阅读说明：本技术 化合物的微生物生产 (Microbial production of compounds ) 是由 安德鲁·P·克莱因 C·D·里夫斯 C·J·帕栋 V·F·福尔摩斯 于 2020-03-13 设计创作，主要内容包括：提供了经修饰宿主细胞,其通过工程化改造以在一种外源试剂存在下减少产物表达至不可检测水平,而在另一外源试剂存在下提高该产物的表达。修饰的酵母菌株宿主细胞不表达可检测水平的用于生成产物的前体或底物。该产物可为大麻素或其前体,底物可为己酸盐/酯。还提供了采用该经修饰宿主细胞制备产物的方法。经修饰宿主细胞可为酵母菌株,例如酿酒酵母(S.cerevisiae)。(Modified host cells are provided that are engineered to reduce expression of a product to undetectable levels in the presence of one exogenous agent, and to increase expression of the product in the presence of another exogenous agent. The modified yeast strain host cell does not express detectable levels of the precursor or substrate used to produce the product. The product may be a cannabinoid or a precursor thereof and the substrate may be a hexanoate salt. Methods of making products using the modified host cells are also provided. The modified host cell may be a yeast strain, such as saccharomyces cerevisiae (s.)

1. A host cell comprising a heterologous genetic pathway for the production of a heterologous product and which is regulated by an exogenous agent, wherein the host cell does not produce a precursor required for the production of the product.

2. The host cell of claim 1, wherein the exogenous agent comprises a gene expression modulator.

3. The host cell of claim 2, wherein the exogenous agent reduces production of a heterologous product.

4. The host cell of claim 3, wherein the exogenous agent is glucose and the expression of the one or more enzymes encoded by the heterologous genetic pathway is under the control of a glucose-repressible promoter.

5. The host cell of claim 2, wherein the exogenous agent increases production of a heterologous product.

6. The host cell of claim 5, wherein the exogenous agent is galactose and expression of the one or more enzymes encoded by the heterologous genetic pathway is under the control of a GAL promoter.

7. The host cell of claim 1, wherein the heterologous genetic pathway comprises a galactose-responsive promoter, a maltose-responsive promoter, or a combination of both.

8. The host cell of claim 1, wherein the heterologous product is a cannabinoid or a cannabinoid precursor.

9. The host cell of claim 8, wherein the cannabinoid or cannabinoid precursor is CBDA, CBD, CBGA, or CBG.

10. The host cell of claim 8, wherein the genetic pathway encodes at least two enzymes selected from the group consisting of: hexanoyl-CoA synthase (HCS), tetrone synthase (TKS) and olivo-late cyclase (OAC).

11. The host cell of claim 8, wherein the precursor required to produce the product is a hexanoate salt.

12. The host cell of claim 1, wherein the heterologous genetic pathway comprises a nucleic acid construct comprising at least three protein coding regions.

13. The host cell of claim 1, wherein the host cell is a yeast cell or a yeast strain.

14. The host cell of claim 13, wherein the yeast cell is saccharomyces cerevisiae.

15. A mixture comprising the host cell of any one of claims 1-14 and a culture medium.

16. The mixture of claim 15, wherein the culture medium comprises an exogenous agent that reduces production of a heterologous product.

17. The mixture of claim 16, wherein the exogenous agent is glucose, maltose, or lysine.

18. The mixture of claim 15, wherein the culture medium comprises: (i) an exogenous agent that increases production of a heterologous product; and (ii) a precursor required for the preparation of the heterologous product.

19. The mixture of claim 18, wherein the exogenous agent is galactose.

20. The mixture of claim 18, wherein the precursor required for the production of the heterologous product is a caproate.

21. A method of reducing expression of a heterologous product, comprising culturing the host cell of any of claims 1-14 in a medium comprising an exogenous agent, wherein the exogenous agent reduces expression of the heterologous product.

22. The method of claim 21, wherein the exogenous agent is glucose, maltose, or lysine.

23. The method of claim 21, wherein culturing the host cell in a medium comprising the exogenous agent results in production of less than 0.001mg/L of the heterologous product.

24. A method of increasing expression of a heterologous product, comprising culturing the host cell of any of claims 1-14 in a medium comprising an exogenous agent, wherein the exogenous agent increases expression of the heterologous product.

25. The method of claim 24, wherein the exogenous agent is galactose.

26. The method of claim 24, further comprising culturing the host cell in the presence of a precursor required for the production of the heterologous product.

27. The method of claim 24, wherein the precursor required for the production of the heterologous product is a caproate.

28. A host cell comprising a heterologous genetic pathway for the production of cannabinoids and which is regulated by an exogenous agent, wherein the host cell comprises a hexanoate salt at a level which does not produce cannabinoids in an amount of more than 10 mg/L.

29. The host cell of claim 28, wherein the exogenous agent down-regulates expression of a heterologous genetic pathway.

30. The host cell of claim 29, wherein the exogenous agent is glucose and the expression of the one or more enzymes encoded by the heterologous genetic pathway is under the control of a glucose-repressible promoter.

31. The host cell of claim 28, wherein the exogenous agent upregulates expression of a heterologous genetic pathway.

32. The host cell of claim 31, wherein the exogenous agent is galactose and expression of the one or more enzymes encoded by the heterologous genetic pathway is under the control of a GAL promoter.

33. The host cell of claim 28, wherein the genetic pathway encodes at least two enzymes selected from the group consisting of: hexanoyl-CoA synthase (HCS), tetrone synthase (TKS) and olivo-late cyclase (OAC).

34. The host cell of claims 28-33, wherein the host cell is a yeast cell or yeast strain.

35. The host cell of claim 34, wherein the yeast is saccharomyces cerevisiae.

36. A method of reducing cannabinoid expression comprising culturing the host cell of any of claims 28-30 and 33-35 in a medium comprising an exogenous agent, wherein the exogenous agent reduces expression of a cannabinoid or a precursor thereof.

37. The method of claim 36, wherein the exogenous agent is glucose, maltose, or lysine.

38. The method of claim 36, wherein culturing the host cell in a medium comprising the exogenous agent results in the production of less than 0.001mg/L cannabinoid or a precursor thereof.

39. A method of increasing cannabinoid expression, comprising culturing the host cell of any of claims 31-35 in a medium comprising an exogenous agent, wherein the exogenous agent increases expression of a cannabinoid or a precursor thereof.

40. The method of claim 39, wherein the exogenous agent is galactose.

41. The method of claim 39, further comprising culturing the host cell in a medium comprising hexanoate.

42. The method of any one of claims 36-41, wherein the cannabinoid is CBDA, CBD, CBGA, or CBG.

43. The method of any one of claims 36-41, wherein the host cell is a yeast cell or a yeast strain.

44. The method of claim 43, wherein the yeast is Saccharomyces cerevisiae.

Background

In the production of biomolecules from organisms, there is usually an externally controlled gene switch, and it is advantageous to switch between a high growth, low production mode (suitable for biomass production and easy handling) and a low growth, high production mode (suitable for advantageous manufacturing). For most fermentatively produced biomolecules, a single switch mechanism is sufficient, even though it achieves yields of up to 10-20% in the low production mode. For some biomolecules, e.g. cannabinoids, regulatory requirements have created a need for very low or undetectable production in low production states.

A number of strong single mechanism switches are found in nature, including the galactose regulatory system in yeast and the arabinose regulatory system in bacteria. Most are based on normal physiological responses, where genes are activated when an organism senses a threat or resource. There are also several systems that respond to molecules that are not uncommon in the context of organisms-tetracycline, IPTG, indigo-which have been used in biotechnological applications.

Summary of The Invention

In one aspect, a modified, engineered or recombinant host cell is provided, comprising a heterologous genetic pathway for the production of a heterologous product, and which is regulated by an exogenous agent, wherein the host cell does not produce the precursor required for the production of the product. In some embodiments, the exogenous agent comprises a gene expression modulator.

In some embodiments, the exogenous agent reduces the production of the heterologous product. In some embodiments, the exogenous agent that reduces production of the heterologous product is glucose, and expression of one or more enzymes encoded by the heterologous genetic pathway is under the control of a glucose-repressible promoter.

