite production assays. The metabolite profile and titer can be analyzed using liquid chromatography and tandem mass spectrometry (LC-MS/MS). To separate the cells from the medium for analysis, the fermentation culture can be pelleted by centrifugation at 3,500 Xg for 5 minutes at 12 ℃ and an aliquot of 100-200. mu.L of the supernatant can then be removed for direct analysis. Metabolite production can be analyzed by LC-MS/MS using any suitable HPLC apparatus paired with a triple quadrupole mass spectrometer, such as Agilent 1260 infinite Binary HPLC and Agilent 6420 triple quadrupole mass spectrometer. Chromatography can be performed using a C18 reverse phase column such as a Zorbax eclipse plus C18 column (2.1X 50mm, 1.8 μm; Agilent Technologies) with 0.1% v/v formic acid in water as mobile phase solvent A and 0.1% v/v formic acid/acetonitrile as solvent B. The column was operated at 40 ℃ with a constant flow rate of 0.4mL/min and a sample injection volume of 5. mu.L. Compound separation can be performed using the following gradient: 0.00-0.75 min, 1% B; 0.75-1.33 minutes, 1-25% B; 1.33-2.70 minutes, 25-40% B; 2.70-3.70 minutes, 40-60% B; 3.70-3.71 minutes, 60-95% B; 3.71-4.33 minutes, 95% B; 4.33-4.34 minutes, 95-1% B; 4.34-5.00 min, equilibrated with 1% B. The LC eluent was directed to the MS at 0.01-5 minutes, operating in positive mode electrospray ionization (ESI), source gas temperature 350 deg.C, gas flow rate 11L/min and atomizer pressure 40 psi. Metabolites can be quantified by integrating peak areas based on Multiple Reaction Monitoring (MRM) parameters and standard curves.

And (4) fluorescent microscopy. Individual colonies of yeast strains transformed with plasmids encoding biosynthetic enzymes fused to fluorescent protein reporter genes were inoculated into 1mL YNB-DO medium and allowed to grow overnight at 30 ℃ and 250 rpm. The overnight culture was pelleted by centrifugation at 500 Xg for 4 minutes and resuspended in 2mL YNB-DO medium with 2% w/v dextrose, then grown for an additional 4-6 hours at 30 ℃ and 250rpm to reach exponential phase and allow for complete folding of the expressed fluorescent protein. Approximately 5-10 μ L of the culture was then spotted onto glass microscope slides and covered with glass coverslips, and then imaged using a suitable inverted fluorescence microscope with a 60X oil immersion objective. Fluorescence excitation can be performed using a xenon arc lamp and the following filter settings: GFP, ET470/40X excitation filter and ET525/50 emission filter; mCherry, ET572/35X excitation filter and ET632/60 emission filter. The emitted light is captured by a CCD camera and subsequent image analysis may be performed in any suitable scientific image analysis software, such as imagej (nih).

Novel gene variants were identified from transcriptome databases. Sequence alignment-based searches of transcriptome and genomic databases can be used to identify novel genes and variants thereof. For example, orthologs of N-methylputrescine oxidase from Nicotiana tabacum (NtMPO1) were identified in the 1000Plants Project database using a tBLASTn search of the transcriptome of Datura and belladonna (see Matasci, N. et al Data access for the 1,000Plants (1KP) Project. Gigascience 3,17 (2014)). The coding sequences of the putative genes identified using these search strategies can then be optimized for yeast expression and then cloned into expression vectors as previously described.

And (5) analyzing an enzyme structure. By examining homology models constructed using any suitable homology modeling or de novo structure prediction software (such as RaptorX or Rosetta), structural features of heterologous enzymes that may present problems during expression in yeast, such as large unstructured regions, can be analyzed. The resulting protein model can be visualized using any three-dimensional molecular viewing software, such as pymol (schrodinger) or UCSF kernel. The affinity of an enzyme for a particular substrate may be analyzed using any suitable ligand docking simulation software, such as AutoDock, swissdock, GOLD, or Glide.

Protein expression in yeast was analyzed by western blot. For immunoblot analysis of yeast expressed proteins, the appropriate strain is transformed with an expression vector containing the epitope tagged protein of interest. Three days after transformation, transformed colonies were inoculated into 2mL YNB-DO medium and grown to stationary phase overnight (. about.16-20 h) at 30 ℃ and 460 rpm. Cells were pelleted at 3,000 Xg for 5 minutes and resuspended in 200. mu. L H₂In O, mixed with 200. mu.L of 0.2M NaOH and incubated for 5 minutes at room temperature to allow cell wall glycoprotein hydrolysis. Cells were pelleted at 3,000 Xg for 5 minutes and resuspended in 75. mu. L H ₂O, mixed with 25. mu.L of 4 XNuPAGE LDS sample buffer (Thermo Fisher), and boiled at 95 ℃ for 3 minutes to lyse the cells. The suspension was pelleted by centrifugation at 16,000 Xg for 5 minutes to removeInsoluble debris was removed and the supernatant was transferred to a pre-cooling tube. For analysis under reducing conditions, protein lysates were mixed with β -mercaptoethanol (final concentration 10%) and incubated at 70 ℃ for 10 min. Approximately 20-40. mu.g of total protein was loaded onto NuPAGE Bis-Tris 4-12% acrylamide gel (Thermo Fisher) with Precision Plus Dual Color protein molecular weight markers (BioRad). Electrophoresis was performed at 150V for 90 minutes in 1 XNuPAGE MOPS SDS running buffer. Proteins were transferred to nitrocellulose membranes at 15V for 15 min using a Trans-Blot Semi-Dry apparatus (BioRad) and NuPAGE transfer buffer (Thermo Fisher) according to the manufacturer's instructions. For reducing conditions, NuPAGE antioxidant (Thermo Fisher) was added to the running buffer and transfer buffer to a final concentration of 1X. The membrane with the transfer protein was washed in Tris buffered saline with Tween (TBS-T; 137mM NaCl, 2.7mM KCl, 19mM Tris base, 0.1% Tween 20, pH 7.4) for 5 minutes and then blocked with 5% skim milk in TBS-T for 1h at room temperature. The membrane was incubated with HRP-conjugated antibody in TBS-T with 5% milk overnight at 4 ℃, washed 3 times with TBS-T for 5 minutes each, and visualized using a Western Pico PLUS HRP substrate (Thermo Fisher) and a suitable imager.

Experiment of

A specific set of genetic modifications provides a biosynthetic process in Saccharomyces cerevisiae for the production of TA from simple, inexpensive raw materials or precursor molecules. Methods for constructing novel strains capable of producing early TA molecular putrescine, N-methyl putrescine, 4-methylaminobutyraldehyde, N-methylpyrrolidinium (NMPy), tropinone, tropine, phenyllactic acid (PLA) and 1-O- β -phenyllactoyl glucose (PLA glucoside) from non-TA precursors or simple starting materials are described. NMPy is a natural precursor of all known TA molecules. Methods for manipulating the yeast biosynthetic pathway to regulate and optimize the production of amino acid-derived TA precursors are also described. Methods for constructing novel strains capable of producing non-pharmaceutical TAs such as pseudotropine alkaloids and calanthe extracts from simple raw materials are described. In addition, methods are described for constructing novel strains capable of producing pharmaceutically acceptable TAs such as hyoscyamine, anisodamine and scopolamine from non-TA precursors or simple starting materials. In addition, methods are described for constructing novel strains capable of producing non-native TA, such as cinnamoyl tropine, from non-TA precursors or simple starting materials.

Example 1 engineering of a platform Yeast Strain for high level putrescine production

The tropine moiety of TA is derived from the amino acid arginine through the polyamine molecule putrescine. For the purpose of increasing the intracellular concentration of TA precursor molecules including putrescine, NMP, 4MAB and NMPy, strains of saccharomyces cerevisiae with increased flux through the arginine and polyamine biosynthetic pathways were developed. These strains combine genetic modifications for increasing carbon and nitrogen flux overall from central metabolism to arginine and polyamine biosynthesis, and include the introduction of key heterologous enzymes to additionally produce the TA precursor putrescine. Genetic modifications are employed, including the introduction of feedback inhibition mitigating mutations in the genes encoding the native biosynthetic enzymes and regulatory proteins, fine-tuning of the transcriptional regulation of the native biosynthetic enzymes, deletion or disruption of the genes encoding enzymes that deviate the precursor molecule from the intended pathway, and the introduction of heterologous enzymes for the conversion of endogenous molecules into TA precursor molecules.

1.1) the biosynthetic pathway in the engineered strain incorporates overexpression of native yeast genes involved in arginine metabolism and polyamine biosynthesis (FIG. 4).

1.1.1) examples of overexpressing native genes in yeast include, but are not limited to: glutamate N-acetyltransferase (Arg2p), which catalyzes the first step in the biosynthesis of arginine from glutamate; arginase (Car1p) which removes the guanidinium group of arginine to produce ornithine in the mitochondrial matrix; a mitochondrial membrane transporter (Ort1p) that transports ornithine from outside the mitochondrial matrix to the cytosol; an ornithine decarboxylase (Spe1p) which decarboxylates cytosolic ornithine to putrescine; and polyamine oxidase (Fms1p) which dealkylates spermine and spermidine to putrescine.

1.1.2) the effect of overexpression of these native enzymes on putrescine production was examined by co-transforming yeast strains with different combinations of three low copy plasmids each expressing one of SPE1, ORT1, CAR1, ARG2, FMS1 or Blue Fluorescent Protein (BFP) as a negative control. After 48 hours of growth in selective medium, the titer of putrescine accumulated in the extracellular medium of the co-transformed cells was quantified by LC-MS/MS (FIG. 5). Overexpression of SPE1 alone resulted in a 13.4 fold increase in putrescine titer to 23 mg/L. While co-overexpression of CAR1 or ARG2 with SPE1 resulted in 27% and 12% increase in putrescine yield relative to SPE1 alone, overexpression of ORT1 with SPE1 resulted in a 35% decrease in putrescine titer compared to SPE 1. Overexpression of any three of SPE1, CAR1, ARG2, and FMS1 together increased extracellular putrescine titer to 34-35 mg/L.

1.2) the biosynthetic pathway in the engineered strain incorporates expression of heterologous enzymes from the polyamine production pathway found in organisms other than yeast to further increase putrescine production (FIG. 4).

1.2.1) in addition to the ornithine dependent pathway found in most plants, animals and fungi, whereby putrescine is synthesized by debriding of arginine and subsequent decarboxylation of ornithine, many bacteria and plants express an alternative pathway by which arginine is first decarboxylated by Arginine Decarboxylase (ADC) to agmatine. In plants, the guanidine group of agmatine is subsequently converted to urea by An Iminohydrolase (AIH) to produce N-carbamoylputrescine (NCP), from which the amide group is then removed by an amidase (CPA) to give putrescine (see Patel, J. et al Dual functional of plant amides providing a third route for putrescine Synthesis. plant Sci.262, 62-73 (2017)). Some bacteria have evolved Agmatine Urea Hydrolase (AUH), which is capable of removing the guanidine group directly from agmatine to produce putrescine without the intermediate of N-carbamoylation (see Klein, R.D. et al, Reconstation of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. microbiology 145(Pt 2,301-7 (1999)).

1.2.2) to reconstruct the heterologous putrescine biosynthetic pathway in yeast, the following enzymes can be used: ADC, AIH, CPA and AUH. As an example of engineered strains with these enzymatic activities, previously shown active in Saccharomyces cerevisiae, ADC (Avena sativa; AsADC) from oat (see Klein, R.D. et al Reconsistition of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae. Microbiology 145(Pt 2,301-7 (1999)), AIH (AtAIH) from Arabidopsis thaliana, two CPA orthologs from tomato (Solanum lycopersicum; SlCPA) and Arabidopsis thaliana (AtCPA), and two AUH from Escherichia coli (speB) and Arabidopsis thaliana (AtARGAH2) were selected for expression in yeast.

1.2.3) to establish the functionality of each heterologous enzyme in yeast, the three-step (arginine → agmatine → NCP → putrescine) or two-step (arginine → agmatine → putrescine) putrescine pathway was reconstituted in a stepwise manner by expressing AsADC, AtAIH and SlCPA or AtCPA; or co-transforming a wild-type yeast strain with a low copy plasmid of AsADC and speB or AtARGAH 2. To eliminate the effects on cell growth and metabolite production caused by different auxotrophic levels, all transformations were performed using three low copy plasmids containing different auxotrophic markers, instead of a blank or absent plasmid, using BFP as a negative control. Analysis of the relative accumulation of agmatine, NCP and putrescine in the extracellular medium after 48 hours of growth of transformed cells in selective medium by LC-MS/MS showed that all enzymes remained active in yeast except for SlCPA and AtARGAH2 (fig. 6, 7). Reconstitution of a plant-specific pathway comprising AsADC, AtAIH and AtCPA was able to produce putrescine at titers of 23mg/L, a 22-fold increase over wild-type titers. When combined with AsADC and AtAIH, orthologous cpa (slpha) from tomato is able to produce putrescine at titers of 4.5mg/L, similar to the putrescine levels in AsADC and AtAIH expressing cells. Reconstitution of the bacterial short path (bacterial short path) by AsADC and E.coli urea hydrolase (speB) enabled production of putrescine at a titer of 34mg/L, 32-fold higher than wild-type.

1.3) biosynthetic pathways in engineered strains incorporate overexpression of native yeast genes involved in arginine and polyamine biosynthesis and expression of heterologous biosynthetic enzymes from polyamine production pathways found in organisms other than yeast to further increase putrescine production.

1.3.1) optimal triplet (top-reforming tripartite) of overexpressed native genes to be used for putrescine biosynthesis (SPE1, ARG2, CAR 1; 1.1.2) and optimal heterologous putrescine pathway (AsADC, speB; 1.2.3) by co-transforming a wild-type yeast strain with a low copy plasmid encoding SPE1, AsADC and speB and a low copy plasmid encoding ARG2 and CAR 1. After 48 hours the putrescine titer in the medium of the transformed cells was measured by LC-MS/MS analysis. The resulting strain produced putrescine at a titer of 47mg/L (FIG. 10).

1.4) polyamine biosynthesis in yeast is regulated by several mechanisms (FIG. 8). Biosynthetic pathways in engineered strains incorporate disruption of one or more of these regulatory mechanisms to reduce feedback inhibition of putrescine production.

