Hydrolysis of steviol glycosides by β -glucosidase
阅读说明：本技术 通过β-葡糖苷酶水解甜菊醇糖苷 (Hydrolysis of steviol glycosides by β -glucosidase ) 是由 毛国红 J.E.维克 M.贝滕 余晓丹 于 2018-06-29 设计创作，主要内容包括：本披露提供了利用β-葡糖苷酶的水解活性从多种甜菊醇糖苷产生提高量的莱鲍迪苷M(Reb M)的方法。更特别地,本披露提供了从含有目的底物的破碎的重组细胞(例如,重组微生物细胞)产生所希望的甜菊醇糖苷的改善的方法。在该方法中,此类细胞(例如,微生物)已经被修饰以携带能够产生目的甜菊醇糖苷的基因,这些目的甜菊醇糖苷包括莱鲍迪苷A(Reb A)、莱鲍迪苷E(Reb E)、和/或甜菊苷。在该实施例中,该方法包括以下步骤：从破碎的细胞(例如,微生物细胞)获得材料,并且在废产物中存在的甜菊苷上进行酶促水解以增强Reb M的产生。另外提供了通过本文所述的方法产生的甜菊醇糖苷组合物。(The present disclosure provides methods of producing enhanced amounts of rebaudioside M (Reb M) from a variety of steviol glycosides using the hydrolytic activity of β -glucosidase more particularly, the present disclosure provides improved methods of producing desired steviol glycosides from disrupted recombinant cells (e.g., recombinant microbial cells) containing a substrate of interest in which such cells (e.g., microorganisms) have been modified to carry genes capable of producing the steviol glycosides of interest, including rebaudioside A (Reb A), rebaudioside E (Reb E), and/or stevioside.)
1. A method of altering glycosylation of a steviol glycoside, the method comprising:
a) providing a recombinant microorganism, algae, or plant cell, which is modified to produce a first substrate;
b) disrupting the recombinant microorganism, algae or plant cell to release its cellular cytosol, wherein such cytosol contains the first substrate;
c) obtaining cytosol from the recombinant microorganism, algae or plant cell;
d) exposing the cytosol to β -glucosidase wherein the β -glucosidase has hydrolytic activity for a time sufficient to produce a second desired substrate by removing at least one glucosyl group from the first substrate, and
e) collecting the second target substrate.
2. The method of claim 1, wherein the first substrate is a steviol glycoside.
3. The method of claim 2, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 position of the steviol glycoside.
4. The method of claim 2, wherein the hydrolytic activity acts to remove glucosyl groups from the C13 position of the steviol glycoside.
5. The method of claim 2, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 position of the steviol glycoside or the C13 position of the steviol glycoside.
6. The method of claim 2, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 position of the steviol glycoside and the C13 position of the substrate.
7. The method of claim 3, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 position of rubusoside to produce steviol-13-glucoside.
8. The method of claim 3, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 position of stevioside to produce a steviol bioside.
9. The method of claim 3, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 position of Reb E to produce steviol biosides.
10. The method of claim 3, wherein the hydrolytic activity acts to remove a glucosyl group from the C19 position of Reb I to produce Reb A.
11. The method of claim 3, wherein the hydrolytic activity acts to remove a glucosyl group from the C19 position of Reb A to produce Reb B.
12. The method of claim 4, wherein the hydrolytic activity acts to remove glucosyl groups from the C13 position of steviol-13-glucoside to produce steviol.
13. The method of claim 4, wherein the hydrolytic activity acts to remove a glucosyl group from the C13 position of Reb D to produce Reb B or Reb a.
14. The method of claim 5, wherein the hydrolytic activity acts to remove glucosyl groups from the C19 and C13 positions of Reb G to produce steviol-13-glucoside.
15. The method of claim 1, wherein the second substrate of interest is Reb M.
16. The method of claim 1, wherein the second substrate of interest is Reb B.
17. The method of claim 1, wherein the second substrate of interest is Reb a.
18. The method of any one of claims 1 to 17, further comprising using β -galactosidase or pectinase to increase the speed of enzymatic hydrolysis.
19. The method of any one of claims 1 to 18, further comprising:
expressing a steviol glycoside in the transformed cell system;
culturing the cell system in a culture medium; and
producing said second target substrate.
20. The method of any one of claims 1 to 19, wherein the period of time during which β -glucosidase is exposed to the first substrate is at least 6 hours.
21. The method of any one of claims 1 to 19, wherein the period of time during which β -glucosidase is exposed to the first substrate is at least 12 hours.
22. The method of any one of claims 1 to 19, wherein the period of time during which β -glucosidase is exposed to the first substrate is at least 18 hours.
23. The method of any one of claims 1 to 19, wherein the period of time during which β -glucosidase is exposed to the first substrate is at least 24 hours.
24. The method of claim 1, wherein the second substrate of interest is a steviol glycoside.
25. The method of any one of claims 1 to 24, wherein the β -glucosidase has an amino acid sequence having at least 90% identity to SEQ ID No. 1.
26. The method of any one of claims 1 to 24, wherein the β -glucosidase has an amino acid sequence having at least 95% identity to SEQ ID No. 3.
27. The method of any one of claims 1 to 26, wherein the recombinant microorganism, algae, or plant cell is selected from the group consisting of: bacteria, yeast, filamentous fungi, cyanobacterial algae, and plant cells.