In some embodiments, the exogenous agent increases the production of the heterologous product. In some embodiments, the exogenous agent that increases production of the heterologous product is galactose and expression of one or more enzymes encoded by the heterologous genetic pathway is under the control of a GAL promoter.

In some embodiments, the heterologous genetic pathway comprises a galactose-responsive promoter, a maltose-responsive promoter, or a combination thereof.

In some embodiments, the heterologous product is a cannabinoid or a cannabinoid precursor. In some embodiments, the cannabinoid or cannabinoid precursor is cannabidiolic acid (CBDA), Cannabidiol (CBD), cannabigerolic acid (CBGA), or Cannabigerol (CBG).

In some embodiments, the genetic pathway encodes at least two enzymes selected from the group consisting of: hexanoyl-CoA synthase (HCS), tetrone synthase (TKS) and Olivil Acid Cyclase (OAC).

In some embodiments, the precursor required to prepare the product is a hexanoate salt.

In some embodiments, the heterologous genetic pathway comprises a nucleic acid construct comprising at least three protein coding regions.

In some embodiments, the host cell is a yeast cell or a yeast strain. In some embodiments, the yeast cell is saccharomyces cerevisiae (s.

In another aspect, a mixture is provided, the mixture comprising a host cell as described herein and a culture medium. In some embodiments, the medium comprises an exogenous agent that reduces production of the heterologous product. In some embodiments, the exogenous agent that reduces production of the heterologous product is glucose, maltose, or lysine.

In some embodiments, the culture medium comprises: (i) an exogenous agent that increases production of a heterologous product; and (ii) a precursor required for the preparation of the heterologous product. In some embodiments, the exogenous agent that increases production of the heterologous product is galactose. In some embodiments, the precursor required for the preparation of the heterologous product is a hexanoate salt.

In another aspect, a method of reducing expression of a heterologous product is provided, the method comprising culturing a host cell described herein in a medium comprising an exogenous agent, wherein the exogenous agent reduces expression of the heterologous product. In some embodiments, the exogenous agent that reduces expression of the heterologous product is glucose, maltose, or lysine. In some embodiments, culturing the host cell strain in a medium comprising an exogenous agent results in the production of less than 0.001mg/L of the heterologous product.

In another aspect, a method of increasing expression of a heterologous product is described, the method comprising culturing a host cell described herein in a medium comprising an exogenous agent, wherein the exogenous agent increases expression of the heterologous product. In some embodiments, the exogenous agent that increases expression of the heterologous product is galactose.

In some embodiments, the method further comprises culturing the host cell in the presence of a precursor required for the production of the heterologous product. In some embodiments, the precursor required for the preparation of the heterologous product is a hexanoate salt.

In some embodiments described herein, the heterologous product is a cannabinoid or a cannabinoid precursor. In some embodiments, the cannabinoid or cannabinoid precursor is CBDA, CBD, CBGA, or CBG.

In another aspect, a host cell is provided that comprises a heterologous genetic pathway for cannabinoid production, and that pathway is regulated by an exogenous agent. In some embodiments, the host cell does not contain precursors required for the production of cannabinoids, or does not contain precursors required for the production of cannabinoids in amounts exceeding a predetermined level (e.g. greater than 10 mg/L). In some embodiments, the host cell comprises a level of hexanoate that is insufficient to produce an amount of cannabinoid in excess of 10 mg/L. In some embodiments, the cannabinoid is CBDA, CBD, CBGA, or CBG.

In some embodiments, the exogenous agent down-regulates expression of the heterologous genetic pathway. In some embodiments, the exogenous agent that down-regulates expression of the heterologous genetic pathway is glucose. In some embodiments, expression of one or more enzymes encoded by the heterologous genetic pathway is under the control of a glucose-repressible promoter.

In some embodiments, the exogenous agent upregulates expression of the heterologous genetic pathway. In some embodiments, the exogenous agent that upregulates expression of the heterologous genetic pathway is galactose. In some embodiments, expression of the one or more enzymes encoded by the heterologous genetic pathway is under the control of a GAL promoter.

In some embodiments, the genetic pathway encodes at least two enzymes selected from the group consisting of: hexanoyl-CoA synthase (HCS), tetrone synthase (TKS) and Olivil Acid Cyclase (OAC).

In some aspects or embodiments described herein, the host cell can be a yeast cell or a yeast strain. In some aspects or embodiments described herein, the yeast cell is saccharomyces cerevisiae.

In another aspect, there is provided a method of reducing cannabinoid expression, the method comprising culturing a host cell described herein in a medium comprising an exogenous agent, wherein the exogenous agent reduces expression of a cannabinoid or a precursor thereof. In some embodiments, the exogenous agent that reduces expression of a cannabinoid or a precursor thereof is glucose, maltose, or lysine. In some embodiments, culturing the host cell in a medium comprising an exogenous agent results in the production of less than 0.001mg/L cannabinoid or a precursor thereof.

In another aspect, there is provided a method of increasing cannabinoid expression, the method comprising culturing a host cell described herein in a medium comprising an exogenous agent, wherein the exogenous agent increases expression of a cannabinoid or a precursor thereof. In some embodiments, the exogenous agent that reduces expression of a cannabinoid or a precursor thereof is glucose, maltose, or lysine. In some embodiments, the method further comprises culturing the host cell in a medium comprising hexanoate.

In some embodiments, the cannabinoid or cannabinoid precursor is CBDA, CBD, CBGA, or CBG.

Brief description of the drawings

Figure 1 shows the expression of the cannabinoid precursors, olivetol and olivetol, by the modified host cells described herein, as described in example 1.

FIGS. 2 and 3 show the genetic map of the heterologous nucleic acid transformed into the modified host cell as described in example 1.

Figure 4 shows part of the cannabinoid synthetic pathway referred to herein.

FIG. 5 is a schematic diagram showing the structure and function of a maltose-regulated transcriptional switch used herein.

Figure 6 shows the biochemical pathway of geranyl pyrophosphate (GPP) derived from sugars and the synthesis of Cannabigerol (CBG) and Cannabidiol (CBD) from hexanoic acid.

Figure 7 shows the overall layout of the N-terminal to C-terminal arrangement of CBDA Synthase (CBDAs) surface display constructs.

Fig. 8, 9 and 10 are paired graphs showing normalized biomass (upper panels) and normalized CBDA titer (lower panels) without caproic acid or 2mM caproic acid, each also assayed for Y61508 (fig. 8), Y66316 (fig. 9) and Y66085 (fig. 10) strains with 4% maltose, 2% maltose and 2% sucrose.

Definition of

As used herein, a "genetic pathway" refers to a set of at least two different coding sequences that encode enzymes that catalyze different parts of a synthetic pathway to form a desired product. In the genetic pathway, a first encoded enzyme employs a substrate to form a first product, and the first product further serves as a substrate for a second encoded enzyme to form a second product. In some embodiments, a genetic pathway comprises 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.) in which the product of one encoded enzyme is a substrate for the next enzyme in the synthetic pathway. An example of the cannabinoid synthetic pathway is shown in figure 4.

As used herein, the term "endogenous" refers to a substance or process that occurs naturally in a host cell. Conversely, the term "exogenous" refers to a substance or compound that originates from an organism or outside of a cell. The exogenous material or compound, when introduced into an organism or host cell as described herein, is capable of maintaining its normal function or activity.

The terms "modified", "recombinant" and "engineered" when used to modify a host cell as described herein refer to a host cell or organism that is not naturally occurring, or that expresses a compound, nucleic acid or protein at a level that is different from the level of expression of the naturally occurring cell or organism.

As used herein, the term "genetically modified" refers to a host cell comprising a heterologous nucleotide sequence. The genetically modified host cells described herein do not normally exist in nature.

The term "heterologous compound" refers to a compound that is produced by a cell that does not normally produce the compound, or to a compound that is produced at a level that is different from the level at which the cell normally produces the compound.

The phrase "heterologous" as used herein refers to a substance not normally found in nature. The term "heterologous compound" refers to a compound that is produced by a cell that does not normally produce the compound, or to a compound that is produced at a level that is different from the level at which the cell normally produces the compound. For example, the cannabinoid may be a heterologous compound.