1.4.1) Natural yeast genes involved in the regulation of polyamine biosynthesis and therefore likely to be disrupted to improve intracellular putrescine accumulation include, but are not limited to, the following examples (FIG. 8). Methionine adenosylphosphorylase (Meu1p) catalyzes the driving step in the recycling pathway of decarboxylated S-adenosylmethionine (dcSAM), which constitutes an alkyl donor for the conversion of putrescine to spermidine and spermine catalyzed by spermidine synthase (Spe3p) and spermine synthase (Spe4p) (see Chattopadhyay, m.k., Tabor, c.w. and Tabor, h.methylidophenosine and polyaminobiosynthesis in a Saccharomyces cerevisiae Meu1 Δ mutant, biochem. biophysis. res. com.343, 203-207 (2006)). Methylthioadenosine is known to inhibit the activity of spermidine synthase (see Chattopadhyay, m.k., Tabor, c.w., and Tabor, h. students on the regulation of organic dehydrogenase in yeast: Effect of deletion in the MEU1 gene. proc. natl. acad. sci.102, 16158-16163 (2005)). Polyamine biosynthesis is regulated by an anti-enzyme mediated negative feedback loop that is conserved across fungi and metazoans (see Pegg, A.E. Regulation of organic Chemistry. journal of Biological Chemistry 281, 14529-14532 (2006)). In yeast, the OAZ1 gene comprises two exons separated by a single nucleotide, which together encode anti-enzyme-1, a competitive inhibitor of ornithine decarboxylase (Spe1 p). The polyamine-induced ribosomal frameshift mechanism is only capable of translating full-length antitases at high polyamine levels, thereby imposing feedback inhibition on their biosynthesis. Finally, the uptake of polyamines from the extracellular environment is mediated by signaling pathways involving Agp2p (a plasma membrane permease with affinity for carnitine, spermidine and spermine) and Sky1p (a protein kinase thought to interact with Agp2 p).

1.4.2) constructing yeast single gene disruption strains for each of MEU1, OAZ1, SPE4, SKY1 and AGP2 by inserting a series of tandem nonsense mutations within the first third of each open reading frame in wild-type yeast. To characterize the effect of each regulatory disruption in the context of the native and heterologous putrescine production pathways, either the yeast ODC (SPE1) was overexpressed, or AsADC and speB were co-expressed from low copy plasmids in each single gene disruption strain. After 72 hours of growth, the putrescine titer in the extracellular medium was measured by LC-MS/MS (fig. 9). When the native putrescine production pathway by SPE1 was overexpressed, MEU1 disruption increased the putrescine titer by 68%. Similarly, OAZ1 disruption significantly increased putrescine production by 174% when combined with overexpression of SPE 1. Disruption of OAZ1 resulted in a 21-fold increase in putrescine titer in untransformed cells that did not have an overexpressed native or heterologous putrescine pathway. When overexpressed with SPE1, disruption of SKY1 and AGP2 resulted in 29% and 14% increase in putrescine titer, respectively. When combined with heterologous expression of AsADC and speB, the SKY1 disruption resulted in a 41% reduction in putrescine titres.

1.5) biosynthetic pathway in engineered strains combining MEU1 and OAZ1 regulatory gene knockouts with overexpression of native and heterologous putrescine biosynthetic genes to further increase putrescine production in engineered strains. Additional copies of the native arginine and polyamine biosynthesis genes ARG2, CAR1, and FMS1 were integrated into the genome of the meu1/oaz1 double disrupted strain. This strain was transformed with low copy plasmids expressing SPE1, AsADC and speB. LC-MS/MS analysis of the extracellular medium of this transformed strain showed that after 48 hours of growth in selective medium, the putrescine titer reached 86mg/L (FIG. 10).

Example 2 Yeast strains engineered for NMPy production

A strain of saccharomyces cerevisiae was developed for the production of the TA precursor NMPy by modifying the putrescine overproducing strain developed in example 1. These strains combine genetic modifications aimed at increasing the carbon and nitrogen fluxes from putrescine to NMPy biosynthesis and involve the introduction of key heterologous enzymes to produce the TA precursors NMP, 4MAB and NMPy. Genetic modifications are used, including modification of the N and/or C terminal domains of the enzyme of interest to improve activity in a heterologous host, as well as deletion or disruption of genes encoding enzymes that drive the precursor molecule away from the intended pathway.

2.1) biosynthetic pathways in the engineered strain are capable of producing NMPy from endogenous putrescine. Putrescine is first converted to N-methylputrescine (NMP) by SAM-dependent N-methyltransferase (PMT), which is subsequently oxidized by copper-dependent diamine oxidase (MPO) to 4-methylaminobutyraldehyde (4 MAB). Like many aldehyde compounds, 4MAB is unstable in aqueous solution and spontaneously cyclizes to form NMPy by base-catalyzed nucleophilic attack (fig. 11).

2.1.1) putrescine overproducing strains of example 1.5 containing low copy plasmids expressing SPE1, AsADC and speB to overproduce putrescine were co-transformed with an additional low copy plasmid expressing PMT from belladonna (AbPMT1) and subsequent MPO enzyme from nicotiana tabacum (NtMPO 1). After 48 hours of growth, the accumulation of intermediates in the extracellular medium of transformed cells expressing each successive enzyme between putrescine and NMPy was compared by LC-MS/MS analysis. The direct product of NtMPO1 (4MAB) and its spontaneous cyclization product (NMPy) were generated by expression of AbPMT1 and NtMPO1 (fig. 11) and their precursors NMP and putrescine (fig. 12).

2.1.2) measuring NMP accumulation in the growth medium of putrescine overproducing yeast strains (described in example 1.4.2) with and without disruption of the MEU1 gene by LC-MS/MS analysis. This analysis showed that the previous disruption of MEU1 in the putrescine overproducing strain and its concomitant effect on SAM recycling did not inhibit putrescine N-methylation by AbPMT1 (fig. 13).

2.2) the enzyme, when expressed heterologously, may be localized to a different subcellular compartment than in its original host organism, resulting in a reduced function. Biosynthetic pathways in engineered strains may incorporate modifications to the polypeptide sequences of native and heterologous enzymes to induce localization of these modified enzymes to subcellular compartments other than those in which they are naturally localized. For example, previous studies have shown that, although NtPMT is expressed in the cytosol of tobacco cells, NtMPO1 is localized to the peroxidase cavity (see nanoconsie, m., Kato, k., Shoji, t. and Hashimoto, t. molecular evolution of n-methyl pulesensine oxidase in tobaco. plant Cell physiology.55, 436-444 (2014)).

2.2.1) subcellular localization of NtMPO1 was examined by in silico prediction of enzyme subcellular localization using Sherloc2 utility for signal peptide detection (see Briesemiester, S. et al, Sherloc2: A high-acquisition hybrid method for predicting subellicular localization of proteins. J. protein Res.8, 5363-5366 (2009)). This analysis indicated that NtMPO1 contains a strong yeast consensus Peroxisome Targeting Sequence (PTS) (Ala-Lys-Leu, denoted PTS1) at its C-terminus, indicating that NtMPO1 can localize to the peroxisome when expressed heterologously in yeast (fig. 14).

2.2.2) fluorescence microscopy of wild-type yeast cells expressing AbPMT1 and NtMPO1 with N-or C-terminal GFP-tagged from low-copy plasmids showed that although AbPMT1 is predominantly present in the cytosol, the localization of NtMPO1 in the peroxisomes is dependent on the exposed C-terminal PTS (fig. 15a, 16).

2.2.3) cytosolic expression of NtMPO1 by masking the C-terminal PTS with GFP fusion did not significantly affect extracellular 4MAB or NMPy levels (fig. 15 b).

2.3) biosynthetic pathways in the engineered strains may incorporate orthologs of biosynthetic enzymes other than those listed in Table 1. When expressed in heterologous hosts, different orthologs of the enzyme may exhibit significant differences in activity. Thus, orthologs of biosynthetic enzymes provided herein as examples and listed in table 1 may also be used in engineered non-plant cells to perform the same biochemical transformation.

2.3.1) tBLASTn search of the transcriptome of belladonna and Datura flower in the 1000Plants Project database (see Matasci, N. et al Data access for the 1,000Plants (1KP) Project. Gigascience 3,17(2014)) using the amino acid sequence of NtMPO1 as query sequence and 10^-150E value threshold of (a). Two full-length orthologs, designated AbMPO1 and DmMPO1, were identified Sequences of plants, each sharing 91% sequence identity with NtMPO1 (fig. 17 a).

2.3.2) yeast codon optimized sequences of AbMPO1 and DmMPO1 were obtained and cloned into low copy expression plasmids. To assess their activity, each of the three MPO variants was co-expressed from a low copy plasmid with AbPMT1 in the putrescine-overproducing strain of example 1.5 and the accumulation of 4MAB and NMPy in the extracellular medium was measured by LC-MS/MS after 48 hours of growth in selective medium. DmMPO1 showed comparable levels of 4MAB and NMPy production to the original NtMPO1 variant (fig. 17 b).

2.3.3) differences in activity between orthologous enzymes can generally be attributed, at least in part, to structural differences in their active sites. The template-based homology models for NtMPO1, AbMPO1, and DmMPO1 were constructed based on the crystal structure of pea copper-containing amino oxidase (PDB: 1KSI) using a RaptorX web server (see, e.g., FIG. 1K)M. et al Template-based protein structuring using the Raptorx web server. Nat. Protoc.7, 1511-22 (2012)). Homology models indicated that orthologs had long, unstructured N-and C-terminal tail regions (FIG. 17C).

2.3.4) the activity of truncations of the two active orthologs NtMPO1 and DmMPO1 was tested in engineered yeast. The N-terminal truncations removed the first 84 and 81 residues of the two orthologs, respectively. The C-terminal truncate removed the last 21 residues. A C-terminal truncate was also constructed in which the unstructured tail was removed but the PTS (expressed as ). Each MPO truncate was co-expressed with AbPMT1 from a low copy plasmid in the putrescine overproducing strain of example 1.5 and 4MAB and NMPy accumulation in the medium was quantified by LC-MS/MS after 48 hours of growth. No significant difference in activity was observed between the NtMPO1 truncates (fig. 18). Removal of the C-terminal unstructured region from DmMPO1 while retaining the C-terminal PTS tripeptide resulted in extracellular 4MAB levels relative to that of DmMPO1The wild type DmMPO1 enzyme was increased by 31%.

2.4) biosynthetic pathways in engineered strains incorporate one or more genetic modifications to reduce or eliminate metabolic flux of undesirable side reactions. Biosynthetic enzymes expressed in heterologous hosts may be involved in undesirable side reactions that draw metabolite fluxes away from the biosynthesis of the desired compound. For example, yeast aldehyde dehydrogenases can oxidize heterologous aldehyde molecules (such as 4MAB) to their cognate carboxylic acids. Based on LC-MS/MS analysis, when AbPMT1 andaccumulation of 4MAB acid was observed in the growth medium of the putrescine-overproducing strain of example 1.5 when co-expressed from a low copy plasmid, but not in the absence of MPO enzyme (FIG. 11).

2.4.1) six yeast genes (ALD2-ALD6 and HFD1) have been shown in the literature to encode enzymes with aldehyde dehydrogenase activity (see Datta, S., Annapure, U.S. and Timson, D.J.Difference species of two aldehyde dehydrogenase from Saccharomyces cerevisiae var. boulardardi. biosci.Rep.37, BSR20160529 (2017); and Nakahara, K. et al The Syndrome Gene Encodes a Hexaformal Dehydrogenase of the Sphingosine 1-Phosphate Degradation pathway, mol. cell 46, 461-471 (2012)). The ALD2 and ALD3 genes encode a pair of nearly identical cytosolic Dehydrogenases that catalyze the oxidation of 3-aminopropionaldehyde to beta-alanine in pantothenate biosynthesis (see White, W.H., Skatrud, P.L., Xue, Z. and Toyn, J.H. specialization of Function amplitude dehydrogenation: Genetics 163, 69-77 (2003)). The ALD4, ALD5 and ALD6 genes encode two mitochondria and a cytosolic acetaldehyde dehydrogenase, respectively, in addition to oxidizing acetaldehyde to acetate during fermentative growth on glucose and ethanol (see Saint-Prix, f.,l, and Dequin, S.functional analysis of the ALD geThe NADP + -dependent Ald6p and Ald5p iso forms a major roll in acetate formation. microbiology 150, 2209-2220 (2004)), which have been demonstrated to oxidize a range of different aliphatic and aromatic aldehydes to carboxylic acids (see Datta, S., Annapure, U.S. and Timson, D.J. Difference properties of two different aliphatic aldehydes from Saccharomyces cerevisiae var. boulardii. biosci. 37, 20160529 (2017)). These individual knockout strains of the four target genes were constructed by inserting a series of tandem nonsense mutations within the first third of their open reading frames in the putrescine overproducing strain of example 1.5. By co-expressing AbPMT1 and low copy plasmids in each single disruption strain And the contribution of each of the four dehydrogenases to 4MAB oxidation was assessed by LC-MS/MS measurement of 4MAB acid accumulation in the medium after 48 hours of growth. A marginal drop in 4MAB acid level was observed with HFD1 and ALD4-6 destruction alone (fig. 19).

2.4.2) although ALD4-6 was considered an essential gene due to its role in acetate and acetyl-CoA production, previous studies have shown that these three genes are at least partially redundant and that the lethal phenotype of double and triple knockouts can be rescued by supplementing the medium with acetate (see Saint-Prix, F.; et al, supra),L, and Dequin, S.functional analysis of The ALD gene family of Saccharomyces cerevisiae during and atmospheric growth on glucose The NADP + -dependent Ald6p and Ald5p of The isoformans plant a major roll in acetate formation. microbiology 150, 2209-2220 (2004); and Luo, Z, Walkey, C.J., Madilao, L.L., Measday, V.and Van Vuuren, H.J.J.functional improvement of Saccharomyces cerevisiae to reduced cellulose acid in wire FEMS Yeast research Res.13, 485-494 (2013)). Construction of a quadruple knock-out yeast strain in which the open reading frame of HFD1 and ALD4-6 was disrupted and represented by a low copy plasmid Reach AbPMT1 andthis strain showed a 45% decrease in 4MAB acid level (fig. 20a) and a concomitant 46% increase in NMPy production (fig. 20b) compared to the non-disrupted strain.

2.4.3) by deleting the tandem ALD2-ALD3 gene from the genome of the quadruple knockout strain of example 2.4.2 and co-expressing AbPMT1 and low copy plasmidsTo construct ALD-null strains. After 48 hours of growth, LC-MS/MS analysis showed that the deletion of ALD2 and ALD3 completely eliminated the 4MAB acid by-product and increased the 4MAB and NMPy yields by 83% and 75%, respectively, compared to all six ALD gene-intact strains (fig. 20a, b).

2.4.4) by integrating the previously plasmid-borne putrescine overproducing gene cassette (SPE1, AsADC, speB) into the genome of the ALD null strain of example 2.4.3 and additionally the AbPMT1 andto construct NMPy-producing yeast strains. LC-MS/MS analysis confirmed that the NMPy yield of the strain after 48 hours of growth in non-selective medium was comparable to that of putrescine-producing genes AbPMT1 and AbPMT, which are essential for expression from low copy plasmidsAnd the ALD-null strain of example 2.4.3 cultured in selective medium was comparable (figure 21).