As used herein, the phrase "heterologous enzyme" refers to an enzyme that does not normally occur naturally in a given cell. The term includes the following enzymes: (a) exogenous to a given cell (i.e., encoded by a nucleotide sequence that does not naturally occur in the host cell or does not naturally occur in a particular environment in the host cell); and (b) naturally occurring in the host cell (i.e., the enzyme is encoded by a nucleotide sequence that is endogenous to the cell), but is produced in a non-natural amount in the host cell (e.g., greater than or less than the amount that is naturally occurring).

As used herein, "heterologous genetic pathway" refers to a genetic pathway that is not normally found or naturally occurring in an organism or cell.

As used herein, the phrase "operably linked" refers to a functional linkage between nucleic acid sequences such that the linked promoter and/or regulatory region functionally controls the expression of the coding sequence.

As used herein, the term "producing" generally refers to the production of an amount of a compound by a genetically modified host cell provided herein. In some embodiments, the production is expressed as a yield of the compound produced by the host cell. In other embodiments, production is expressed as the production capacity of the host cell to produce the compound.

As used herein, the term "productivity" refers to the production of a compound by a host cell, expressed as the amount (by weight) of non-catabolic compound produced per volume of fermentation broth (broth) in which the host cell is cultured, as a function of time (per hour).

As used herein, the term "promoter" refers to a class of synthetic or naturally derived nucleic acids that is capable of activating, increasing or promoting expression of a DNA coding sequence, or inactivating, reducing or inhibiting expression of a DNA coding sequence. A promoter may include one or more specific transcriptional regulatory sequences to further enhance or inhibit expression of a coding sequence and/or to alter spatial and/or temporal expression of a coding sequence. The promoter may be located 5' (upstream) of the coding sequence under its control. The promoter may also initiate transcription in the downstream (3') direction, the upstream (5') direction, or be designed to initiate transcription in both the downstream (3') direction and the downstream (3') direction. The distance between the promoter and the coding sequence to be expressed may be substantially the same as the distance between the promoter and the native nucleic acid sequence it controls. As is known in the art, this distance can be accommodated without loss of promoter function. The term also includes a regulated promoter that is generally capable of effecting transcription of a nucleic acid sequence in a permissive environment (e.g., under microaerobic fermentation conditions or in the presence of maltose), but that stops transcription of the nucleic acid sequence in a non-permissive environment (e.g., under aerobic fermentation conditions or in the absence of maltose). Promoters used herein may be constitutive, inducible, or repressible.

The term "yield" refers to the production of a compound by a host cell and refers to the amount of the compound produced per amount of carbon source consumed by the host cell (by weight).

The term "about" when modifying a value or range herein includes normal variations encountered in the art, including the endpoints of the value or range plus (+) or minus (-) 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%). Accordingly, the value 10 includes all values from 9-11. All numerical ranges set forth herein include the endpoints of the ranges (unless otherwise stated) and all numbers between the endpoints, to the first significant digit.

Detailed Description

Provided herein are recombinant or modified host cells useful for the production of heterologous products, and methods of using the host cells. Recombinant or modified host cells comprise a heterologous genetic pathway that can be differentially regulated by one or more exogenous agents. Recombinant host cells provide the following benefits: under one set of conditions, the expression of the heterologous product is reduced to a very low, preferably undetectable level, while under a second set of conditions, the heterologous product is allowed to be strongly expressed. In some embodiments, the host cell is engineered to express a heterologous enzyme in the cannabinoid pathway. In some embodiments, the host cell is a yeast cell.

Modified host cells comprising heterologous genetic pathways

In one aspect, provided herein are host cells comprising a heterologous genetic pathway for the production of a heterologous product. In some embodiments, the heterologous genetic pathway comprises a genetic regulatory element, e.g., a nucleic acid sequence, that is regulated by an exogenous agent. In some embodiments, the exogenous agent functions to regulate expression of a heterologous genetic pathway. Thus, in some embodiments, the exogenous agent can be a gene expression modulator.

In some embodiments, the exogenous agent can be used as a carbon source for the host cell. For example, the same exogenous agent may both regulate expression of a heterologous genetic pathway and provide a carbon source for growth of the host cell. In some embodiments, the exogenous agent is glucose. In some embodiments, the exogenous agent is galactose. In some embodiments, the exogenous agent is maltose.

In some embodiments, the genetic regulatory element is a nucleic acid sequence, such as a promoter. In some embodiments, the genetic regulatory element is a glucose-responsive promoter or a glucose-repressed promoter. In some embodiments, glucose negatively regulates expression of the heterologous genetic pathway, thereby reducing production of the heterologous product. Exemplary glucose-repressible promoters include: pMAL11, pMAL12, pMAL13, pMAL21, pMAL22, pMAL31, pMAL32, pMAL33, pCAT8, pHXT2, pHXT4, pMTH1 and pSUC 2.

Table 1: exemplary glucose repressible promoter sequences

Promoters	Sequence of
		pMAL11	SEQ ID NO:
pMAL12	SEQ ID NO:
		pMAL13	SEQ ID NO:
pMAL21	SEQ ID NO:
		pMAL22	SEQ ID NO:
pMAL31	SEQ ID NO:
		pMAL32	SEQ ID NO:
pMAL33	SEQ ID NO:
		pCAT8	SEQ ID NO:
pHXT2	SEQ ID NO:
		pHXT4	SEQ ID NO:
pMTH1	SEQ ID NO:
		pSUC2	SEQ ID NO:

In some embodiments, the genetic regulatory element is a galactose-responsive promoter. In some embodiments, galactose positively regulates expression of a heterologous genetic pathway, thereby increasing production of a heterologous product. In some embodiments, the galactose-responsive promoter is a GAL1 promoter. In some embodiments, the galactose-responsive promoter is a GAL10 promoter. In some embodiments, the galactose-responsive promoter is a GAL2, GAL3, or GAL7 promoter. In some embodiments, the heterologous genetic pathway comprises a galactose-responsive regulatory element as described by Westfall et al (PNAS (2012) vol.109: E111-118). In some embodiments, the host cell lacks the gal1 gene and is unable to metabolize galactose, but galactose is still capable of inducing the galactose regulatory gene.

Table 2: exemplary GAL promoter sequences

Promoters	Sequence of
		pGAL1	SEQ ID NO:
pGAL10	SEQ ID NO:
		pGAL2	SEQ ID NO:
pGAL3	SEQ ID NO:
		pGAL7	SEQ ID NO:
pGAL4	SEQ ID NO:

In some embodiments, the galactose regulatory system used to control gene expression is reconfigured such that it is no longer induced in the presence of galactose. Instead, the gene is expressed only in the presence of a repressor in the culture medium, which may be lysine in some strains or maltose in others.

In some embodiments, the genetic regulatory element is a maltose-responsive promoter. In some embodiments, maltose negatively regulates expression of the heterologous genetic pathway, thereby increasing production of the heterologous product. In some embodiments, the maltose-responsive promoter is selected from the group consisting of: pMAL1, pMAL2, pMAL11, pMAL12, pMAL31, and pMAL 32. The maltose genetic regulatory element may be designed to both activate the expression of certain genes and repress the expression of other genes depending on the presence or absence of maltose in the medium. Maltose regulation of gene expression as well as maltose-responsive promoters are described in U.S. patent publication No. 2016/0177341, which is incorporated herein by reference. Genetic regulation of Maltose Metabolism is described in Novak et al, "Maltose Transport and Metabolism in S.cerevisiae" ("Maltose Transport and Metabolism in Saccharomyces cerevisiae", Food Technol. Biotechnol.42(3) 213- "218 (2004)).

Table 3: exemplary MAL promoter sequences

Promoters	Sequence of
		pMAL1	SEQ ID NO:
pMAL2	SEQ ID NO:
		pMAL11	SEQ ID NO:
pMAL12	SEQ ID NO:
		pMAL31	SEQ ID NO:
pMAL32	SEQ ID NO:

In some embodiments, the heterologous genetic pathway is regulated by a combination of maltose and galactose regulators.

In some embodiments, the heterologous genetic pathway is regulated by lysine. Regulation of the LYS gene is described, for example, in beller et al (eur.j. biochem.261,163-170 (1999)).

In some embodiments, the recombinant host cell does not contain or express very low levels (e.g., undetectable amounts) of precursors required to form the heterologous product. In some embodiments, the precursor is a substrate for an enzyme in a heterologous genetic pathway.

Cannabinoid pathways

In another aspect, the host cell comprises a heterologous genetic pathway for the production of a cannabinoid or a precursor of a cannabinoid. In some embodiments, the precursor is a substrate in the cannabinoid pathway. In some embodiments, the precursor is hexanoyl-CoA synthase (HCS), tetrone synthase (TKS), or Olivil Acid Cyclase (OAC). In some embodiments, the precursor, substrate, or intermediate in the cannabinoid pathway is a hexanoate, olive alcohol, or olive alcohol acid. In some embodiments, the precursor is a hexanoate salt. In some embodiments, the host cell contains insufficient amounts of precursors, substrates, or intermediates to produce a cannabinoid or cannabinoid precursor. In some embodiments, the host cell comprises a level or amount of hexanoate that is insufficient to produce cannabinoid in an amount in excess of 10 mg/L. In some embodiments, the heterologous genetic pathway encodes at least two enzymes selected from the group consisting of: hexanoyl-CoA synthase (HCS), tetrone synthase (TKS) and olivo-late cyclase (OAC). The cannabinoid pathway is described in Keasling et al (WO 2018/200888).

In some embodiments, the host cell is a yeast strain. In some embodiments, the yeast strain is a Y27600, Y27602, Y27603, or Y27604 strain.

Yeast strains

In some embodiments, yeasts useful in the present methods comprise: yeasts which have been deposited with the microbiological depositary (e.g. IFO, ATCC, etc.) and belong to the following genera: saccharomyces (Aciclococcus), Saccharomyces (Ambrosozyma), Strongylocentrotus (Arthroascus), Bipolar Yeast (Arxiozyma), Ashbya (Ashbya), Saccharomyces (Babjevia), Bentonium (Benstonia), Saccharum glucanum (Botryoascus), (Botryozyma), Brettanomyces (Brettanomyces), Marylaria (Bullera), Bullera (Bulleromyces), Candida (Candida), Saccharomyces (Citeromyces), Corynebacterium (Clavispora), Cryptococcus (Cryptococcus), Sphaeromyces nigrospora (Cystofildium), Debaryomyces (Debaryomyces), Dikkera (Dekkera), Saccharomyces bisporus (Cryptomyces), Saccharomyces cerevisiae (Gekkonii), Saccharomyces cerevisiae (Gekkoniella), Saccharomyces cerevisiae (Gekkoniensis), Saccharomyces (Gekkoniella), Saccharomyces (Gekkoniensis), Saccharomyces (Gekkoniella), Saccharomyces (Gekkonidis), Saccharomyces (Gekkoniella), Saccharomyces (Gekkoniella), Gekkoniella (Gekkoniella), Gekkoniella, Gekkonie, and/or Gekkonie, or (C, or Gekkonie, or C, or (C, or Gekkonie, or C, or (C, or, There are Hansenula (Hanseniaspora), Hansenula (Hansenula), Rhodotorula (Hasegawaea), Gloeostereum (Holtermannia), Hornobacillus (Hormoascus), Pichia marinus (Hypopichia), Issatchenkia (Issatchenkia), Kloeckera (Kloeckera), Kluyveromyces (Kluyveromyces), Kondoa (Kuraishia), (Kurtzmanomyces), Saccharomyces bailii (Leucosporium), Lipomyces (Lipomyces), Lodoromyces (Loddermomyces), Malassezia (Malassezia), Metschnikowia (Maackia), Murakia (Murraya), Murraya (Phaxomyces), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Rhodotorula (Pichia pastoris), Rhodotorula (Rhodotorula rubra), Rhodotorula (Rhodotorula rubrum), Rhodotorula (Rhodotorula farinosa), Rhodotorula (Rhodotorula), Rhodotorula (Rhodotorula) and Rhodotorula farinosa), Rhodotorula (Rhodotorula) are included in the genus Rhodotorula), Rhodotorula, Rhodo, Saccharomyces multocida (Saccharomyces), Saccharomyces sartorius (Saitoella), Saccharomyces sakaguchi (Sakaguchi), Zygosaccharomyces (Sarurnospora), Schizosaccharomyces pombe (Schizosaccharomyces), Schizosaccharomyces schwanensis (Schwanniomyces), Saccharomyces schwannioides (Schwanniomyces), Sporidiobolus (Sporidiobolus), Sporobolomyces (Sporobolomyces), Protorula (Sporosaccharomyces), Stephanocasculus (Stephanocasculus), Brevibacterium (Sterigmatomyces), Trichosporoides (Steriginosorus), Saccharomyces paragypticus (Symbiostachys), Saccharomyces axomyces (Sympodiomyces), and Trichosporomyces syngenes (Symphomycosis), there are torulospora (Torulaspora), (Trichosporiella), Trichosporon (Trichosporon), Trigonopsis (Trigonopsis), Loranthomycetes (Tsuchiyaea), Artonella (Udenomyces), Walthomyces (Waltromyces), Wilknella (Wickerhamamia), Wickera (Wickeramiella), Williamsis (Williapisis), Yamadazyma (Yamadazyma), Yarrowia (Yarrowia), Zygosaccharomyces (Zygosaccharomyces), Zygosaccharomyces (Zygogligowioposis), and Zygosazyma (Zygozymea), and the like.

In some embodiments, the strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), Schizosaccharomyces africana (Schizosaccharomyces pombe), brussel's yeast (Dekkera bruxellensis), Kluyveromyces lactis (originally called Saccharomyces cerevisiae), Kluyveromyces markouyveri (Kluyveromyces marxianus), arabidopsis adenine (Arxula adeninivorans), or Hansenula polymorpha (also known as Pichia anserina). In some embodiments, the host microorganism is a strain of the genus Candida, such as Candida lipolytica (Candida lipolytica), Candida utilis (Candida guilliermondii), Candida krusei (Candida kruseii), Candida pseudotropicalis (Candida pseudotropicalis), or Candida betulina (Candida utilis).

In a particular embodiment, the strain is saccharomyces cerevisiae. In some embodiments, the host cell is a strain of saccharomyces cerevisiae selected from the group consisting of: baker's yeast, CEN.PK, CEN.PK2, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1 and AL-1. In some embodiments, the strain of saccharomyces cerevisiae is selected from the group consisting of: PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1. In a particular embodiment, the strain of Saccharomyces cerevisiae is PE-2. In another specific embodiment, the strain of Saccharomyces cerevisiae is CAT-1. In another specific embodiment, the strain of Saccharomyces cerevisiae is BG-1.

In some embodiments, the strain is a microorganism suitable for industrial fermentation. In particular embodiments, the microorganism is adapted to survive high solvent concentrations, high temperatures, extended substrate applications, nutrient limitation, osmotic stress due to sugar and salt, acidity, sulfite and bacterial contamination or combinations thereof, all known stress conditions in industrial fermentation environments.

In some embodiments, the yeast strain is Y27598, Y27599, Y27600, Y27601, Y27602, Y27603, Y27604, or Y25618 strain. Exemplary yeast strains are shown in table 4 below.

Table 4: yeast strains

Mixture of

In another aspect, a mixture of a host cell as described herein and a medium as described herein is provided. In some embodiments, the culture medium comprises an exogenous agent described herein. In some embodiments, the medium comprises an exogenous agent that reduces production of the heterologous product. In some embodiments, the exogenous agent that reduces production of the heterologous product is glucose or maltose.

In some embodiments, the medium comprises an exogenous agent that increases production of the heterologous product. In some embodiments, the exogenous agent that increases production of the heterologous product is galactose. In some embodiments, the culture medium comprises precursors or substrates required for the production of the heterologous product. In some embodiments, the precursor required for the preparation of the heterologous product is a hexanoate salt. In some embodiments, the culture medium comprises: exogenous agents that increase the production of the heterologous product and precursors or substrates required to produce the heterologous product. In some embodiments, the exogenous agent that increases production of the heterologous product is galactose and the precursor or substrate required for preparation of the heterologous product is hexanoate.