Example 3 engineered Yeast strains for production of tropine from monosaccharides and nutrients

Type III polyketide synthases (PKS) and cytochrome P450 are able to convert NMPy to tropinone via the TA precursor MPOB. Tropinone can be reduced by a stereospecific reductase, denoted as tropinone reductase 1(TR1), to produce tropine (see Kim, n., estoda, o., Chavez, b., Stewart, c., and D' Auria, j.c. tropane and grandane alloy biosyntheses: a Systematic analysis. molecules 21, (2016)) (fig. 22).

3.1) the biosynthetic pathway in the engineered strain incorporates pyrrolidone synthase, tropinone synthase CYP82M3, one or more cytochrome P450 reductases, and tropinone reductase 1 to convert NMPy to tropine.

3.1.1) obtaining a yeast codon optimized DNA sequence encoding belladonna pyrrolidone synthase (AbPYKS), tropinone synthase (AbCYP82M3) and Datura stramonium ketoreductase 1(DsTR 1). A set of yeast codon-optimized sequences for four different CPRs was also obtained, including three plant CPRs from arabidopsis thaliana, Escholzia californica (California poppy) and Papaver somniferum (opium poppy), and native yeast CPR (NCP1) for expression in yeast, as the P450 enzyme requires NADP ⁺The Cytochrome P450 Reductase (CPR) partner undergoes a sustained electron exchange. Yeast strains were constructed by integrating DsTR1 into the genome of the NMPy-producing strain of example 2.4.4 and expressing AbPYKS, AbCYP82M3 and each of the four CPR from low copy plasmids. To verify the enzyme activity and identify potential bottlenecks, the culture medium of the transformed strains was monitored for accumulation of NMPy, MPOB, tropinone and tropine by LC-MS/MS after 48 hours of growth (fig. 23). Under the assay conditions, a comparable level of de novo tropin production was observed with all four CPR partners (175-.

3.2) the presence of metabolic bottlenecks defined as biosynthetic enzymes or spontaneous steps whose low activity limits the flux through a part of the biosynthetic pathway can lead to a sub-optimal production of the desired TA and precursors.

3.2.1) for example, analysis of TA intermediate accumulation in the culture medium of the engineered strain of example 3.1.1 showed that most of the MPOB produced by AbPYKS remained unconsumed by AbCYP82M3, although the accumulation of the product tropinone of AbCYP82M3 was minimal (figure 24).

3.2.2) integration of the tropine biosynthesis genes into the yeast genome can improve the production of tropine by achieving a more stable expression of AbCYP82M 3. A tropicalizing platform strain was constructed by integrating atr1 together with AbPYKS and AbCYP82M3 into the genome of the NMPy-producing strain of example 3.1.1. After 48 hours, the accumulation of both tropine and coumarine by the integrated strain was compared to plasmid-based expression of the same gene by LC-MS/MS analysis (FIG. 28). Genomic expression of AbPYKS, AbCYP82M3, and AtATR1 increased the tropin titer nearly threefold (565. mu.g/L) relative to plasmid-based expression (189. mu.g/L). The engineered strain also showed a 2.6-fold increase in the accumulation of gulconine.

3.3) accumulation of by-products in the biosynthetic pathway of engineered strains can lead to sub-optimal production of the desired TA and precursors.

3.3.1) for example, analysis of the accumulation of TA intermediates in the medium of the engineered strain of example 3.1.1 showed that the derivative of NMPy, coumarine, was accumulated in large amounts with titers almost four times greater than that of tropine (775-900. mu.g/L). In the relevant literature, spontaneous decarboxylation accumulation of gulconine by MPOB has been observed (see Bedevitz, M.A., Jones, A.D., D' Auria, J.C., and Barry, C.S. tropinone synthesis via an enzymatic synthesis and P450-mediated cyclization. Nat. Commun.9,5281(2018)) (FIG. 22). As another example, LC-MS/MS analysis of the growth medium of the engineered strain of example 3.1.1 showed that due to decarboxylative condensation with NMPy, gulconine also accumulated in the negative control strain lacking AbPYKS and AbCYP82M3 (fig. 22).

3.3.2) modulation of growth temperature can be used to reduce the accumulation of byproducts in the biosynthetic pathway of engineered strains to increase flux towards the desired TA and precursors. In one example, the effect of temperature on spontaneous coumarine production was evaluated by using the following kinetic principles: that is, the rates of enzymatic and spontaneous reactions decrease at lower temperatures. Since belladonna and other TA producing solanaceae plants are suitable for optimal growth in colder climates, growth of yeast strains expressing solanaceae genes at 25 ℃ can improve enzyme folding and/or activity, enabling an enzymatically produced yield of tropine comparable to growth at 30 ℃ while reducing the rate of spontaneous guguline production. Cultures of the tropene-producing strain of example 3.2.2 were grown in nonselective defined media at 30 ℃ and 25 ℃ and compared for accumulation of tropene and coumarine after 48 hours by LC-MS/MS analysis of the growth media. The tropine titer was minimally affected by the decrease in temperature. The accumulation of gulicine was reduced by 42% at 25 ℃ compared to 30 ℃ resulting in a 60% increase in the ratio of tropane to gulicine produced (figure 25).

3.3.3) reducing or eliminating adverse side reactions can be used to increase metabolite flux towards the desired TA and TA precursors in the biosynthetic pathway of engineered strains. In one example, flux towards the TA precursor tropine can be increased by reducing the production of gulconine resulting from spontaneous decarboxylating condensation with acetate. The effect of removing fed acetate from the medium of the NMPy-producing strain of example 2.4.4 on the production of both coumarine and tropine was evaluated. The effect of ablating acetate auxotrophy in the engineered strain of example 2.4.4 was assessed by expressing functional copies of ALD4 and ALD6 on low copy plasmids and then monitoring the accumulation of guline and 4MAB acid by LC-MS/MS analysis after 48 hours of growth. Although reconstitution of ALD4 or ALD6 was able to achieve growth on selective media in the absence of fed acetate (fig. 26a), addition of ALD4 resulted in a 5-fold increase in accumulation of 4MAB acid, while ALD6 produced no significant increase (fig. 26 b). Furthermore, elimination of acetate feed in the case of ALD4 or ALD6 resulted in a 38% and 59% reduction in the accumulation of gulconine, respectively (fig. 26 b).

3.3.4) functional copies of the ALD6 gene were reintegrated into the tropicalizing strain of example 3.2.2 at the previously disrupted ALD6 locus. The effect of this integration on the accumulation of all metabolites between NMPy and the tropine was measured by LC-MS/MS analysis after 48 hours of growth in non-selective medium. Restoration of acetate metabolism by Ald6p resulted in a 2.7-fold increase in tropine titer and a 1.6-fold increase in coumarine accumulation (figure 28). Furthermore, integration of ALD6 resulted in a significant increase in NMPy and tropinone yields and an increase in consumption of MPOB (fig. 27).

3.3.5) expression of the respective biosynthetic enzyme genes between putrescine and tropine from the low copy plasmids in the engineered strains of example 3.3.4 (i.e., AbPMT1,AbPYKS and AbCYP82M3) and in selective mediumAfter 48 hours of growth, the production of TA intermediate was compared to that of the same strain expressing BFP by LC-MS/MS. Expression of additional copies of AbPYKS resulted in a 4.3 fold increase in NMP accumulation and a 1.3 fold increase in tropin yield (figure 29). Expression of additional copies of AbPMT1 resulted in a significant increase in the yield of all TA precursors between NMP and tropinone, and a 2.4-fold increase in tropine yield (fig. 29). Thus, additional copies of PMT (AbPMT1 and DsPMT1) and pyks (abpyks) were integrated into the genome of the tropicalis strain of example 3.3.4 (CSY1249) at the PAD1 locus. The resulting engineered strain (CSY1251) was grown in non-selective medium at 25 ℃ for 48 hours, resulting in the production of tropine at a titer of 3.4mg/L, 2.2-fold greater than the tropine-producing strain in example 3.3.4 (CSY1249) (FIG. 30).

Example 4: a yeast engineered to produce a pseudotropine alkaloid from L-arginine.

Yeast strains can be engineered to produce non-medicinal TA from early amino acid precursors (such as L-arginine). For example, the platform yeast strain described in example 3 can be further engineered to produce pseudotropine alkaloids from L-arginine (fig. 1).

A platform yeast strain that produces tropinone from L-arginine (see description in example 3) can be further engineered to incorporate a stereospecific reductase, such as tropinone reductase 2(TR 2; EC 1.1.1.236), to convert the biosynthetic tropinone to pseudotropine. An expression cassette containing the coding sequence of a strong constitutive promoter such as TDH3 and a TR2 variant (e.g., TR2 from datura grandiflora (DsTR2)) can be integrated into the genome of a tropinone-producing platform yeast strain. The resulting strain can be further engineered to produce hydroxylated derivatives of pseudotropine, e.g., calycanthus essence, by integrating one or more expression cassettes containing a strong constitutive promoter (such as PGK1) and a hydroxylating enzyme (such as cytochrome P450 acting on the pseudotropine scaffold). By incorporating multiple P450 enzymes, each acting at different positions of the pseudotropine scaffold, a variety of warfarin and its derivatives can be biosynthesized. The engineered strain can then be cultured in non-selective synthetic complete media at 30 ℃ or 25 ℃ for 48 to 96 hours, and the accumulation of pseudotropine alkaloids in the media can then be analyzed by LC-MS/MS.

Example 5: yeast engineered to overproduce pyruvate and related TA precursors.

Yeast strains can be engineered to overproduce phenylpyruvate, which represents the precursor of the acyl donor molecule required to produce a medicinal TA (fig. 2), in order to increase the carbon and nitrogen flux from central metabolism towards the desired TA and TA precursors. Yeast strains can be engineered to overproduce pyruvate by incorporating genetic modifications including, but not limited to, fine-tuning of transcriptional regulation of natural biosynthetic enzymes, deletion or disruption of genes encoding enzymes that deviate precursor molecules from the intended pathway, and introduction of heterologous enzymes for conversion of endogenous molecules to TA precursor molecules.

In one example, yeast strains can be engineered to increase production of pyruvate by incorporating additional copies of the native genes encoding biosynthetic enzymes that produce pyruvate from amino acids or other central metabolites. These additional copies may be under the control of a strong constitutive promoter, such as GPD, TEF1, or PGK 1. Examples of natural gene targets include, but are not limited to, the aromatic acid aminotransferases ARO8 and ARO9, and the dehydratase PHA 2. In one instance, one or more additional copies of ARO8 may be incorporated into the engineered strain under the control of a strong constitutive promoter. In one instance, one or more additional copies of ARO9 may be incorporated into the engineered strain under the control of a strong constitutive promoter. In another case, one or more additional copies of PHA2 can be incorporated into the engineered strain under the control of a strong constitutive promoter. In one embodiment of the invention, one or more additional copies of one or more genes selected from the group comprising ARO8, ARO9, and PHA2 may be incorporated into the engineered strain under the control of a unique strong constitutive promoter.

Example 6: yeasts engineered to produce acyl donors for TA scaffold biosynthesis from L-phenylalanine or L-tyrosine.

Yeast strains can be engineered to produce a variety of phenylalanyl donor compounds from L-phenylalanine and L-tyrosine, including PLA, cinnamic acid, coumaric acid, ferulic acid, benzoic acid, and coenzyme a thioesters, and glycoside derivatives of these compounds, which can be esterified with tropines, pseudotropines, or derivatives thereof to biosynthesize medicinal TA, non-medicinal TA, and non-natural TA (fig. 1-3).

6.1) since wild-type yeast only produce traces of PLA, the production of this TA precursor must be increased to allow sufficient accumulation of downstream TA. To increase PLA production, heterologous phenylpyruvate reductase (PPR) can be expressed in the engineered host cell. PPR orthologs from E.coli, Lactobacillus, belladonna and Fluoroway yeast, as well as Lactate Dehydrogenase (LDH) with reporter activity on 3-phenylpyruvate from Bacillus and Lactobacillus were screened for activity in yeast by expressing each enzyme from a low copy plasmid in CSY1251 and measuring PLA production by LC-MS/MS after 72h growth in selective medium (Table 1). All LDH candidates as well as PPR from lactobacillus plantarum, escherichia coli and belladonna gave a moderate (1.3 to 3.5 fold) improvement in PLA production relative to controls, while expression of PPR from fluorogenic yeast led to nearly 80 fold increase in PLA yield to-250 mg/L (fig. 31). Therefore, WfPPR was selected for integration into CSY1251 to make strain CSY 1287.

6.2) As another example, yeast strains can be engineered to produce cinnamic acid and coumaric acid, which are phenylpropanes, which can be used as acyl donor compounds for esterification with tropine or pseudotropine to form unnatural TA from L-phenylalanine and L-tyrosine, respectively. Yeast can be engineered to produce cinnamic acid from L-phenylalanine by incorporating a deaminase, such as phenylalanine ammonia lyase (PAL; EC 4.3.1.24). Similarly, yeast can be engineered to produce coumaric acid from L-tyrosine by incorporating a deaminase, such as tyrosine ammonia lyase (TAL; EC 4.3.1.23). Yeast strains were engineered to produce cinnamic acid from L-phenylalanine by transformation with a low copy CEN/ARS plasmid with a TRP1 selective marker, the TEF1 promoter and the coding sequence for the PAL variant from Arabidopsis (AtPAL 1). The resulting low copy plasmid containing strains in appropriate amino acid deletion solution (-Ura) synthesis complete medium at 30 degrees C growth. After 48 hours of growth, the media were analyzed for cinnamic acid content by LC-MS/MS analysis (FIG. 32).

6.3) in belladonna, PLA was activated by glucosylation using UDP-glucosyltransferase 84A27(AbUGT) to transfer acyl groups to a tropine (see Qiu, F. et al, Functional genes analysis systems resources two novel genes required for littoraine biosyntheses. New Phytol., nph.16317 (2019)). Since plant UGTs are involved in the biosynthesis of various phenylpropanes and generally exhibit a broad substrate range (see Ross, j., Li, y., Lim, e. -k., d.j. bowles, high plant glycerol transfer. genome biol.2, 3004.1-3004.6 (2001)), it is necessary to select UGTs with sufficiently high activity for the desired acyl donor.

6.3.1) for example, the activity of AbUGT on different phenylalanyl donors (including the classical substrate PLA) was assessed by expressing AbUGT from a low copy plasmid in CSY1251 and measuring the conversion of three phenylalanyl donors (PLA, cinnamic acid, ferulic acid) to their respective glycosides. Although AbUGT glucosylated-60% and 90% cinnamic acid and ferulic acid, respectively, glucosylation of PLA was the lowest among the substrates tested, with conversion < 3% (figure 33).