Method for producing host cell

In another aspect, methods of making the modified host cells described herein are provided. In some embodiments, the method comprises transforming a host cell with a heterologous nucleic acid construct described herein that encodes a protein expressed by a heterologous genetic pathway described herein. Methods for transforming host cells are described in "Laboratory Methods in Enzymology: DNA" (Methods of Enzymology experiments: DNA), Jon Lorsch code, volume 529, (2013); U.S. Pat. No. 9,200,270 to Hsieh, Chung-Ming, et al, and references cited therein.

Method for producing heterologous products

In another aspect, methods of producing the heterologous products described herein are provided. In some embodiments, the method reduces expression of the heterologous product. In some embodiments, the method comprises culturing a host cell comprising a heterologous genetic pathway described herein in a culture medium comprising an exogenous agent, wherein the exogenous agent reduces expression of the heterologous product. In some embodiments, the exogenous agent is glucose or maltose. In some embodiments, the method results in less than 0.001mg/L of heterologous product. In some embodiments, the heterologous product is a cannabinoid or a cannabinoid precursor.

In some embodiments, the method reduces the expression of a cannabinoid product or a precursor thereof. In some embodiments, the methods comprise culturing a host cell comprising a heterologous cannabinoid pathway described herein in a culture medium comprising an exogenous agent, wherein the exogenous agent reduces expression of a cannabinoid or a precursor thereof. In some embodiments, the exogenous agent is glucose or maltose. In some embodiments, the methods produce less than 0.001mg/L of cannabinoid or a precursor thereof.

In some embodiments, the method increases expression of the heterologous product. In some embodiments, the method comprises culturing a host cell comprising a heterologous genetic pathway described herein in a culture medium comprising an exogenous agent, wherein the exogenous agent increases expression of the heterologous product. In some embodiments, the exogenous agent is galactose. In some embodiments, the method further comprises culturing the host cell in the presence of a precursor or substrate required for the production of the heterologous product.

In some embodiments, the method increases expression of a cannabinoid product or a precursor thereof. In some embodiments, the methods comprise culturing a host cell comprising a heterologous cannabinoid pathway described herein in a culture medium comprising an exogenous agent, wherein the exogenous agent increases expression of a cannabinoid or a precursor thereof. In some embodiments, the exogenous agent is galactose. In some embodiments, the method further comprises culturing the host cell in the presence of a precursor or substrate required for the production of the heterologous cannabinoid product or a precursor thereof. In some embodiments, the precursor required for the production of the heterologous cannabinoid product or a precursor thereof is a hexanoate salt. In some embodiments, the combination of the exogenous agent and the precursor or substrate required to produce the heterologous cannabinoid product or precursor thereof produces a yield of cannabinoids that is greater than if the exogenous agent alone were used.

In some embodiments, the cannabinoid or precursor thereof is cannabidiolic acid (CBDA), CBD, cannabigerolic acid (CBGA), or CBG.

Nucleic acids

Due to the inherent degeneracy of the genetic code, other polynucleotides encoding substantially identical or functionally equivalent polypeptides may also be used to clone and express polynucleotides encoding the protein components of the heterologous genetic pathways described herein.

It is understood by those of ordinary skill in the art that it may be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code contains redundantly 64 possible codons, but most organisms generally use only a portion of these codons. The codons that are most frequently used in a species are called optimal codons, while those that are infrequent are classified as rare or low-availability codons. Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes referred to as "codon optimization" or "species codon bias control".

Optimized coding sequences comprising codons preferred by a particular prokaryotic or eukaryotic host can be prepared (Murray et al, 1989, Nucl Acids Res.17:477-508), for example, to increase translation speed or to produce recombinant RNA transcripts with desired characteristics (e.g., longer half-life) compared to transcripts produced with non-optimized sequences. Translation stop codons can also be modified to reflect host preferences. For example, typical stop codons for Saccharomyces cerevisiae and mammals are UAA and UGA, respectively. A typical stop codon for monocotyledons is UGA, whereas insects and E.coli usually use UAA as a stop codon (Dalphin et al, 1996, Nucl Acids Res.24: 216-8).

It will be appreciated by those skilled in the art that due to the degenerate nature of the genetic code, a variety of DNA molecules differing in nucleotide sequence may be used to encode a given enzyme of the disclosure. References herein to natural DNA sequences encoding the aforementioned biosynthetic enzymes are merely illustrative of embodiments of the disclosure, and the disclosure includes DNA molecules of any sequence that encodes the amino acid sequences of the enzyme proteins and polypeptides employed in the methods of the disclosure. In a similar manner, polypeptides are generally able to tolerate substitutions, deletions and insertions of one or more amino acids in their amino acid sequence without loss or significant loss of the desired activity. The present disclosure includes polypeptides having different amino acid sequences than the proteins described herein, so long as the modified or variant polypeptide has the enzymatic anabolic or catabolic activity of the reference polypeptide. In addition, the amino acid sequences encoded by the DNA sequences set forth herein are intended to be illustrative of embodiments of the present disclosure only.

Furthermore, homologues of enzymes useful in the compositions and methods provided herein are also included in the present disclosure. In some embodiments, two proteins (or regions of proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences may be aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may not be considered for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, 60%, even more typically at least 70%, 80%, 90% or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position (as used herein, "identity" of amino acids or nucleic acids is equivalent to "homology" of amino acids or nucleic acids). The percent identity between two sequences is related to the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced and the length of each gap for optimal alignment of the two sequences.

When "homologous" is used with a protein or peptide, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. "conservative amino acid substitution" refers to the substitution of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. The manner in which this adjustment is made is well known to those of ordinary skill in the art (see, e.g., Pearson W.R.,1994, Methods in Mol Biol 25: 365-89).

The following six groups each contain amino acids that are conservative substitutions for each other: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), alanine (a), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

Sequence homology (also referred to as percent sequence identity) of polypeptides is typically determined using sequence analysis software. A typical algorithm for comparing a molecular sequence to a database containing a large number of sequences obtained from different organisms is the computer program BLAST. When searching databases containing sequences from a large number of different organisms, the amino acid sequences are typically compared.

In addition, any gene encoding the aforementioned enzyme (or any other regulatory element described herein (or controlling or regulating its expression)) can be optimized by gene/protein engineering techniques, such as directed evolution or rational mutagenesis, as is known to those of ordinary skill in the art. This effect allows one of ordinary skill in the art to optimize the expression and activity of the enzyme in the host cell (e.g., yeast).

In addition, genes encoding these enzymes can be identified from other fungal and bacterial species and expressed to regulate this pathway. A variety of organisms can be the source of these enzymes, including but not limited to: yeasts (Saccharomyces spp.), including Saccharomyces cerevisiae and Saccharomyces uvarum (s.uvarum); kluyveromyces spp, including Kluyveromyces thermotolerans (k. thermotolerans), Kluyveromyces lactis (k. lactis), and Kluyveromyces marxianus; pichia pastoris (Pichia spp.); hansenula sp (Hansenula spp.), including Hansenula polymorpha (H.polymorpha); candida (Candida spp.), trichosporium (trichosporium spp.); zygosaccharomyces (Yamadazyma spp.), including zygosaccharomyces pedunculatus (y. spp. stipitis), torula tourbilla pretoriensis (torula pretoriensis), Issatchenkia orientalis (Issatchenkia orientalis); schizosaccharomyces (Schizosaccharomyces spp.), including Schizosaccharomyces pombe (s.pombe); cryptococcus (Cryptococcus spp.); aspergillus (Aspergillus spp.); neurospora (Neurospora spp.); or Ustilago sp. Genetic sources of anaerobic fungi include, but are not limited to: verbena pyricularis (Piromyces spp.), Verbena rhizogenes (Orpinomyces spp.) or Verbena neoformans (Neocallimastix spp.). Useful sources of proribozymes include, but are not limited to, E.coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus, Clostridium, Corynebacterium (Corynebacterium spp.), Pseudomonas, lactococcus, Enterobacter, and Salmonella.