6.3.2) can assess the activity of orthologs of AbUGT from other TA-producing solanaceae plants on PLA and other phenylpropanes. In this example, a tBLASTn search was used to obtain transcripts encoding UGT84a27 from the transcriptome of datura stramonium (BsUGT) and datura flower (DmUGT) in the 1000 plantats database. Yeast codon optimized sequences encoding these orthologous UGTs were screened for activity by expressing the AbUGT, BsUGT, DmUGT or BFP negative controls from low copy plasmids in CSY 1251. After 72h growth in selective medium supplemented with 500 μ M PLA, Cinnamic Acid (CA) or Ferulic Acid (FA) as glucose receptors, glucoside production in the culture of the transformed strain was measured by LC-MS/MS. All three UGT orthologs showed significant glycosylation of CA (34-65% conversion) and FA (85-90% conversion) and showed only trace activity on PLA (< 3% conversion), and AbUGT showed the maximum PLA conversion (2.7%) (fig. 33, 34).

6.3.3) given the disproportionate difference in the activity of AbUGT on structurally similar substrates cinnamate, ferulate and PLA, a structure-guided rational mutagenesis approach can be implemented to engineer the active site of AbUGT to improve activity on PLA. In this example, a homology model of the UDP-glucose-binding AbUGT was first constructed based on the crystal structure of the Arabidopsis thaliana UDP-glucosyltransferase UGT74F2 (PDB: 5V2K) using a RaptorX web server (FIG. 35). Next, docking of D-PLA at the active site was simulated using the Maestro/GlideXP software suite. Based on the energy minimization coupled mode, the aryl ring of D-PLA may be stabilized by pi-stacking interactions with F130, while its alpha-hydroxy and carboxylate groups are stabilized by hydrogen bonding with Q151 and H24, respectively, such that the nucleophilic carboxylate oxygen is located at the electrophilic C1 carbon of UDP-glucoseInside (fig. 35). D-PLA is also adjacent to residues L205 and I292, neither of which appears to interact with either substrate. This indicates that (i) the F130 mutation to tyrosine can retain pi stacking with the aryl ring of D-PLA while providing additional hydrogen bonding to stabilize the alpha-hydroxy oxygen of D-PLA, which is not present in cinnamate and ferulate; (ii) the mutation of L205 to phenylalanine can increase the pi stacking stability of D-PLA and F130Y; and (iii) mutation of I292 to glutamine would generate two additional stable hydrogen bonds with D-PLA and UDP-glucose (fig. 35). F130Y, L205F, and I292Q point mutants of AbUGT were screened for activity by expressing each mutant, wild type AbUGT or BFP control from low copy plasmids in CSY1251 and measuring glucoside production by LC-MS/MS after 72h growth in selective media supplemented with 500 μm PLA, CA or FA. Although the F130Y and I292Q mutations significantly reduced UGT activity on CA, all three mutants exhibited relative to wild-type AbUGT: ( <3% conversion) was rather low (and statistically indistinguishable) activity on PLA (figure 36).

6.3.4) based on the results described in sections 6.1 and 6.3, strain CSY1288 was constructed by integrating yeast codon optimized WfPPR and AbUGT into the genome of CSY1251, verified by verifying PLA production (66mg/L) and minimal PLA glucoside accumulation (fig. 37).

6.4) since poor activity of AbUGT on PLA may limit the flux of TA precursors towards downstream TAs, the flux of phenylalanine to PLA glucoside can be increased by incorporating genetic modifications that promote UDP-glucose accumulation and reduce glycoside degradation.

6.4.1) UDP-glucose is essential for the formation of storage polysaccharides, cell wall glucans and glycoproteins, and therefore its biosynthesis is tightly regulated (see Nishizawa, M., Tanabe, M., Yabuki, N., Kitada, K., Toh-e, A. Pho85kinase, a yeast cycle-dependent kinase, regulation of the expression of UGP1 encoding UDP-glucose pyrophosphorylase. Yeast.18, 239-249 (2001)). During growth on glucose, yeast directly produces glucose-6-phosphate along two major metabolic pathways, glycolysis and starch biosynthesis. Since citrate is an allosteric inhibitor of the glycolysis rate-limiting enzyme phosphofructokinase (see Li, Y. et al, Production of recombinant A from stereoside Catalyzed by the Engineered Saccharomyces cerevisiae. appl. biochem. Biotechnol.178, 1586-1598 (2016)), partial inhibition of glycolysis by supplementation with citrate may increase UDP-glucose availability and glucoside Production (FIG. 38). Strain CSY1288, encoding genomic WfPPR and AbUGT for endogenous PLA glucoside production, was cultured in medium supplemented with 2% citrate and 500 μ M CA or FA and glucoside production was compared by LC-MS/MS after 72h growth. Supplementation with citrate reduced glucosylation of PLA, CA and FA by 83%, 56% and 78%, respectively (fig. 39).

6.4.2) overexpression of PGM2 and UGP1 whose gene products catalyze the isomerization of glucose-6-phosphate to glucose-1-phosphate and the conversion of glucose-1-phosphate to UDP-glucose, respectively, can be used to increase UDP-glucose supply.

6.4.2.1) additional copies of PGM2 and UGP1 were expressed from low copy plasmids in CSY1288 and PLA glucoside production was measured after 72h of growth in selective media. Although PGM2 overexpression produced no improvement relative to the control, overexpression of UGP1 resulted in a-1.8 fold increase in PLA glucoside production (fig. 40), which supports an increase in UDP-glucose pool (pool) improving the utilization of AbUGT for PLA.

6.4.2.2) native glucosidases can act on PLA and other TA precursor glucosides to reduce accumulation, as other heterologous glucosides have been shown to hydrolyze in this manner in yeast (see Schmidt, S., Rainieri, S., Witte, S., Matern, U., Martens, S., Identification of a Saccharomyces cerevisiae glucose enzymes which are hydrolytically fluconoid glucosides, Appl. environ. Microbiol.77, 1751-1757 (2011); see also Wang, H.et al, Engineering Saccharomyces cerevisiae with the deletion of end carbohydrates for the production of flavor carbohydrates. Microb. cell fact.15, 1-12 (2016)). In this example, three native glucosidase genes-EXG 1, SPR1, and EGH 1-were disrupted in CSY1288, and PLA glucoside production was measured after the disruption mutants were grown for 72h in non-selective medium. EGH1 disruption increased PLA glucoside production more than one-fold (fig. 41), indicating that hydrolysis by Egh1p constitutes a significant loss of TA precursor flux.

Example 7: yeast engineered to convert ritodrine to hyoscyaldehyde

The yeast strain may be engineered to convert ritodrine to hyoscyaldehyde (fig. 2). For example, the tropine-and PLA glucoside-producing yeast strains described in example 6 may be further engineered to express cytochrome P450 CYP80F1(EC 1.14.19.-) to catalyze the rearrangement of ritodrine to hyoscyaldehyde, and to express cytochrome P450 reductase (CPR; EC 1.6.2.4) to support the activity of the P450 enzyme. The yeast strain was engineered to convert the fed ritodrine to hyoscyaldehyde by using a low copy CEN/ARS plasmid with the LEU2 selection marker, TDH3 promoter, and the coding sequence from the CYP80F1 variant from belladonna (AbCYP80F 1); and transforming the yeast strain with a low copy CEN/ARS plasmid having a TRP1 selectable marker, a TEF1 promoter, and a coding sequence for Cytochrome P450 Reductase (CPR) from saccharomyces cerevisiae (NCP1) or arabidopsis thaliana (atrr 1). The resulting strain containing the low copy plasmid was grown at 30 ℃ in synthetic complete medium with the appropriate amino acid deletion solution ((-Leu-Trp)) supplemented with 1mM ritorine. After 48 hours of growth, the media was analyzed for the content of scopolamine by LC-MS/MS analysis (fig. 42).

Example 8: a yeast engineered to convert scopolamine to scopolamine.

The yeast strain may be engineered to convert scopolamine to scopolamine (fig. 2). For example, the yeast strain described in example 7 may be further engineered to incorporate an enzyme having hydroxylase activity at the 6 β -position of the scopolamine to form scopolamine and an enzyme having dioxygenase activity at the 6 β -hydroxyl position of the scopolamine to form scopolamine, or both activities (EC1.14.11.11). The yeast strain is engineered by transforming it with a low copy CEN/ARS plasmid having the LEU2 selection marker, the TDH3 promoter, and the coding sequence for hyoscyamine 6 β -hydroxylase/dioxygenase (H6H) from datura stramonium (DsH6H), scopolia acutangula (AaH6H), woody stramonium (BaH6H) or datura flower (DmH6H) to convert the fed scopolamine to scopolamine. The resulting strains containing low copy plasmids were grown at 30 ℃ in synthetic complete medium with the appropriate amino acid deletion solution (-Leu) supplemented with 1mM hyoscyamine. After 72 hours of growth, the media was analyzed for scopolamine content by LC-MS/MS analysis (fig. 43). Strains expressing the H6H variant from datura showed the greatest conversion of the fed scopolamine to scopolamine, although all tested variants showed H6H activity in vivo. The cofactor requirements were further optimized by supplementing the medium of the engineered yeast strains with different cofactors and analyzing the medium by LC-MS/MS after 72 hours of growth. This analysis determined that supplementation with ferrous iron increased the conversion of scopolamine to scopolamine (fig. 44).

Example 9 identification of a scopolamine dehydrogenase candidate and reduction of scopolamine to scopolamine

In order to identify a TA alcohol-aldehyde interconversion suitable for performing the methods disclosed herein, in particular a dehydrogenase that reduces scopolamine to scopolamine, a scopolamine dehydrogenase (HDH) open reading frame was identified from publicly available plant RNA sequencing data.

9.1) tissue specific abundance (fragment per kilobase contig per million mapped reads, FPKM) and putative protein structure and function annotations for each of the 43,861 unique transcripts identified from the belladonna transcriptome were obtained from Michigan State University Medicinal Plant Genomics resources. Transcripts encoding hyoscyamine dehydrogenase candidates were identified based on clustering of tissue-specific expression profiles with decoy genes (bait gene) CYP80F1 (ritopressin mutase) and H6H (hyoscyamine 6 β -hydroxylase/dioxygenase) before and after the dehydrogenase step in the TA biosynthetic pathway, respectively, using the following computational filter algorithm.

First, a complete list of 43,861 transcripts was filtered to find those with annotations for any of the following Protein Family (PFAM) IDs: PF00106, PF13561, PF08659, PF08240, PF00107, PF00248, PF00465, PF13685, PF13823, PF13602, PF16884, PF 00248; or annotate the keyword with any of the following functions: alcohol dehydrogenase, aldehyde reductase, short chain, aldehyde/ketone. In addition, any transcript with functional annotation containing the keywords putrescine, tropinone, and tropine was included in the filter as a positive control TA-associated gene to verify clustering with the decoy gene. Next, mean tissue-specific expression profiles were generated for CYP80F1 and H6H decoy genes. For each of the two bait genes, a linear regression model was constructed to represent the bait gene expression profiles (in FPKM) as a linear function of each candidate gene profile and to calculate the correlation p-value for each candidate. Candidates identified using each of the two decoy genes were pooled and duplicates were removed. The combined p-value for each candidate was calculated as the sum of the log10 p-values of the association with each of the two bait genes. Transcripts matching known dehydrogenases in the TA biosynthetic pathway (i.e., tropinone reductase I and II) were removed, and the remaining candidates were ranked by combining p-values and by distance from the decoy gene via hierarchical clustering of tissue-specific expression profiles (fig. 45).

9.2) almost all candidates identified in example 9.1 showed the same secondary root-specific expression pattern as observed for known TA biosynthetic genes (Secondary root-specific expression pattern). BLASTp searches of the resulting-30 candidates against the UniPROT/SwissPROT database found that many transcripts lacked terminal or internal sequence regions. To solve this problem, De novo transcript alignment from RNA-seq assembly for the deletion of the deletion platform for reference generation and analysis, nat. protocol.8, 1494-512 (2013) was repeated from deposited original RNAseq reads using the Trinity software package, and all missing sequence fragments of the twelve HDH candidates were reconstructed by BLAST alignment of incomplete sequence regions against the newly assembled transcriptome (table 2).

9.3) the lost HDH activity was identified by screening the candidates produced in examples 9.1 and 9.2 in yeast.

9.3.1) lack of true commercial standards for hyoscyaldehyde and insufficient yields for chemical synthesis required co-expression of HDH candidates with the upstream biosynthetic enzyme cytochrome P450 ritoprinomutase (CYP80F1) -for in vivo activity screening by fed ritoprene (see example 7). Since ritoprine shows similar chromatographic and mass spectral properties to the HDH product hyoscyamine, the HDH screening strain (CSY1292) was constructed by integrating yeast codon-optimized AbCYP80F1 and DsH6H (see example 8) into the genome of CSY1251, thereby enabling the detection of ritoprine (m/z) fed by the three-step biosynthetic pathway (fig. 2) ⁺290) Resulting scopolamine (m/z)⁺304) To screen HDH candidates.

9.3.2) the yeast codon-optimized sequence encoding each of the 12 HDH candidates was expressed from a low copy plasmid in strain CSY1292 and scopolamine production was measured after 72h growth in medium supplemented with 1mM ritoprene. One of the twelve candidates, HDH2 (termed AbHDH), showed a 35% reduction in the level of scopolamine and a measurable accumulation of scopolamine (7.2 μ g/L), indicating that it encodes lost HDH activity (figure 46).

9.4) structural and phylogenetic analyses provide further insight into the catalytic mechanism and evolutionary history of HDH.

9.4.1) homology model of AbHDH isConstructed based on the crystal structure of microplasminol dehydrogenase (PtSAD; PDB: 1YQD) (FIG. 47). AbHDH is a member of the zinc-dependent alcohol dehydrogenase (ZADH) family in the medium chain dehydrogenase/reductase (MDR) superfamily. A typical feature of this family is that AbHDH exhibits a double-lobular structure with a highly conserved nucleotide binding domain and a more variable substrate binding domain. Alignment of residues S216, T217, S218, and K221 within the AbHDH nucleotide binding domain with phosphate stabilizing residues S214, T215, S216, and K219 in PtSAD indicates that AbHDH is an NADPH-dependent oxidoreductase. Similarly, a characteristic feature of ZADH is that AbHDH appears to bind to the structure Zn using a quadruplet of cysteine residues (C105, C108, C111 and C119) close to the protein surface ²⁺And binds catalytic Zn in the active site²⁺。

9.4.2) the catalytic mechanism of AbHDH was elucidated by docking the substrate hyoscyaldehyde molecule into the active site using the Maestro/Glide software package (fig. 47). The most advantageous mode of binding positions the aldehyde group of the substrate in catalyzing Zn²⁺And NADPH hydride donorsWithin. Docking results and general mechanism of ZADH (see Bomati, E.K., Noel, J.P., Structural and kinetic basis for substrate selection in Populus tremuloides lateral alcohol reduction. plant cell.17, 1598-1611 (2005)) indicate the following catalytic mechanism of AbHDH. Catalytic Zn in the active site in the absence of substrate²⁺Are stabilized by C52, H74, C168 and water molecules that are positioned by polar interaction with S54 and displaced upon binding with hyoscyaldehyde. Nucleophilic attack formation of aldehyde carbonyl by dihydronicotinamide hydride and catalysis of Zn²⁺And possibly through NADP⁺Is protonated by shuttling with a proton between the ribose group of S54.

9.5) to confirm whether orthologous oxidoreductases catalyze hyoscyamine biosynthesis in other TA-producing solanaceae plants, tBLASTX search was used to identify variants of the AbHDH coding sequence from the transcriptome of Datura and Datura stramonium (FIG. 48). HDH activity of two identified orthologs (DiHDH, DsHDH) was verified by co-expressing the yeast codon-optimized sequence with an additional copy of flux-limiting DsH6H from a low copy plasmid in CSY1292 and measuring scopolamine production in medium supplemented with 1mM ritoprene. DsHDH showed the highest substrate consumption and product accumulation for the tested variants (figure 49).

9.6) integrating the over-expressed medicinal TA biosynthesis branch comprising the optimal enzyme variant and the flux limiting enzyme into the platform yeast strain. Strain CSY1294 was constructed by integrating yeast codon optimized WfPPR and AbUGT, DsHDH and a second copy of DsH6H into CSY 1292. Scopolamine production from the fed ritopram was verified in CSY1294 (figure 50).

Example 10: yeast engineered to perform esterification of acyl donor and acceptor used to generate TA scaffolds.

Yeast strains can be engineered to express enzymes that catalyze the esterification of activated acyl donor compounds and acyl acceptor compounds to produce various TA scaffolds (fig. 2, 3). Activation of the acyl donor group can be achieved by engineering the acyl donor-producing yeast strain to incorporate an enzyme that attaches a chemical moiety with high group transfer potential, such as coenzyme a (coa) or glucose (glucoside), to the carboxyl group of the acyl donor, as described in example 6. Examples of acyl donor activating enzymes that can be used for this capability include CoA ligase and UDP-glycosyltransferase. Examples of esterases that may be used to catalyze the esterification of activated acyl donor compounds and acyl acceptors such as tropes and pseudotropins are acyltransferases, including serine carboxypeptidase-like acyltransferase (SCPL-AT) and BAHD-type acyltransferases. When expressed in heterologous hosts, such as yeast, the coding sequences for such acyltransferases may be modified to increase their activity.

10.1) in plants in which SCPL-AT is normally found naturally, the coding sequence for SCPL-AT includes an N-terminal signal peptide which directs the nascent polypeptide to the Endoplasmic Reticulum (ER). Once localized to the ER, SCPL-AT polypeptides are transported through the golgi via the secretory transport pathway to the vacuolar lumen where they are found to exhibit activity. During this ER to vacuole transport, they undergo several post-translational modifications (fig. 51), including but not limited to signal peptide cleavage, N-glycosylation, removal of internal propeptide sequences and disulfide bond formation (see Stehle, f., Stubbs, m.t., Strack, d. and Milkowski, c.heterologous expression of a serine carboxypeptidase-ligation and ligation of the kinetic mechanism, FEBS Journal,275, (2008)). However, since the intracellular trafficking pathway and the pattern of post-translational modifications vary from organism to organism, expression of SCPL-AT in a heterologous host may lead to incorrect subcellular localization and/or incorrect post-translational modifications for activity. For example, the coding sequence for SCPL-AT, such as ritodrine synthase (LS) (Table 1), can be modified to increase activity when expressed in yeast.

10.1.1) the signal peptide sequence can influence the processing and localization of SCPL-AT in yeast.

10.1.1.1) presence of the putative N-terminal signal peptide in AbLS indicates that it follows the expected SCPL ER to vacuole transport pathway in plants. The localization of AbLS in yeast was examined by expressing N-terminal and C-terminal GFP fusions of AbLS from low copy plasmids in CSY 1294. Fluorescence microscopy revealed that the N-terminal fusion (GFP-AbLS) co-localized with the tonoplast stain FM4-64 (FIG. 52). Fluorescence of the C-terminal fusion (AbLS-GFP) was not detected, consistent with reports that the native C-terminal is crucial for stability of SCPL acyltransferase (see Stehle, f., Stubbs, m.t., Strack, d. and Milkowski, C. heterologous expression of a serine carboxypropylase-like enzyme and catalysis of the kinetic mechanism, FEBS Journal,275, (2008)).

10.1.1.2) Vacuolar isolation of SCPL-AT in yeast (Vacuolar sequencing) may prevent access to the cytosol substrate pool, since yeast may lack the necessary Vacuolar membrane (tonoplastic) transporter present in plants for exchange of secondary metabolites with the cytosol. To determine whether forced localization of AbLS to other yeast compartments-possibly by improving access to cytosolic metabolites-could achieve activity, the wild-type N-terminal SP sequence was replaced with a set of N-terminal signal sequences taken from yeast proteins targeting the vacuolar cavity (Prc1p and Pep4p), the vacuolar membrane facing the cavity (Dap2p), the trans-golgi network (Och1p), the ER membrane facing the cavity (Mns1p) and the mitochondrial matrix (Cit1p) (fig. 53). Wild-type SP was also completely removed and for another variant, a classical peroxisome targeting sequence (PTS1) was attached to the C-terminus. These chimeric AbLS variants were expressed from high copy plasmids in CSY1294 and after 96h growth in selective medium transformants were screened for activity by LC-MS/MS. For any of the variants, no production of ritodrine or downstream intermediates was observed (fig. 53).

10.1.2) incorrect post-translational processing of SCPL-AT in yeast may prevent expression of the active enzyme.

10.1.2.1) protein N-glycosylation patterns vary from yeast to plant, and previous reports indicate that the correct N-glycosylation of various plant enzymes is critical to their folding, stability and/or activity (see Kar, B., Verma, P., den Haan, R., Sharma, A.K., Effect of N-linked glycosylation on the activity and stability of a β -glycosylation from Putrajiva roxburghi, int.J.biol.Macromol.112, 490-498 (2018); see also Podzimek, T. et al, N-glycosylation of tomato nuclear TBN1 produced in N.benthamiana and its effect on the enzyme activity plant Sci.276, 152-161 (2018); see also Strasser, R., Plant protein glycosylation. glycobiology.26, 926-939 (2016)). In silico analysis of the AbLS polypeptide predicted four N-glycosylation sites (N152, N320, N376, N416) and no O-glycosylation of the protein was detected in nicotiana benthamiana (fig. 54). Wild-type AbLS with a C-terminal HA-tag, each of the four N → Q mutants (in which the N to Q mutation eliminates N-glycosylation (23)), or the quadruple N → Q mutant were expressed in CSY1294 and nicotiana benthamiana, and the glycosylation profiles were compared by western blotting. Whereas the wild-type AbLS, N → Q single mutant and quadruple mutant all appeared as a single band in nicotiana benthamiana, indicating a monoglycosylation state, only the quadruple N → Q mutant produced a single band in yeast; all other variants appeared as double or triple bands, indicating a combination of multiple glycosylation states (fig. 55). However, since the heavily concentrated parts of the two wild-type AbLS bands (densers) in yeast show partial overlap with the heavily concentrated parts of wild-type AbLS in tobacco, at least some fractions of the AbLS expressed by yeast must be in the correct glycosylation state, and mis-glycosylation is unlikely to explain the complete lack of AbLS activity in yeast. (FIGS. 54-55).

10.1.2.2) a subset of SCPL acyltransferases, including sinapoyl glucose from Arabidopsis thaliana choline sinapoyl transferase (AtSCT) and avenin synthase from Avena sativa (AsSCPL1), have been shown to contain an internal propeptide linker that is proteolytically removed to yield an active heterodimer linked by a disulfide bond (see Shirley, A.M., Chappel, C., Biochemical catalysis of serine lactoglucanase: Choline serine lactoyltransferase, a serine carboxypeptidase-like protein kinase enzymes from an acyl transferase in molecular synthesis, J.biol.Chem.278, 19870-19877 (2003); see also Mugford, S.T. et al, A server carboxypeptidase-like acyl transfer enzyme is required for synthesis of antimicrobial compounds and release resistance in plants cell.21, 2473-2484 (2009)). Comparison of the amino acid sequence of AbLS with those of the previously characterized plant serine carboxypeptidase and SCPL acyltransferase revealed the presence of an internal sequence of 25 to 30 residues that aligns with the highly variable propeptide of atcct, AsSCPL1 and wheat carboxypeptidase 2(TaCBP2), indicating that AbLS also undergoes endoproteolytic cleavage to form heterodimers (fig. 56). Furthermore, homology models of AbLS indicate that the predicted internal propeptide blocks the active site, thus requiring removal of activity (fig. 57). However, the wild-type AbLS expressed in nicotiana benthamiana does not appear to undergo proteolytic cleavage, as the expected-20-25 kDa C-terminal fragment was not detected by western blotting under disulfide-reducing conditions (fig. 54, 55). Since the putative propeptide does not appear to be cleaved or removed in plants, AbLS may adopt a native conformation in plants that offsets the propeptide from the active site, but differences in the biochemical environment of the yeast secretory pathway and/or vacuole prevent this offset, thereby blocking activity.

To address this failure mode, a split Ab was constructedLS control, in which the N and C terminal domains flanking the putative propeptide linker are expressed independently, with or without a separate signal peptide. In addition, AbLS variants were constructed in which the putative propeptide was replaced with flexibility (GGGS)_n(SEQ ID NO:26) linker, internal propeptide from AtSCT previously demonstrated to be cleaved in yeast (see Shirley, A.M., Chapple, C., Biochemical engineering of serine cellulose, a mineral carbohydrate cellulose transfer, a mineral carbohydrate enzyme kinase, a protein enzyme substrate synthesis, J.biol.Chem.278, 19870-19877 (2003)), or synthetic linkers containing a polyarginine site cleaved by trans-Golgi protease Kex2p (see Chen, X.Zaro, J.L. useful, W.C., Fusion proteins, design, additive, J.L. useful, W.C., Fusion proteins, design, supplement, repair, tissue, repair, K.1667, P.7. C., 3.7. D.7. section, Biostrain, 14. C., 3. 7. C., 3. 7. D.7. section, 3. 7. section, 7. C., 3. 7. section, 3. 7. D.7. section, see. Each of split AbLS control and propeptide/linker variants was expressed from a low copy plasmid in CSY1294 and after 96h growth in selective medium transformants were screened for LS activity by LC-MS/MS. For any of these variants, no production of ritodrine or downstream TA was observed.

To address the problem of protein expression, each of the above AbLS variants with C-terminal HA tags was expressed from a low copy plasmid in CSY1294 and the apparent protein size was compared to split AbLS controls by western blotting (figure 58). Neither the AtSCT nor the polyarginine linker produced the 20-25kDa C-terminal fragment expected from proteolytic cleavage. In the latter case, failure of cleavage of the polyarginine AbLS variant indicates that the protein becomes arrested in the secretory pathway upstream of the trans-golgi network (TGN; also referred to as late golgi in yeast), probably due to the severe growth defect observed in CSY1294 expressing wild-type or golgi targeted (Och1p SP fused) AbLS.

10.1.3) functional expression of SCPL-AT in yeast can be achieved by engineering N-terminal fusions that alter sorting from TGN. Transport of soluble Yeast proteins from TGN to vacuoles requires recognition of a typical N-terminal signal sequence by the vacuolar Protein Sorting (Vps) cargo transporter, while integral Membrane proteins that reach Yeast TGN appear to be sorted by default to the Vacuole (see Stack, J.H., Receptor-media Protein Sorting to the Vacuole in Yeast: circles for Protein Kinase, Lipid Kinase and GTP-Binding proteins. Annu.Rev.cell. biol.11, 1-33 (1995); see also Roberts, C.J., Nothwehr, S.F., Steps, T.H., Membrane Protein Sorting in the first gene Sorting pathway: Evidence of the Vacuole Membrane sample solution, 119.83-1992). Thus, conversion of SCPL-AT into a transmembrane protein could resolve the barrier in TGN sorting by masking the SP with an N-terminally fused soluble domain.

10.1.3.1) in one example, AbLS variants are constructed with a set of N-terminally fused soluble domains, including fluorescent proteins from jellyfish (GFP, BFP, mVenus) and the coral of Lentinus edodes (mCherry, DsRed) family; a small ubiquitin-related modifier (Smt3p) with a mutated protease cleavage Site (SUMO); and the upstream enzyme in the TA pathway, AbUGT. These variants and wild-type AbLS were expressed from low copy plasmids in CSY1294 and screened for ritodrine synthase activity after 96h growth in selective medium. All of the N-terminally fused AbLS variants showed measurable accumulation of scopolamine and scopolamine. Fusing the fluorescent protein derived from jellyfish GFP with the AbLS to cause the yield of hyoscyamine and scopolamine to be 1 mug/L and 0.1 mug/L respectively; whereas the fusion of the Cordiculus shiitake derived fluorescent protein resulted in significantly higher TA yields, with the maximum titers obtained by the DsRed fusion (10.3. mu.g/L hyoscyamine, 0.87. mu.g/L scopolamine) (FIG. 59). The enhancement of AbLS activity appears to be related to the oligomerization state of the N-terminal domain, with scopolamine production increasing in order from monomer (GFP, BFP, mVenus, mCherry, SUMO) to homodimer (AbUGT) to homotetramer (DsRed) domain.

10.2) to produce a strain capable of completing TA biosynthesis, yeast codon optimized DsRed-ABLS and a second copy of UGP1 were integrated into the genome of CSY1294 at the disrupted EGH1 site to produce CSY 1296. CSY1296 showed the production of de novo scopolamine and scopolamine with titers of 10.2. mu.g/L and 1.0. mu.g/L, respectively.

Example 11 use of heterologous transporters to alleviate intracellular substrate transport limitations

Since the enzymes that perform TA biosynthesis are distributed in multiple subcellular compartments (cytosol, ER membrane, peroxisomes, vacuole, mitochondria) and yeast is unlikely to have transporters found in plants that are capable of moving TA biosynthetic intermediates between different compartments, intracellular metabolite transport may limit TA production.

11.1) Intercompartmental transport limitations can be addressed by functional expression of plant transporters in non-plant host cells. Vacuolar compartmentalization of DsRed-AbLS (FIG. 60) requires inward transport of cytosolic tropins and PLA glucosides to the vacuolar cavity and outward transport of vacuolar ritolin to the cytosol. Several Multidrug and toxin efflux (MATE) transporters responsible for Vacuolar alkaloid and glycoside sequestration have been identified in plants of the Solanaceae family, including three species with observed or predicted activity for TA (see Morita, M. et al, vacuum transport of nucleotides, medium prepared by multiple and toxin composition evolution (MATE) transporter in Nicotiana tabacum. Proc. Natl. Acad. Sci. U.S. A.106, 2447-2452 (2009); see also Shoji, T. et al, Multi drug and toxin composition evolution-type expressed in vaccine sequence of toxicity in bacterial strain (708, plant 149, 718-2009)). In one example, nicotiana tabacum jasmonate inducible alkaloid transporter 1(NtJAT1) and two mats (ntmat 1, ntmat 2) were expressed from low copy plasmids in CSY1296 and measured for accumulation of TA after 96h growth in selective medium. Expression of NtJAT1 and NtMATE2 improved TA production, with the former resulting in 74% and 18% increase in scopolamine and scopolamine titers, respectively (fig. 61).