Techniques known to those of ordinary skill in the art may be applied to identify other homologous genes and homologous enzymes. Typically, similar (analogous) genes and/or similar enzymes, which will have functional similarity, can be identified by functional analysis. Techniques known to those of ordinary skill in the art may be applied to identify similar genes and similar enzymes. For example, to identify homologous or analogous ADA genes, proteins or enzymes, techniques may include, but are not limited to: the genes were cloned by PCR using primers based on published sequences of the ADA genes/enzymes or by degenerate PCR using degenerate primers designed to amplify conserved regions between the ADA genes. Also, one of ordinary skill in the art can employ techniques to identify homologous or similar genes, proteins, or enzymes having functional identity or similarity. Techniques include examining the catalytic activity of an enzyme in a cell or cell culture by performing an in vitro enzyme assay on the activity (e.g., as described herein or in "Branched-Chain Amino Acids Methods Enzymology" by Kiritani, K. (Branched Chain Amino acid method, Enzymology, 1970); then separating out the enzyme with the activity by purification; determining the protein sequence of the enzyme by Edman (Edman) degradation or the like; designing PCR primers for possible nucleic acid sequences; amplifying the DNA sequence by PCR; to identify homologous or similar (similar) genes and/or homologous or similar enzymes, similar genes and/or similar enzymes, or proteins, the technique further comprises comparing data regarding the candidate genes or enzymes to a database such as BRENDA, KEGG, or metacycc.

In some embodiments, the protein or polypeptide encoded by the nucleic acid sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the protein or enzyme encoded by the heterologous genetic pathway described herein. In some embodiments, the nucleic acid sequence encodes a protein or polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of an HCS, TKS or OAC.

Culture and fermentation process

Materials and methods for maintenance or growth of microbial cultures are well known to those skilled in the art of microbiology or zymology (see, e.g., Bailey et al, "Biochemical Engineering Fundamentals," basis of Biochemical Engineering, "second edition, McGraw Hill Press, New York, 1986). Depending on the specific requirements of the host cell, fermentation and process, appropriate media, pH, temperature and the need for aerobic, micro-aerobic or anaerobic conditions must be considered.

The method of producing the heterologous product described herein can be performed in a suitable medium (e.g., supplemented or unsupplemented with pantothenate) in a suitable vessel, which can include, but is not limited to, a cell culture plate, flask, or fermentor. Furthermore, these processes can be performed on any fermentation scale known in the art to support the industrial production of microbial products. Any suitable fermentor may be employed including stirred tank fermentors, airlift fermentors, bubble fermentors, or any combination thereof. In a particular embodiment using Saccharomyces cerevisiae as host cell, the strain may be grown in a fermenter, as described in detail by Kosaric et al in Ullmann's Encyclopedia of Industrial Chemistry, sixth edition, volume 12, pages 398, 473, Wiley-VCH Press, Inc. (Wiley-VCH Verlag GmbH & Co. KDaA), Germany, and Haemam.

In some embodiments, the culture medium is any medium capable of feeding (i.e., maintaining growth and viability) a genetically modified microorganism that produces a heterologous product therein. In some embodiments, the medium is an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources. Such media may also include appropriate salts, minerals, metals, and other nutrients. In some embodiments, the carbon source and various essential cell nutrients are added to the fermentation medium in increments or continuously, and each required nutrient is maintained substantially at the minimum level required for efficient assimilation by the growing cells, e.g., according to a predetermined cell growth curve based on cellular metabolism or respiratory function for the conversion of the carbon source to biomass.

Suitable conditions and suitable media for culturing the microorganisms are well known in the art. In some embodiments, a suitable medium is also supplemented with one or more additional agents, e.g., an inducing agent (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressing agent (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selecting agent (e.g., for selecting a microorganism comprising a genetic modification).

In some embodiments, the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or a combination of one or more thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, collagen, cellulose, chitosan, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol.

The concentration of the carbon source (e.g.glucose) in the culture medium should be such that it promotes cell growth but not so high as to inhibit the growth of the microorganism used. Typically, the cultivation is carried out with a carbon source (e.g., glucose) at levels added to achieve the desired growth and biomass levels. Production of heterologous products can also occur under these culture conditions, but at undetectable levels (detection limit of about <0.1 g/l). In other embodiments, the concentration of the carbon source (e.g., glucose) in the medium is greater than about 1g/L, preferably greater than about 2g/L, and more preferably greater than about 5 g/L. Furthermore, the concentration of the carbon source (e.g., glucose) in the medium is generally less than about 100g/L, preferably less than about 50g/L, and more preferably less than about 20 g/L. It should be noted that the indicated concentrations of culture components may refer to the concentrations of the components in the initial and/or ongoing process. In some cases, it is desirable to allow carbon source depletion of the medium during culture.

Assimilable nitrogen sources that can be used in a suitable medium include, but are not limited to: simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, plant and/or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptones, yeast extract, ammonium sulfate, urea, and amino acids. The concentration of the nitrogen source in the medium is generally greater than about 0.1g/L, preferably greater than about 0.25g/L, and more preferably greater than about 1.0 g/L. However, the addition of nitrogen sources above a certain concentration to the medium is detrimental to the growth of the microorganism. Thus, the concentration of the nitrogen source in the medium is less than about 20g/L, preferably less than about 10g/L, and more preferably less than about 5 g/L. Furthermore, in some cases it is desirable to allow the medium to undergo nitrogen source depletion during the culture.

The effective medium may contain other compounds such as inorganic salts, vitamins, trace metals or growth promoters. Such other mixtures can also be present in the carbon, nitrogen or mineral sources in the effective medium, or added specifically to the medium.

The culture medium may also comprise a suitable source of phosphoric acid. Such phosphoric acid sources include inorganic phosphoric acid sources and organic phosphoric acid sources. Preferred sources of phosphoric acid include, but are not limited to, phosphates such as sodium and potassium dihydrogen phosphate, or disodium and dipotassium hydrogen phosphate, ammonium phosphate, and mixtures thereof. The concentration of the source of phosphoric acid in the medium is generally greater than about 0.1g/L, preferably greater than about 2.0g/L, and more preferably greater than about 5.0 g/L. However, phosphate added to the medium above a certain concentration is detrimental to the growth of the microorganisms. Thus, the phosphate concentration in the medium is generally less than about 20g/L, preferably less than about 15g/L, more preferably less than about 10 g/L.

Suitable media may also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources providing similar amounts of magnesium concentration may also be used. The magnesium concentration in the medium is generally greater than about 0.5g/L, preferably greater than about 1.0g/L, and more preferably greater than about 2.0 g/L. However, magnesium added to the medium above a certain concentration is detrimental to the growth of the microorganism. Thus, the magnesium concentration in the medium is generally less than about 10g/L, preferably less than about 5g/L, more preferably less than about 3 g/L. Furthermore, in some cases it is desirable to allow the medium to undergo magnesium source depletion during the culture.

In some embodiments, the medium may also include a biologically acceptable chelating agent, such as a dihydrate of trisodium citrate. In this case, the concentration of the chelating agent in the medium is greater than about 0.2g/L, preferably greater than about 0.5g/L, and more preferably greater than about 1 g/L. However, chelating agents added to the medium above a certain concentration are detrimental to the growth of the microorganisms. Thus, the concentration of the chelating agent in the medium is generally less than about 10g/L, preferably less than about 5g/L, more preferably less than about 2 g/L.

The medium may also initially include a biologically acceptable acid or base to maintain the desired pH of the medium. Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and mixtures thereof. Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.

The culture medium may also include a biologically acceptable source of calcium, including but not limited to calcium chloride. Typically, the concentration of the calcium source (e.g., calcium chloride dihydrate) in the medium is from about 5mg/L to about 2000mg/L, preferably from about 20mg/L to about 1000mg/L, and more preferably from about 50mg/L to about 500 mg/L.

The culture medium may also include sodium chloride. Typically, the concentration of sodium chloride in the medium is from about 0.1g/L to about 5g/L, preferably from about 1g/L to about 4g/L, more preferably from about 2g/L to about 4 g/L.

In some embodiments, the culture medium may further comprise trace metals. Such trace metals may be added to the medium as a stock solution, which may be prepared separately from the remainder of the medium for convenience. The amount of such trace metal solutions added to the medium is generally greater than about 1ml/L, preferably greater than about 5ml/L, more preferably greater than about 10 ml/L. However, trace metals added to the medium above a certain concentration are detrimental to microbial growth. Therefore, the amount of such trace metal solutions added to the medium is generally less than about 100ml/L, preferably less than about 50ml/L, more preferably less than about 30 ml/L. It should be noted that, in addition to the addition of the trace metal to the stock solution, the various components may be added separately, each within the component amount ranges specified independently for the above ranges of the trace metal solution.

The culture medium may include other vitamins such as pantothenate, biotin, calcium, pantothenate, inositol, pyridoxine hydrochloride, and thiamine hydrochloride. Such vitamins may be added to the medium as a stock solution, which may be prepared separately from the rest of the medium for convenience. However, the addition of vitamins to the medium above a certain concentration is detrimental to the growth of the microorganism.

The fermentation processes described herein may be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell-cycle, continuous, and semi-continuous. In some embodiments, the fermentation is performed in fed-batch mode. In this case, some components of the medium are depleted during the cultivation, including pantothenate in the production phase of the fermentation. In some embodiments, the culture can be supplemented with relatively high concentrations of such components, for example at the beginning of the production phase, in order to support growth and/or production for a period of time before addition is required. By adding these components at the level consumed by the culture, the preferable ranges of these components are maintained throughout the culture. The level of the components in the medium can be monitored by, for example, periodically sampling the medium and analyzing the concentration. Alternatively, once standard culture procedures have been developed, additions may be made at specific times throughout the culture process, at time intervals corresponding to known levels. As will be appreciated by those skilled in the art, as the cell density of the culture medium increases, the rate of consumption of nutrients during culture increases. In addition, to avoid the introduction of foreign microorganisms into the culture medium, the addition is performed using aseptic addition methods known in the art. In addition, a small amount of antifoaming agent may be added during the culture.

The temperature of the medium can be any temperature suitable to genetically modify the growth of the cells and/or produce the compound of interest. For example, the medium may be subjected to and maintained at a temperature in the range of about 20 to about 45 ℃ prior to inoculating the medium with the inoculum, preferably at a temperature in the range of about 25 to about 40 ℃, more preferably in the range of about 28 to 32 ℃.

The pH of the medium can be controlled by adding an acid or base to the medium. In this case, when ammonia is used for pH control, it can also be conveniently used as a nitrogen source in the medium. Preferably, the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.

In some embodiments, the concentration of a carbon source, such as glucose, in the culture medium is monitored during the culturing. The glucose concentration of the culture medium can be monitored using known techniques, for example, using a glucose oxidase assay or high pressure liquid chromatography, which can be used to monitor the glucose concentration in the supernatant (e.g., the cell-free component of the culture medium). As previously mentioned, the carbon source concentration should be kept below the level at which inhibition of cell growth occurs. Although such a concentration may vary from organism to organism, with respect to glucose as a carbon source, when the glucose concentration is more than about 60g/L, cell growth is inhibited and can be easily determined by an experiment. Therefore, when glucose is used as the carbon source, it is preferable to supply glucose to the fermentor and keep it below the detection limit. Alternatively, the glucose concentration in the medium is maintained from about 1g/L to about 100g/L, more preferably from about 2g/L to about 50g/L, and more preferably from about 5g/L to about 20 g/L. Although the carbon source concentration can be maintained within a desired level by, for example, adding a substantially pure glucose solution, it is acceptable and may be preferred to maintain the carbon source concentration of the medium by adding an aliquot of the original medium. The use of equal portions of the original medium is advantageous because it also allows the concentration of other nutrients (e.g., nitrogen and phosphorus sources) in the medium to be maintained simultaneously. Likewise, by adding small aliquots of trace metal solution, trace metal concentrations can be maintained in the medium.

Examples

Example 1: host cells engineered with the cannabinoid synthetic pathway

Yeast are engineered to express a portion of the cannabinoid synthetic pathway. As shown in FIG. 4, hexanoyl-CoA synthase (HCS), tetrone synthase (TKS) and olivate cyclase (OAC) initiate olivate synthesis with hexanoate as substrate. HCS uses hexanoate as a substrate to form hexanoyl-CoA, which then serves as a substrate for TKS to form malonyl-CoA, which further serves as a substrate for OAC to form olivinic acid. Each of the sequences encoding HCS, TKS and OAC is inserted into a s.cerevisiae cell, each under the control of a GAL promoter. Therefore, only when yeast is grown in the presence of galactose, the synthesis of each of these enzymes is induced. As shown in table 4, fig. 2 and fig. 3, several constructs (and resulting yeast strains) were prepared, some of which expressed only a subset of HCS, TKS and OAC, while others and yeast strains contained at least one copy each of HCS, TKS and OAC under the control of the GAL promoter. Table 4, fig. 2 and fig. 3 help to understand what strains the data shown in fig. 1 are tested for.

In the case of the cannabinoid pathway, hexanoate salts can be dosed to provide the hexanoyl-coa substrate required for the production of the cannabinoid polyketide precursor (see fig. 4). Wild-type yeast produce very low levels of hexanoate and therefore the yield of cannabinoids is greatly reduced if not dosed. Figure 1 shows the levels of the cannabinoid precursors, olivine and olivine acid, produced by various yeast strains engineered to express pathway genes (HCS, TKS and OAC) in switchable fashion and grown under three conditions. In the first two cases, no hexanoate was added to the strain and the carbon source was glucose (gluc; closed pathway expression) or galactose (gal; open pathway expression). In the third case (rightmost), galactose was used as the carbon source, which activated the pathway genes and hexanoate was given to the yeast. It can be seen that when galactose is the carbon source and caproate is given to the yeast, a large amount of cannabinoid precursors is produced. On the other hand, when glucose was the carbon source, thus shutting down the expression of the cannabinoid pathway, and no hexanoate was administered, cannabinoid production was below the detection limit of the test (<0.001 mg/L). This example shows the use of two orthogonal switching systems (galactose-induced pathway expression and addition of hexanoate) to ensure complete shut-down of production of olivil and olivil acid. Similar orthogonal switching systems that combine the necessary exogenous supply pathway precursors with a gene switch (e.g., inducible or repressible promoters) can be used to control other heterologous pathways introduced into the yeast.

Example 2: yeast transformation method

In an optimized lithium acetate (LiAc) transformation, each DNA construct was integrated into Saccharomyces cerevisiae (CEN. PK113-7D) using standard molecular biology techniques. Briefly, cells were shaken (200rpm) overnight in yeast extract peptone dextran (YPD) medium at 30 ℃ and diluted to OD in 100mL YPD₆₀₀Is 0.1, and is grown to OD₆₀₀Is 0.6-0.8. For each transformation, 5mL of culture was collected by centrifugation, washed in 5mL of sterile water, spun down again, resuspended in 1mL of 100mM LiAc, and transferred to a microcentrifuge tube. Cells were spun down (13000 × g) for 30 seconds, the supernatant removed, and cells resuspended in a transformation mixture consisting of: mu.L of 50% PEG, 36. mu.L of 1M LiAc, 10. mu.L of boiled salmon sperm DNA and 74. mu.L of donor DNA. For transformations requiring expression of the endonuclease F-Cph1, the donor DNA includes a plasmid carrying the F-CphI gene expressed under the yeast TDH3 promoter. This will cleave the F-CphI endonuclease recognition site in the landing plateau to facilitate integration of the target gene of interest. After heat shock at 42 ℃ for 40 min, cells were recovered overnight in YPD medium and then plated on selective medium. DNA integration was confirmed by colony PCR with primers specific for integration.

Example 2: production of a base strain comprising a gene switch system suitable for rapid genetic engineering to produce non-catabolic compounds.

In order to produce a strain that can be rapidly engineered to produce any natural compound, several engineering steps were performed on the original yeast isolate cen. pk113-7D. First, meganuclease proteins are integrated into the chromosome in order to achieve nuclease-based engineering in subsequent rounds of transformation. Second, seven chromosomal sites were engineered to obtain nucleotide sequences that enable efficient integration of future DNA constructs using validated nucleases. Third, a maltose responsive gene switch was added to control gene expression driven by GAL promoter (pGALx). The obtained strain Y46850 was used as a base plate into which a design for biosynthesis of a natural compound was rapidly prototyped.

The invention and use of maltose responsive gene switches was previously described in WO 2016210350; US 201615738555; and US201615738918, each incorporated herein by reference in its entirety. Briefly, the gene switch enables the heterologous, non-catabolic pathway to switch between on and off states depending on maltose and temperature (fig. 5). When the strain is grown in the presence of maltose and at temperatures ≦ 28 ℃, the expression of all pGALx driver genes will be turned off, allowing cellular resources to be turned on for biomass production, i.e. growth. In contrast, when the strain was grown at temperatures ≧ 30 ℃ without maltose, the expression of all pGALx driver genes would be turned on, enabling high-yield conversion of the administered sucrose to non-catabolic products.