11.2) to assess the subcellular localization of these transporters and to determine the possible mechanism of action, fluorescence microscopy was performed on CSY1296 expressing a C-terminal GFP fusion of NtJAT1 or NtMATE2 from low copy plasmids. The analysis supported that NtJAT1 was almost completely localized to the vacuolar membrane (co-localized with DsRed-AbLS), whereas NtMATE2 was partitioned between the vacuolar membrane and the plasma membrane (fig. 60), suggesting that both transporters may act to eliminate vacuolar substrate transport limitation, which may also improve cellular TA outward transport.

Example 12: yeast engineered to produce non-native TA from L-phenylalanine or L-tyrosine and L-arginine

In addition to being engineered to produce both medicinal and non-medicinal TAs that occur naturally in organisms, yeast can also be engineered to produce non-natural TAs (fig. 3). For example, yeast can be engineered to express biosynthetic pathways to produce acyl donor compounds that are not naturally incorporated into TA by plants.

12.1) in one example, the torotropic yeast strain platform described in example 3 can be further engineered to produce the acyl donor compound cinnamic acid (as described in example 6) and express a cinnamic acid activating enzyme and an esterifying enzyme to produce a non-native TA, such as cinnamoyl tropine.

12.1.1) cinnamate can be produced from phenylalanine by a phenylalanine ammonia lyase, such as PAL1 from Arabidopsis (AtPAL 1). Since EcCS requires a coenzyme a (CoA) -activated acyl donor, 4-coumarate-CoA ligases with defined activity on cinnamates, such as 4CL5(At4CL5) from arabidopsis thaliana (see Eudes, a. et al, explicit members of the BAHD acyl transferase family to synthesis multiple hydrochinonates and benzoate conjugates in yeast. microbial Cell Factories,15, (2016), can be expressed to achieve cinnamoyl-CoA biosynthesis in yeast. The torotropic strain platform described in example 3 was transformed with a low copy plasmid capable of producing cinnamic acid as described in example 6.

12.1.2) the engineered strain of example 12.1.1 was further modified to produce cinnamoyl tropine by transformation with a high copy 2 μ plasmid with URA3 selectable marker, HXT7 and PMA1 promoters, and the coding sequence for the 4-coumarate-CoA ligase variant from arabidopsis (At4CL5) and cocaine synthase (EcCS) from coca. Plasmids containing low and high copiesThe resulting strain was grown in a synthetic complete medium with the appropriate amino acid deletion solution (-Ura-Trp) at 25 ℃. After 72 hours of growth, cinnamoyl tropine in the medium was analyzed by LC-MS/MS analysis (fig. 62). Tandem MS/MS and fragment analysis were used to detect and verify the identity of cinnamoyl tropine. Parent Mass (m/z) with cinnamoyl tropine ⁺272) revealed a new peak at a retention time of 3.684 minutes and produced a fragment whose mass appears to match the conversion of a true cinnamoyl tropine standard (fig. 62 a). The most abundant mass transition, m/z +272 → 124, with the predominant m/z produced during fragmentation of scopolamine⁺The 124-tropine fragment (see Bedewitz, M.A. et al, A Root-Expressed L-Phenylalanine: 4-hydroxypropylpyrazoluvate Aminotranferase Is Required for Tropaene Alkaloid Biosynthesis in Atropa belladonna.the Plant Cell,26, (2014)).

12.1.3) based on the 272 → 124LC-MS/MS shift of cinnamoyl tropine described in example 12.1.2, a Multiple Reaction Monitoring (MRM) LC-MS/MS method was developed to measure de novo cinnamoyl tropine production. Cinnamoyl tropine accumulated to a fairly high level in the extracellular medium of the engineered strain of example 12.1.2, but not in the absence of AtPAL1, At4CL5, and EcCS (fig. 62 b). The titer of de novo produced cinnamoyl tropine was estimated to be 6.0. mu.g/L based on the standard curve.

Example 13: growth medium modification to increase TA and TA precursor production

Production titers of TA precursor and TA can be increased by modifying the media composition. For example, the type of medium can be varied in terms of medium basis (e.g., yeast peptone, yeast nitrogen base), carbon source (e.g., glucose, maltodextrin), and nitrogen source (e.g., amino acids, ammonium sulfate, urea). The type of medium may also vary in the relative proportions of the components, such as the concentration of carbon source and nitrogen source, or the concentration of the individual amino acids.

13.1) Totropina producing yeast strains (as described in example 3) were initially grown in defined media with different carbon sources (i.e.YNB with ammonium sulfate and all amino acids) and the yield of the tropina was determined after 48 hours of growth at 25 ℃. The highest yield of tropine was observed with 2% galactose (fig. 63 a). However, some engineered strains fail to grow or grow severely slowed down with certain carbon sources (such as glycerol, arabinose, and sorbitol), possibly due to the inability to assimilate these carbon sources into central metabolism.

13.2) Totropina producing yeast strains (as described in example 3) were cultured in defined medium with 2% dextrose for growth and supplemented with 2% of another carbon source, and the tropina production was determined after 48 hours of growth at 25 ℃. The highest yield of tropine was observed with 2% dextrose and 2% glycerol (fig. 63 b). Glycerol is a non-sugar carbon source that can contribute to higher yields of TA precursors and TA by several mechanisms, including stabilization of the cytosolic membrane, improvement of the folding and stability of heterologous proteins, and the NADPH co-factor required to regenerate the activity of cytochrome P450 and some short chain dehydrogenases/reductases (see Li, y. et al complex biology of noscapine and halogenated alkaloids in yeast. proc. natl. acad. sci. u.s.a.2018,115(17) E3922-E3931).

13.3) improvements in de novo medicinal TA biosynthesis in engineered yeast can be achieved by alleviating flux bottlenecks and transport limitations.

13.3.1) improved TA production was achieved by over-expression of bottleneck enzymes and media optimization. Since the production of trope in CSY1296 (-mg/L) is unlikely to limit the flux to scopolamine (-ug/L), a metabolic bottleneck limiting scopolamine production was identified by expressing additional copies of each heterologous enzyme between phenylpyruvic acid and scopolamine in CSY1296 from low copy plasmids and measuring the production of TA and intermediates (figure 2). Additional copies of WfPPR and DsH6H resulted in 64% and 89% increase in scopolamine and scopolamine titers, respectively, suggesting that these enzymes are the major limiting factors for pathway flux (fig. 64).

13.3.2) an improved scopolamine producing strain was constructed by integrating NtJAT1 and a second copy of WfPPR and DsH6H into CSY 1296. The resulting strain CSY1297 showed a corresponding 2.4-fold and 7.1-fold increase in accumulation of scopolamine and scopolamine relative to CSY1296 (figure 65).

Notwithstanding the additional clauses, the present disclosure may be defined by the following clauses:

clause 1. an engineered non-plant cell that produces a tropane alkaloid product, a precursor to a tropane alkaloid product, or a derivative of a tropane alkaloid product.

Clause 2. the cell of clause 1, wherein the cell is a microbial cell.

Clause 3. the cell of clause 1 or 2, wherein the engineered cell comprises a plurality of heterologous coding sequences for encoding a plurality of enzymes, wherein at least one of the enzymes is selected from the group consisting of: arginine decarboxylase, agmatine urea hydrolase, agmatine enzyme, putrescine N-methyltransferase, N-methylputrescine oxidase, pyrrolidone synthase, tropinone synthase, cytochrome P450 reductase, tropinone reductase, phenylpyruvate reductase, 3-phenyllactic acid UDP-glucosyltransferase 84A27, ritoprene synthase, ritoprinogenase, hyoscyamine dehydrogenase, hyoscyamine 6 beta-hydroxylase/dioxygenase, and cocaine synthase.

Clause 4. the cell of any one of clauses 1-3, wherein endogenous arginine metabolism is improved in the cell.

Clause 5. the cell of any one of clauses 1-4, wherein endogenous phenylalanine and phenylalanine metabolism is improved in the cell.

The cell of any one of claims 1-5, wherein an endogenous polyamine regulatory mechanism is disrupted in the cell.

Clause 7. the cell of any one of clauses 1-6, wherein endogenous acetate metabolism is improved in the cell.

Clause 8. the cell of any one of clauses 1-7, wherein endogenous glycoside metabolism is improved in the cell.

Clause 9. the cell of any one of clauses 1-8, wherein the cell produces a tropane alkaloid product, a precursor of a tropane alkaloid product, or a derivative of a tropane alkaloid product selected from the group consisting of: hyoscyamine, atropine, anisodamine, scopolamine, calycanthin, cocaine or non-natural tropane alkaloids.

Clause 10. the cell of any one of clauses 1-9, wherein the engineered cell comprises a plurality of heterologous coding sequences encoding a plurality of enzymes comprising one or more soluble protein domains fused to the N-terminus of a serine carboxypeptidase-like acyltransferase domain.

Clause 11. the cell of any one of clauses 1-10, wherein transport of the TA, TA precursor, and/or TA derivative across the intracellular membrane or across the plasma membrane is improved in the cell.

Clause 12. the cell of any one of clauses 1-11, wherein the engineered cell comprises a plurality of heterologous coding sequences for encoding a plurality of transporters, wherein at least one of the transporters is selected from the group consisting of: multidrug and toxin efflux transporters, nitrate/peptide family transporters, ATP-binding cassette transporters, and multi-effect drug-resistant transporters.

Clause 13. a method for producing a tropane alkaloid, a precursor to a tropane alkaloid product, or a derivative of a tropane alkaloid product, comprising

(a) Culturing the cell according to any of clauses 1-12 under conditions suitable for protein production;

(b) adding a starter compound to the cell culture; and

(c) recovering the precursor of the tropane alkaloid or tropane alkaloid product from the culture.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Thus, the foregoing merely illustrates the principles of the invention. It will thus be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention is embodied by the appended claims.