The maltose switch is a GAL 80-based switch in which a maltose-responsive promoter drives expression of GAL80 (pMALx > GAL 80). One challenge with the GAL 80-based switch is that mutations that reactivate GAL80p activity during fermentation will shut down biosynthetic production, an event favored by natural selection. Two main approaches were developed to reduce GAL80 reactivation. The first method is as follows: when maltose is depleted and the temperature is ≥ 30 ℃, UBR 1-targeted degradation determinant (degron, D) is fused with temperature-sensitive GAL80(GAL80ts1) to accelerate degradation of GAL80 protein. The second method is as follows: the GAL80 protein was further destabilized by fusing a Maltose Binding Protein (MBP) based degron to the C-terminus of the GAL80 protein. The GAL80p-MBP mutant fusion protein is stable when maltose is present; however, when maltose is depleted, GAL80 protein is rapidly degraded. Another benefit of using MBP mutants is that strains with D _ GAL80ts1_ MBP exhibit significantly lower "leakage" of GAL gene expression during growth under off-state conditions.

Example 3: production of strains capable of producing cannabigerolic acid (CBGA)

A set of genes capable of producing cannabinoid CBGA was introduced into strain Y46850 (table 5 and fig. 6) by three steps. In the first step, constructs were integrated into the chromosomal locus to express three heterologous genes AAE, TKS and OAC from cultivated marijuana (Cannibis sativa), as well as the Zymomonas mobilis (Zymomonas mobilis) PDC gene and the two endogenous saccharomyces cerevisiae ACS1 and ALD6 genes, all using the pGALx promoter. Second, the constructs were integrated into the chromosomal locus to express seven endogenous genes of the s.cerevisiae mevalonate pathway (ERG10, ERG13, catalytic domain of HMG1, ERG12, ERG8, MVD1, and IDI 1). Third, the construct is integrated into the chromosomal locus to express the Streptomyces acutus (GPPS) and cultivated hemp CBGa synthase (CBGAS) genes. The CBGAS gene requires extensive N-terminal engineering to express it in a catalytically active form and not inhibit yeast growth. This engineering description has been made separately (pending patent application for the engineering of DPL1-PT4 and the occurrence of TM78-hop chimerism). The resulting strain Y61508 was able to produce CBGA when given a mixture of sucrose and caproic acid, as described in the yeast culture conditions section below.

Notably, the genes involved in hexanoic acid production have not been engineered into this strain. The endogenous yeast metabolism produces negligible amounts of hexanoic acid or hexanoyl-CoA, which means that these strains rely on the exogenous supply of hexanoic acid to produce cannabinoids (fig. 6).

Table 5:

example 4: production of strains capable of producing cannabidiolic acid (CBDA)

Cannabidiolic acid synthase (CBDAS) is an oxidative cyclase that generates carbon-carbon bonds that fold the geranyl portion of CBGA into a six-membered ring. CBDAS belongs to the Berberine-Bridge Enzyme (Berberine-Bridge Enzyme) family, utilizes molecular oxygen with a double covalent bond in the active site to flavin mononucleotide, and also produces one molecule of hydrogen peroxide (H) per reaction cycle₂O₂). CBDAS in cannabis has disulfide bonds, is glycosylated, and is naturally secreted into the apoplastic space of the trichome, which is thought to be involved by H₂O₂Generated to prevent self-toxicity. Another challenge for functional expression of CBDAS in yeast is its narrow pH range ≈ 4.5-5.

Yeast surface display is a classical molecular biology technique in which the protein of interest is located on the outer surface of the yeast cell, allowing the protein to interact directly with the culture medium. The surface display meets the requirements for CBDAS activity because the surface proteins are glycosylated (from golgi) and the pH of the fermentation medium is low. Surface display is preferred over secretion because pumping proteins into liquid media may cause foaming problems. To design a protein construct for CBDAS surface display, we selected yeast cell wall mannoprotein (manoprotein) CWP2 to provide a signal sequence and SAG1 as the carrier protein (fig. 7 and table 5). This construct was integrated into the near isogenic sibling of strain Y61508 to produce strain Y66085.

Example 5: conditions for Yeast culture

For conventional strain characterization in 96-well plate format, yeast colonies were picked into 96-well "pre-culture plates" of 1.1-mL/well capacity, each well filled with 360 μ L of pre-culture medium. The composition of the preculture medium was as follows: bird Seed medium (BSM, originally described by van Hoek et al (2000)) having a pH of 5.05, Biotechnology and Bioengineering, Vol.68, p.517-523, contained 14g/L sucrose, 7g/L maltose, 3.75g/L ammonium sulfate and 1g/L lysine. Cells were incubated in a high volume microtiter plate incubator at 28 ℃ for 3 days with shaking at 1000rpm and 80% humidity until the culture reached carbon depletion.

The grown saturated culture was subcultured by extracting 14.4. mu.L from the saturated culture and diluting to 96 well "production plates" with a volume of 2.2-mL per well, and adding 360. mu.L of production medium per well. The production medium consisted of BSM (pH 5.05) containing 40g/L sucrose, 3.75g/L ammonium sulfate and 2mM hexanoic acid, cells in the production medium were cultured for 3 days at 30 ℃, 1000rpm and 80% humidity in a high volume microtiter plate shaker prior to extraction and analysis.

Example 6: analytical methods for cannabinoid extraction and titer determination

At the end of the plate incubation, methanol was added to each well to a final concentration of 67% (v/v) methanol. An impermeable seal was added and the plate was shaken at 1000rpm for 30 seconds to lyse the cells and extract the cannabinoids. The plate was centrifuged at 200 Xg for 30 seconds to form pellet cell debris. The 300. mu.L of clarified sample was transferred to a 96-well empty plate with a capacity of 1.1mL and sealed with a foil seal. The sample plates were stored at-20 ℃ until analysis.

Cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA) were separated using a Thermo Vanqish series UPLC-UV system with an Accupore Polar Premium 2.6 μm C18 column (100X 2.1 mm). The mobile phase was a gradient mobile phase of 5mM ammonium formate containing 0.1% aqueous formic acid and 0.1% formic acid in acetonitrile at a flow rate of 1.2 ml/min. Calibration curves were prepared gravimetrically in the extraction solvent using pure standards.

Example 7: validation of two orthogonal switching systems

For some biomolecules, such as cannabinoids, regulatory requirements have created a need for very low or undetectable production in low production states during the growth phase required for strain propagation. To this end, a genetically encoded maltose-responsive switch was combined with the dependence of cannabinoid biosynthesis on the exogenous supply of hexanoic acid.

When strain Y61508 was grown in the absence of maltose but in the presence of hexanoic acid, the highest CBGA titers were observed, with the lowest biomass accumulation (fig. 8), consistent with entry of cellular resources into this non-catabolic pathway. When sucrose was replaced with maltose, CBGA titer decreased and biomass accumulation increased. When exogenous hexanoic acid was no longer supplied, CBGA titer also decreased and biomass accumulation increased. Very similar results were observed for the different CBGA-producing strain Y66316 (FIG. 9).

Importantly, the highest biomass accumulation and lowest CBGA titer were observed when these strains were grown in the presence of 4% maltose without exogenous supply of hexanoic acid. Under these conditions, cannabinoid production was below the detection limit of the assay (<0.001 mg/L). This example shows the use of two orthogonal switching systems to ensure complete shut down of cannabinoid production and direct cellular resources rather than biomass accumulation, i.e., growth.

To extend this finding, we tested CBDA producing strain Y66085 under the same conditions. Also, the lack of maltose and exogenous supply of hexanoic acid completely transformed the cells to cannabinoid production at the expense of growth (fig. 10). By replacing sucrose with maltose or removing exogenously supplied caproic acid, CBDA titers are reduced and biomass is increased. This example shows that the use of two orthogonal switching systems extends to a number of strains engineered to produce different cannabinoids.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications (including GenBank accession numbers), patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.