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210 215 220

Glu Asp Met Ser Asn Glu Asp Tyr Arg Lys His Leu Pro Arg Phe Gln

225 230 235 240

Ser Glu Asn Leu Glu His Asn Lys Lys Leu Tyr Glu Arg Ile Cys Gln

245 250 255

Thr Ala Ala Arg Met Gly Cys Thr Pro Ser Gln Leu Ala Leu Ala Trp

260 265 270

Val His His Gln Gly Asn Asp Val Cys Pro Ile Pro Gly Thr Thr Lys

275 280 285

Ile Glu Asn Leu Asn Gln Asn Ile Glu Ala Leu Ser Ile Lys Leu Thr

290 295 300

Ser Glu Asp Met Thr Glu Leu Glu Ser Ile Ala Ser Ala Asn Ala Val

305 310 315 320

Gln Gly Asp Arg Tyr Gly Ser Gly Ala Ser Thr Tyr Lys Asp Ser Glu

325 330 335

Thr Pro Pro Leu Ser Ala Trp Lys Val Thr

340 345

<210> 4

<211> 346

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 4

Met Glu Val Lys Asn Lys Tyr Val Ala Ile Lys Ser Asn Ile Asn Gly

1 5 10 15

Ala Pro Gln Glu Ser His Phe Glu Ile Lys Val Glu Asn Leu Ser Leu

20 25 30

Ile Val Glu Pro Asp Ser Lys Glu Val Ile Ile Lys Asn Leu Phe Val

35 40 45

Ser Ile Asp Pro Tyr Gln Leu Asn Arg Met Lys Ser Glu Ser Ser Ser

50 55 60

Gln Ala Ala Ile Ser Tyr Ala Ser Ala Ile Thr Pro Gly Lys Ala Ile

65 70 75 80

Asp Thr Tyr Gly Val Gly Arg Val Leu Val Ser Asp Arg Pro Glu Phe

85 90 95

Lys Lys Asp Asp Leu Val Ala Gly Leu Leu Thr Trp Gly Glu Tyr Thr

100 105 110

Val Val Lys Glu Gly Ser Leu Leu Asn Lys Leu Asp Pro Leu Gly Phe

115 120 125

Pro Leu Ser Asn His Val Gly Val Leu Gly Phe Ser Gly Leu Ala Ala

130 135 140

Tyr Gly Gly Phe Phe Glu Val Cys Lys Pro Lys Pro Gly Glu Lys Val

145 150 155 160

Phe Val Ser Ala Ala Ser Gly Ser Val Gly Asn Leu Val Gly Gln Tyr

165 170 175

Ala Lys Leu Leu Gly Cys His Val Val Gly Ser Ala Gly Ser Gln Glu

180 185 190

Lys Val Lys Leu Leu Lys Glu Thr Leu Gly Phe Asp Asp Ala Phe Asn

195 200 205

Tyr Lys Glu Glu Thr Asp Leu Lys Ser Ala Leu Lys Arg Cys Phe Pro

210 215 220

Gln Gly Ile Asp Val Cys Phe Asp Asn Val Gly Gly Lys Met Leu Glu

225 230 235 240

Ala Ala Val Ala Asn Met Asn Leu Phe Gly Arg Val Ala Ile Cys Gly

245 250 255

Val Ile Ser Glu Tyr Thr Asn Ala Ser Thr Arg Ala Ala Pro Glu Met

260 265 270

Leu Asp Ile Val Tyr Lys Arg Ile Thr Ile Gln Gly Phe Leu Ala Ala

275 280 285

Asp Phe Met Lys Val Tyr Ala Asp Phe Leu Ser Glu Thr Val Glu Tyr

290 295 300

Leu Gln Asp Gly Lys Leu Lys Ala Val Glu Asp Val Ser Glu Gly Val

305 310 315 320

Glu Ser Ile Pro Ser Ala Phe Ile Gly Leu Phe Asn Gly Asp Asn Ile

325 330 335

Gly Lys Lys Ile Val Lys Val Ala Asp Glu

340 345

<210> 5

<211> 296

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 5

Met Leu Arg Ile Arg Ser Arg Ile Ile Ser Ile Ser Arg Ser Leu Ile

1 5 10 15

Leu Arg Gln Thr Ser Ser Asn Lys Phe Ser Thr His Ser Glu Arg Lys

20 25 30

Leu Glu Gly Lys Val Ala Val Ile Thr Gly Ala Ala Ser Gly Ile Gly

35 40 45

Lys Glu Thr Ala Ala Lys Phe Ile Ser His Gly Ala Lys Val Ile Ile

50 55 60

Ala Asp Ile Gln Lys Gln Leu Gly Gln Glu Thr Ala Ser Glu Leu Gly

65 70 75 80

Pro Asn Ala Thr Phe Val Ser Cys Asp Val Thr Lys Glu Ser Asp Ile

85 90 95

Ser Asp Val Val Asp Phe Ala Val Ser Lys His Gly Gln Leu Asp Ile

100 105 110

Met Tyr Asn Asn Ala Gly Ile Ala Cys Arg Thr Thr Phe Ser Ile Val

115 120 125

Asp Leu Asp Leu Ala Gln Phe Asp Arg Ile Met Ala Ile Asn Val Arg

130 135 140

Gly Val Val Ala Gly Ile Lys His Ala Ala Arg Val Met Ile Pro Gln

145 150 155 160

Gly Ser Gly Cys Ile Leu Cys Thr Gly Ser Ile Thr Gly Val Met Gly

165 170 175

Gly Leu Ala Gln Pro Thr Tyr Ser Thr Thr Lys Ser Cys Val Ile Gly

180 185 190

Ile Val Lys Ser Thr Thr Gly Glu Leu Cys Lys His Gly Ile Arg Ile

195 200 205

Asn Cys Ile Ser Pro Phe Ala Ile Pro Thr Ala Phe Ser Leu Asp Glu

210 215 220

Met Lys Glu Tyr Phe Pro Gly Val Glu Pro Glu Gly Leu Val Lys Ile

225 230 235 240

Leu Gln Asn Ala Ser Glu Leu Lys Gly Ala Tyr Cys Glu Pro Ile Asp

245 250 255

Val Ala Asn Ala Ala Ile Phe Leu Ala Ser Glu Asp Ala Lys Phe Ile

260 265 270

Ser Gly Glu Asn Leu Met Val Asp Gly Gly Phe Thr Ser Phe Lys Lys

275 280 285

Leu Asn Leu Ser His Leu Val Gln

290 295

<210> 6

<211> 389

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 6

Met Ala Ser Asn Gly Ile Ser His Val Asn Gly Thr Leu Ala Lys Val

1 5 10 15

Ile Thr Cys Arg Ala Ala Val Ala Tyr Gly Pro Gly Gln Pro Leu Val

20 25 30

Val Glu Gln Val Gln Val Asp Pro Pro Gln Lys Met Glu Val Arg Ile

35 40 45

Lys Ile Leu Phe Thr Ser Ile Cys His Thr Asp Leu Ser Ala Trp Lys

50 55 60

Gly Glu Asn Glu Ala Gln Arg Val Tyr Pro Arg Ile Leu Gly His Glu

65 70 75 80

Ala Ser Gly Val Val Glu Ser Val Gly Glu Gly Val Thr Asp Met Lys

85 90 95

Thr Gly Asp His Val Val Pro Ile Phe Asn Gly Glu Cys Gly Glu Cys

100 105 110

Val Tyr Cys Asn Ser Ser Lys Lys Thr Asn Leu Cys Gly Lys Phe Arg

115 120 125

Val Asn Pro Phe Lys Ser Val Met Ala Asn Asp Gly Lys Cys Arg Phe

130 135 140

Arg Asn Lys Asp Gly Asn Pro Ile Tyr His Phe Leu Asn Thr Ser Thr

145 150 155 160

Phe Ser Glu Tyr Thr Val Val Asp Ser Ala Cys Leu Val Asn Ile Asp

165 170 175

Pro His Ala Pro Leu Asp Lys Met Thr Leu Leu Ser Cys Gly Val Ser

180 185 190

Thr Gly Leu Gly Ala Ala Trp Asn Thr Ala Asp Val Gln Thr Gly Glu

195 200 205

Thr Val Ala Val Phe Gly Leu Gly Ala Val Gly Leu Ala Val Val Glu

210 215 220

Gly Ala Arg Thr Arg Gly Ala Ser Arg Ile Ile Gly Val Asp Ile Asn

225 230 235 240

Ser Glu Lys Arg Ile Lys Gly Gln Ala Ile Gly Ile Thr Asp Phe Ile

245 250 255

Asn Pro Lys Glu Ile Asp Val Pro Val His Glu Lys Ile Arg Glu Met

260 265 270

Thr Gly Gly Gly Val His Tyr Ser Phe Glu Cys Ala Gly Asn Leu Glu

275 280 285

Val Leu Arg Glu Ala Phe Ser Ser Thr His Asp Gly Trp Gly Met Thr

290 295 300

Ile Val Leu Gly Ile His Pro Thr Pro Arg Leu Leu Pro Leu His Pro

305 310 315 320

Met Glu Leu Phe Asp Gly Arg Arg Ile Val Ala Ser Val Phe Gly Asp

325 330 335

Phe Lys Gly Lys Ser Gln Leu Pro Phe Phe Ala Lys Gln Cys Met Ala

340 345 350

Gly Val Val Lys Leu Asp Glu Phe Ile Thr His Glu Leu Pro Phe Glu

355 360 365

Lys Ile Asn Glu Gly Phe Gln Leu Leu Val Asp Gly Lys Ser Leu Arg

370 375 380

Cys Leu Leu His Leu

385

<210> 7

<211> 315

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 7

Met Ala Glu Lys Ile Thr Ser Leu Glu Ser Thr Arg Tyr Ala Val Val

1 5 10 15

Thr Gly Gly Asn Lys Gly Ile Gly Tyr Glu Thr Cys Arg Gln Leu Val

20 25 30

Ser Lys Gly Val Val Val Val Leu Thr Ala Arg Asp Glu Lys Arg Gly

35 40 45

Ile Glu Ala Thr Glu Arg Leu Lys Glu Glu Ser Ser Phe Thr Asp Asp

50 55 60

Gln Ile Met Phe His Gln Leu Asp Val Val Asp Pro Asp Ser Ile Ser

65 70 75 80

Ser Leu Val Asp Phe Ile Asn Thr Lys Phe Gly Arg Leu Asp Ile Leu

85 90 95

Val Asn Asn Ala Gly Val Gly Gly Leu Met Val Glu Gly Asp Val Val

100 105 110

Ile Leu Lys Asp Leu Ile Glu Gly Asp Phe Val Ser Val Ser Thr Glu

115 120 125

Asn Glu Glu Glu Gly Asp Thr Glu Lys Ser Ile Glu Gly Ile Val Thr

130 135 140

Asn Tyr Glu Leu Thr Lys Gln Cys Val Glu Thr Asn Phe Tyr Gly Ala

145 150 155 160

Lys Arg Met Ser Glu Ala Phe Ile Pro Leu Leu Gln Leu Ser Asn Ser

165 170 175

Pro Thr Ile Val Asn Val Ala Ser Phe Leu Gly Lys Leu Lys Leu Leu

180 185 190

Cys Asn Glu Trp Ala Ile Lys Val Leu Ser Asn Ala Asn Asn Leu Thr

195 200 205

Glu Asp Arg Val Asp Glu Val Val Asn Glu Phe Leu Lys Asp Phe Thr

210 215 220

Glu Lys Ser Ile Glu Ala Lys Gly Trp Pro Thr Tyr Phe Ala Ala Tyr

225 230 235 240

Lys Val Ser Lys Ala Ala Met Ile Ala Tyr Thr Arg Val Leu Ala Thr

245 250 255

Lys Tyr Pro Asn Phe Arg Ile Asn Ser Val Cys Pro Gly Tyr Cys Lys

260 265 270

Thr Asp Leu Thr Ala Asn Thr Gly Ser Leu Thr Ala Glu Glu Gly Ala

275 280 285

Glu Ser Leu Val Lys Leu Ala Leu Leu Pro Asn Asp Gly Pro Ser Gly

290 295 300

Leu Phe Phe Tyr Arg Lys Asp Val Ala Ala Leu

305 310 315

<210> 8

<211> 276

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 8

Met Ala Ser Val Ser Phe Leu Ser Thr Ile Gly Lys Arg Leu Glu Gly

1 5 10 15

Lys Val Ala Met Val Thr Gly Gly Ala Ser Gly Ile Gly Glu Ala Ile

20 25 30

Ala Lys Leu Phe Tyr Glu His Gly Ala Lys Val Ala Ile Ala Asp Val

35 40 45

Gln Asp Glu Leu Gly Asn Ser Val Ser Asn Ala Leu Gly Gly Ser Ser

50 55 60

Asn Ser Ile Tyr Ile His Cys Asp Val Thr Asn Glu Asp Asp Val Gln

65 70 75 80

Glu Ala Val Asp Lys Thr Ile Ser Thr Phe Gly Lys Leu Asp Ile Met

85 90 95

Ile Cys Asn Ala Gly Ile Ser Asp Glu Thr Lys Pro Arg Ile Ile Asp

100 105 110

Asn Thr Lys Ala Asp Phe Glu Arg Val Leu Ser Ile Asn Val Thr Gly

115 120 125

Val Phe Leu Thr Met Lys His Ala Ala Arg Val Met Val Pro Ala Arg

130 135 140

Ile Gly Cys Ile Ile Ser Thr Ser Ser Val Ser Ser Arg Val Gly Ala

145 150 155 160

Ala Ala Ser His Ala Tyr Cys Ser Ser Lys His Ala Val Leu Gly Leu

165 170 175

Thr Lys Asn Leu Ala Val Glu Leu Gly Gln Phe Gly Ile Arg Val Asn

180 185 190

Cys Leu Ser Pro Tyr Ala Met Val Thr Pro Leu Ala Glu Lys Val Ile

195 200 205

Gly Leu Glu Asn Glu Glu Leu Glu Lys Ala Leu Asp Met Val Gly Asn

210 215 220

Leu Lys Gly Val Thr Leu Arg Val Asp Asp Val Ala Lys Ala Ala Leu

225 230 235 240

Phe Leu Ala Ser Asp Asp Ser Lys Tyr Ile Ser Gly His Asn Leu Phe

245 250 255

Ile Asp Gly Gly Phe Thr Val Tyr Asn Pro Gly Leu Gly Met Phe Lys

260 265 270

Tyr Pro Glu Ser

275

<210> 9

<211> 297

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 9

Met Leu Arg Ile Ala Ser Arg Gly Gly Ile Thr Ser Arg Ser Leu Gln

1 5 10 15

Leu Leu Gln Thr Phe Asn Lys Glu Phe Ser Thr His Ile Glu Arg Lys

20 25 30

Leu Glu Gly Lys Val Ala Leu Ile Thr Gly Ala Ala Ser Gly Ile Gly

35 40 45

Lys Glu Thr Ala Ala Lys Phe Ile Asn Asn Gly Ala Lys Val Ile Ile

50 55 60

Ala Asp Val Gln Lys Gln Leu Gly Gln Glu Thr Ala Ser Gln Leu Gly

65 70 75 80

Pro Asn Ala Thr Phe Val Leu Cys Asp Val Thr Lys Glu Ser Asp Val

85 90 95

Ser Asn Ala Val Asp Phe Ala Val Ser Asn His Gly Gln Leu Asp Ile

100 105 110

Met Tyr Asn Asn Ala Gly Ile Ile Cys Arg Thr Pro Arg Asn Ile Ala

115 120 125

Asp Leu Asp Leu Asp Ala Phe Asp Arg Val Met Ala Ile Asn Val Arg

130 135 140

Gly Met Met Ala Gly Ile Lys His Ala Ala Arg Val Met Ile Pro Arg

145 150 155 160

Lys Ala Gly Ser Ile Leu Cys Thr Ala Ser Ile Thr Gly Thr Met Gly

165 170 175

Gly Leu Ala Gln Pro Thr Tyr Ser Thr Thr Lys Ser Cys Val Ile Gly

180 185 190

Met Met Arg Ser Val Thr Ala Glu Leu Cys Gln Asn Gly Ile Arg Ile

195 200 205

Asn Cys Ile Ser Pro Phe Ala Ile Pro Thr Pro Phe Tyr Ile Asp Glu

210 215 220

Met Lys Ser Tyr Tyr Pro Gly Val Glu Pro Glu Val Leu Val Lys Met

225 230 235 240

Leu Tyr Arg Ala Ser Glu Leu Asn Gly Ala Tyr Cys Glu Pro Val Asp

245 250 255

Val Ala Asn Ala Ala Val Phe Leu Ala Ser Asp Asp Ala Lys Tyr Val

260 265 270

Ser Gly Gln Asn Leu Val Ile Asp Gly Gly Phe Thr Ser Tyr Lys Ser

275 280 285

Leu Asn Phe Pro Met Ser Asp Gln Glu

290 295

<210> 10

<211> 279

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 10

Met Gly Ile Pro Ser Ser Val Thr Pro Ile Val Arg Arg Leu Glu Gly

1 5 10 15

Lys Val Ala Val Ile Thr Gly Gly Ala Ser Gly Ile Gly Glu Ala Ala

20 25 30

Thr Arg Leu Phe Val Lys His Gly Ala Lys Val Val Val Ala Asp Val

35 40 45

Arg Asp Asp Leu Gly Arg Ala Leu Cys Lys Glu Leu Gly Ser Asn Asp

50 55 60

Thr Ile Ser Phe Ala His Cys Ser Val Thr Asp Glu Asn Asp Val Gln

65 70 75 80

Asn Ala Ile Asp Gly Ala Val Ser Arg Tyr Gly Met Leu Asp Ile Met

85 90 95

Phe Asn Asn Ala Gly Ile Thr Gly Asn Met Lys Asp Pro Ser Ile Leu

100 105 110

Ala Thr Asp Tyr Lys Asn Phe Lys Asn Val Phe Asp Val Asn Val Tyr

115 120 125

Gly Ala Phe Leu Gly Ala Arg Ile Ala Ala Lys Ala Met Ile Pro Thr

130 135 140

Lys Gln Gly Ser Ile Leu Phe Thr Ala Ser Ile Ala Ser Val Ile Gly

145 150 155 160

Gly Ile Ala Ser Pro Ile Thr Tyr Ala Ser Ser Lys His Ala Val Val

165 170 175

Gly Leu Thr Asn His Leu Ala Val Glu Leu Gly Gln Tyr Gly Ile Arg

180 185 190

Val Asn Cys Ile Ser Pro Tyr Thr Val Ala Thr Pro Leu Val Arg Glu

195 200 205

Ile Leu Gly Lys Met Asp Lys Glu Lys Ala Glu Glu Val Ile Met Glu

210 215 220

Thr Ala Asn Leu Lys Gly Lys Ile Leu Glu Pro Glu Asp Ile Ala Glu

225 230 235 240

Ala Ala Val Tyr Leu Gly Ser Asp Glu Ser Lys Tyr Val Ser Gly Ile

245 250 255

Asn Leu Val Ile Asp Gly Gly Tyr Ser Lys Thr Asn Pro Leu Ala Ser

260 265 270

Met Val Met Gln Asn Tyr Ile

275

<210> 11

<211> 327

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 11

Met Glu Ser Lys Ser Gly Glu Gly Lys Ile Val Cys Val Thr Gly Ala

1 5 10 15

Ser Gly Phe Ile Ala Ser Trp Leu Val Lys Leu Leu Leu His Arg Gly

20 25 30

Tyr Thr Val Asn Ala Thr Val Arg Asn Leu Lys Asp Thr Ser Lys Val

35 40 45

Ala His Leu Leu Gly Leu Asp Gly Ala Asn Glu Arg Leu His Leu Phe

50 55 60

Lys Ala Glu Leu Leu Glu Glu Gln Ser Phe Asp Ala Ala Val Asp Gly

65 70 75 80

Cys Glu Gly Val Phe His Thr Ala Ser Pro Val Ser Leu Thr Ala Lys

85 90 95

Ser Lys Glu Glu Leu Val Asp Pro Ala Val Lys Gly Thr Leu Asn Val

100 105 110

Leu Arg Ser Cys Ala Lys Ser Pro Ser Val Leu Arg Val Val Ile Thr

115 120 125

Ser Ser Thr Ala Ser Val Ile Cys Asn Lys Asn Met Ser Thr Pro Gly

130 135 140

Ala Val Ala Asp Glu Thr Trp Tyr Ser Asp Pro Glu Phe Cys Glu Glu

145 150 155 160

Arg Glu Glu Trp Tyr Gln Leu Ser Lys Thr Leu Ala Glu Gln Ala Ala

165 170 175

Trp Lys Phe Ala Lys Glu Asn Glu Met Asp Leu Val Thr Leu His Pro

180 185 190

Gly Leu Val Ile Gly Pro Leu Leu Gln Pro Thr Leu Asn Phe Ser Cys

195 200 205

Glu Ala Ile Val Asn Phe Ile Lys Glu Gly Lys Glu Ala Trp Ser Gly

210 215 220

Gly Val Tyr Arg Phe Val Asp Val Arg Asp Val Ala Asn Ala His Ile

225 230 235 240

Leu Ala Phe Glu Val Pro Ser Ala Asn Gly Arg Tyr Cys Leu Val Gly

245 250 255

Val Asn Gly Tyr Ser Ser Leu Val Leu Lys Ile Val Gln Lys Leu Tyr

260 265 270

Pro Ser Ile Thr Leu Pro Glu Asn Phe Glu Asp Gly Leu Pro Leu Thr

275 280 285

Pro His Phe Gln Val Ser Ser Glu Arg Ala Lys Gly Leu Gly Val Lys

290 295 300

Phe Thr Pro Leu Glu Leu Ser Val Lys Asp Thr Val Glu Ser Leu Met

305 310 315 320

Glu Lys Asn Phe Leu His Ile

325

<210> 12

<211> 327

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 12

Met Glu Ser Lys Ser Gly Glu Gly Lys Ile Val Cys Val Thr Gly Ala

1 5 10 15

Ser Gly Phe Ile Ala Ser Trp Leu Val Lys Leu Leu Leu His Arg Gly

20 25 30

Tyr Thr Val Asn Ala Thr Val Arg Asn Leu Lys Asp Thr Ser Lys Val

35 40 45

Ala His Leu Leu Gly Leu Asp Gly Ala Asn Glu Arg Leu His Leu Phe

50 55 60

Lys Ala Glu Leu Leu Glu Glu Gln Ser Phe Asp Ala Ala Val Asp Gly

65 70 75 80

Cys Glu Gly Val Phe His Thr Ala Ser Pro Val Ser Leu Thr Ala Lys

85 90 95

Ser Lys Glu Glu Leu Val Asp Pro Ala Val Lys Gly Thr Leu Asn Val

100 105 110

Leu Arg Ser Cys Ala Lys Ser Pro Ser Val Leu Arg Val Val Ile Thr

115 120 125

Ser Ser Thr Ala Ser Val Ile Cys Asn Lys Asn Met Ser Thr Pro Gly

130 135 140

Ala Val Ala Asp Glu Thr Trp Tyr Ser Asp Pro Glu Phe Cys Glu Glu

145 150 155 160

Arg Lys Glu Trp Tyr Gln Leu Ser Lys Thr Leu Ala Glu Lys Ala Ala

165 170 175

Arg Arg Phe Ala Lys Glu Asn Gly Ile Asp Leu Val Thr Leu His Pro

180 185 190

Gly Leu Val Ile Gly Pro Leu Leu Gln Pro Thr Leu Asn Phe Ser Cys

195 200 205

Glu Ala Ile Val Asn Phe Ile Lys Glu Gly Lys Glu Ala Trp Ser Gly

210 215 220

Gly Val Tyr Arg Phe Val Asp Val Arg Asp Val Ala Asn Ala His Ile

225 230 235 240

Leu Ala Phe Glu Val Pro Ser Ala Asn Gly Arg Tyr Cys Leu Val Gly

245 250 255

Val Asn Gly Tyr Ser Ser Leu Val Leu Lys Ile Val Gln Lys Leu Tyr

260 265 270

Pro Ser Ile Thr Leu Pro Glu Asn Phe Glu Asp Gly Leu Pro Leu Thr

275 280 285

Pro His Phe Gln Val Ser Ser Glu Arg Ala Lys Gly Leu Gly Val Lys

290 295 300

Phe Thr Pro Leu Glu Leu Ser Val Lys Asp Thr Val Glu Ser Leu Met

305 310 315 320

Glu Lys Asn Phe Leu His Ile

325

<210> 13

<211> 365

<212> PRT

<213> belladonna (Atropa belladonna)

<400> 13

Met Ala Ser Glu Lys Ser Leu Glu Glu Lys Gln Ala Glu Asn Thr Phe

1 5 10 15

Gly Trp Ala Ala Met Asp Ser Ser Gly Val Leu Ser Pro Phe Thr Phe

20 25 30

Ser Arg Arg Ala Thr Gly Glu Glu Asp Val Arg Leu Lys Val Leu Tyr

35 40 45

Cys Gly Ile Cys His Ser Asp Leu Gly Cys Ile Lys Asn Glu Trp Gly

50 55 60

Trp Cys Ser Tyr Pro Leu Val Pro Gly His Glu Ile Val Gly Ile Ala

65 70 75 80

Thr Glu Val Gly Ser Lys Val Thr Lys Phe Lys Val Gly Asp Arg Val

85 90 95

Gly Val Gly Cys Met Val Gly Ser Cys Gly Thr Cys Gln Asn Cys Thr

100 105 110

Gln Asn Gln Glu Ser Tyr Cys Pro Glu Val Ile Met Thr Cys Ala Ser

115 120 125

Ala Tyr Pro Asp Gly Thr Pro Thr Tyr Gly Gly Phe Ser Asn Gln Met

130 135 140

Val Ala Asn Glu Lys Phe Val Ile Arg Ile Pro Asn Ser Leu Pro Leu

145 150 155 160

Asp Ala Ala Ala Pro Leu Leu Cys Ala Gly Ser Thr Val Tyr Ser Ala

165 170 175

Met Lys Phe Tyr Gly Leu Cys Ser Gln Gly Leu His Leu Gly Val Val

180 185 190

Gly Leu Gly Gly Leu Gly His Val Ala Val Lys Phe Ala Lys Ala Phe

195 200 205

Gly Met Lys Val Thr Val Ile Ser Thr Ser Leu Gly Lys Lys Glu Glu

210 215 220

Ala Ile Asn Gln Leu Gly Ala Asp Ser Phe Leu Ile Asn Thr Asp Thr

225 230 235 240

Glu Gln Met Gln Gly Ala Met Glu Val Met Asp Gly Ile Ile Asp Thr

245 250 255

Val Ser Ala Leu His Pro Ile Glu Pro Leu Leu Gly Leu Leu Lys Ser

260 265 270

His Gln Gly Lys Leu Ile Ile Val Gly Leu Pro Asn Lys Gln Pro Glu

275 280 285

Leu Pro Val Phe Ser Leu Ile Asn Gly Arg Lys Met Ile Gly Gly Ser

290 295 300

Ala Val Gly Gly Val Lys Glu Thr Gln Glu Met Ile Asp Phe Ala Ala

305 310 315 320

Glu His Asn Ile Thr Ala Asp Ile Glu Ile Val Pro Met Asp Tyr Val

325 330 335

Asn Thr Ala Met Glu Arg Leu Glu Lys Gly Asp Val Lys Phe Arg Phe

340 345 350

Val Ile Asp Val Glu Asn Thr Leu Val Ala Ala Gln Thr

355 360 365

<210> 14

<211> 364

<212> PRT

<213> Maomangaro (Datura innoxia)

<400> 14

Met Ala Ala Glu Lys Leu Ser Glu Glu Glu Ala Val Lys Thr Phe Gly

1 5 10 15

Trp Ala Ala Met Asp Ser Ser Gly Val Leu Ser Pro Phe Glu Phe Ser

20 25 30

Arg Arg Ala Thr Gly Ala Glu Asp Val Arg Leu Lys Val Leu Tyr Cys

35 40 45

Gly Ile Cys His Ser Asp Leu Gly Cys Val Lys Asn Glu Trp Gly Trp

50 55 60

Cys Ser Tyr Pro Leu Val Pro Gly His Glu Ile Val Gly Ile Ala Thr

65 70 75 80

Glu Val Gly Ser Arg Val Thr Lys Phe Lys Val Gly Asp Arg Val Gly

85 90 95

Val Gly Cys Met Val Gly Ser Cys Gly Ser Cys Gln Asn Cys Ser Gln

100 105 110

Asn Leu Glu Ser Tyr Cys Pro Glu Val Ile Met Thr Cys Ala Ser Ala

115 120 125

Tyr Pro Asp Gly Thr Pro Thr Tyr Gly Gly Phe Ser Asn Gln Met Val

130 135 140

Ala Asn Glu Lys Phe Val Ile Gln Ile Pro Glu Lys Leu Pro Leu Asp

145 150 155 160

Ala Ala Ala Pro Leu Leu Cys Ala Gly Ser Thr Val Tyr Ser Pro Met

165 170 175

Lys Phe Tyr Gly Leu Cys Ser Pro Gly Leu His Leu Gly Val Val Gly

180 185 190

Leu Gly Gly Leu Gly His Val Ala Val Lys Phe Ala Lys Ala Phe Gly

195 200 205

Met Lys Val Thr Val Ile Ser Thr Ser Ile Gly Lys Lys Glu Glu Ala

210 215 220

Ile Asn Gln Leu Gly Ala Asp Ser Phe Leu Thr Ser Thr Asp Thr Glu

225 230 235 240

Gln Met Gln Gly Ala Met Glu Thr Met Asp Gly Ile Ile Asp Thr Val

245 250 255

Ser Ala Leu His Pro Ile Glu Pro Leu Val Gly Leu Leu Lys Ser His

260 265 270

Gln Gly Lys Leu Ile Ile Val Gly Leu Pro Asn Lys Gln Pro Glu Leu

275 280 285

Pro Val Phe Ser Leu Ile Asn Gly Arg Lys Met Ile Gly Gly Ser Ala

290 295 300

Val Gly Gly Val Lys Glu Thr Gln Glu Met Ile Asp Phe Ala Ala Lys

305 310 315 320

His Asn Ile Thr Ala Asp Ile Glu Ile Val Arg Met Asp Tyr Val Asn

325 330 335

Thr Ala Met Glu Arg Leu Glu Lys Gly Asp Val Lys Phe Arg Phe Val

340 345 350

Ile Asp Val Glu Asn Thr Leu Val Pro Ala Gln Thr

355 360

<210> 15

<211> 364

<212> PRT

<213> Datura stramonium (Datura stramonium)

<400> 15

Met Ala Ala Glu Lys Leu Glu Glu Arg Lys Arg Trp Glu Thr Phe Gly

1 5 10 15

Trp Ala Ala Met Asp Ser Ser Gly Val Leu Ser Pro Phe Glu Phe Ser

20 25 30

Arg Arg Ala Thr Gly Glu Glu Asp Val Arg Leu Lys Val Leu Tyr Cys

35 40 45

Gly Ile Cys His Ser Asp Leu Gly Cys Ile Lys Asn Glu Trp Gly Trp

50 55 60

Cys Ser Tyr Pro Leu Val Pro Gly His Glu Ile Val Gly Ile Ala Thr

65 70 75 80

Glu Val Gly Ser Arg Val Thr Lys Phe Lys Val Gly Asp Arg Val Gly

85 90 95

Val Gly Cys Met Val Gly Ser Cys Gly Ser Cys Gln Asn Cys Ser Gln

100 105 110

Asn Leu Glu Ser Tyr Cys Pro Glu Val Ile Met Thr Cys Ala Ser Ala

115 120 125

Tyr Pro Asp Gly Thr Pro Thr Tyr Gly Gly Phe Ser Asn Gln Met Val

130 135 140

Ala Asn Glu Lys Phe Val Ile Gln Ile Pro Glu Lys Leu Pro Leu Asp

145 150 155 160

Ala Ala Ala Pro Leu Leu Cys Ala Gly Ser Thr Val Tyr Ser Pro Met

165 170 175

Lys Phe Tyr Gly Leu Cys Ser Pro Gly Leu His Leu Gly Val Val Gly

180 185 190

Leu Gly Gly Leu Gly His Val Ala Val Lys Phe Ala Lys Ala Phe Gly

195 200 205

Met Lys Val Thr Val Ile Ser Thr Ser Ile Gly Lys Lys Glu Glu Ala

210 215 220

Ile Asn Gln Leu Gly Ala Asp Ser Phe Leu Ile Ser Thr Asp Thr Glu

225 230 235 240

Gln Met Gln Gly Ala Met Glu Thr Met Asp Gly Ile Ile Asp Thr Val

245 250 255

Ser Ala Leu His Pro Ile Glu Pro Leu Val Gly Leu Leu Lys Ser His

260 265 270

Arg Gly Lys Leu Ile Ile Val Gly Leu Pro Asn Lys Gln Pro Glu Leu

275 280 285

Pro Val Phe Ser Leu Ile Asn Gly Arg Lys Met Ile Gly Gly Ser Ala

290 295 300

Val Gly Gly Val Lys Glu Thr Gln Glu Met Ile Asp Phe Ala Ala Lys

305 310 315 320

His Asn Ile Thr Ala Asp Ile Glu Ile Val Gly Met Asp Tyr Val Asn

325 330 335

Thr Ala Met Glu Arg Leu Glu Lys Gly Asp Val Lys Phe Arg Phe Val

340 345 350

Ile Asp Val Glu Asn Thr Leu Val Pro Ala Gln Thr

355 360

<210> 16

<211> 238

<212> PRT

<213> Victoria multitubular luminous jellyfish (Aequorea Victoria)

<400> 16

Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val

1 5 10 15

Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu

20 25 30

Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

35 40 45

Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe

50 55 60

Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln

65 70 75 80

His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

85 90 95

Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val

100 105 110

Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

115 120 125

Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

130 135 140

Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly

145 150 155 160

Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val

165 170 175

Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

180 185 190

Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser

195 200 205

Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

210 215 220

Thr Ala Ala Gly Ile Ile His Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 17

<211> 233

<212> PRT

<213> Artificial sequence ()

<220>