28. The method of any one of claims 1 to 26, wherein the recombinant microorganism is selected from the group consisting of: the genus Escherichia; salmonella; bacillus; acinetobacter; streptomyces; corynebacterium genus; campylobacter methylotrophicus; methylomonas sp; rhodococcus genus; pseudomonas sp; (ii) the genus rhodobacter; synechocystis; a genus Saccharomyces; zygosaccharomyces; kluyveromyces; candida genus; hansenula; debaryomyces; mucor genus; a Pichia genus; torulopsis; (ii) Aspergillus; arthrobotlys; the genus Brevibacterium; microbacterium species; arthrobacter; citrobacter sp; the genus Escherichia; klebsiella sp; pantoea; salmonella corynebacterium; clostridium species; and clostridium acetobutylicum.
29. The method of claim 1, wherein the second substrate of interest is Reb E.
30. The method of claim 1, wherein the second substrate is a mixture of Reb E, Reb D4, and Reb M.
31. The method of claim 1, wherein the second substrate of interest is Reb D4.
32. The method of claim 1, wherein the second substrate of interest is a steviol glycoside mixture and the mixture has a higher steviol glycoside content than any other component of the cytosol derived concentrate by weight on a dry weight basis.
33. The method of any one of claims 1 to 32, the second substrate of interest in step e) being a crude product, and step e) further comprising: i) purifying the crude product; and ii) removing the solvent under vacuum to provide a concentrated product.
34. The method of claim 33, wherein the crude product is purified by column chromatography.
35. The method of claim 33, wherein the crude product is purified by acid-base extraction.
36. The process of claim 33, wherein the crude product is purified by vacuum distillation.
37. The method of claim 33, further comprising purifying the concentrated product using semi-preparative HPLC.
Technical Field
Background
In particular, the disclosure relates in part to more efficient production of rebaudioside M ("Reb M") by hydrolyzing specific substrates present in disrupted recombinant cells (e.g., recombinant microbial cells) using β -glucosidase ("B-
Steviol glycosides are natural products isolated from the leaves of Stevia rebaudiana (Stevia rebaudiana) and are widely used as high-intensity, low-calorie sweeteners in foods, feeds and beverages. Naturally occurring steviol glycosides have the same basic diterpene backbone structure (steviol), but differ in the number and structure of carbohydrate residue modifications (e.g., glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the steviol backbone. Steviol glycosides of known structure include stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, and dulcoside a. Rebaudioside M itself has generally been considered safe ('GRAS' state) in terms of commercial utility, but is extremely difficult to obtain from the extraction process and requires significant microbial modification to be produced by microbial bioconversion alone.
Stevioside, rebaudioside a, rebaudioside C, and dulcoside a account for 9. l%, 3.8%, 0.6%, and 0.30%, respectively, of the total weight of steviol glycosides in wild-type stevia rebaudiana leaves on a dry weight basis, while other steviol glycosides (e.g., Reb M) are present in much lower amounts. It is known to use recombinant microbial cells to produce a steviol glycoside of interest, however, in view of the range of biochemical activities of specific enzymes, it is conceivable to produce various steviol glycosides from such microbial strains. In these cases, the disrupted cells of these strains may contain large amounts of stevioside (CAS number 57817-89-7), rebaudioside A (CAS number 58543-16-1), or other steviol glycosides, with Reb M being the desired product. The amounts of these compounds range from about 10% -20% for stevioside and from about 5% -10% for rebaudioside a, with other minor ingredients present in the mixture.
As natural sweeteners, different steviol glycosides have varying degrees of sweetness, "mouthfeel", and specific aftertastes associated with each rebaudioside class tested. The steviol glycosides are significantly more sweet than sugar (i.e., "sucrose"). For example, stevioside is 100-fold more sweet than sucrose, but has a bitter aftertaste as indicated in the taste test, whereas rebaudiosides A and E are 450-fold more sweet than sucrose, and the aftertaste is much better than stevioside, but there is still a noticeable aftertaste. Thus, the taste characteristics of any stevia extract are heavily influenced by the relative amount of steviol glycosides in the extract, which in turn may be influenced by the environmental conditions experienced by the subterranean plants and the extraction method used. These variations in plant yield, weather conditions, and extraction conditions can lead to inconsistent composition of steviol glycosides in sweet chrysanthemum extracts, such that the taste characteristics vary widely between different batches of extracted products. The taste characteristics of stevia extracts can also be affected by plant-derived or environmental-derived contaminants (such as pigments, lipids, proteins, phenols and carbohydrates) remaining in the product after the extraction process. These contaminants typically have an off-taste of their own, which is undesirable for using stevia extracts as sweeteners in consumer products.
Furthermore, the cost of isolating individual or specific combinations of steviol glycosides that are not abundant in stevia extracts is costly and resource intensive. Abiotic production of steviol glycosides from stevioside and Reb a is difficult. Acid hydrolysis of stevioside is cumbersome because under acidic conditions, the produced steviol is rearranged to isosteviol. Given the limited quality and availability of some specific steviol glycosides, commercial supply can be better addressed by bioconversion, where natural enzymes or specific microorganisms can be modified to carry the desired enzymes and use commercially important fermentation processes to specifically increase the production of the glycoside of interest. For example, the bioconversion of stevioside to Reb E by a fermentation pathway using modified microorganisms has been previously reported (see, Mao et al, U.S. patent application publication No. US 2016/0207954, Non-Caloric Sweetener [ Non-Caloric sweeteners ]). Alternatively, other non-biosynthetic means may be used to develop the steviol glycoside of interest.
From a biological perspective, all steviol glycosides are formed by a series of glycosylation reactions of steviol, typically catalyzed by UDP-glycosyltransferase (UGT) enzyme, using uridine 5' -diphosphate glucose (UDP-glucose) as the donor of the sugar moiety. In plants, UGT is a very diverse group of enzymes that transfer glucose residues from UDP-glucose to steviol. In these reactions, stevioside is typically an intermediate in the biosynthesis of various rebaudioside compounds. For example, glycosylation of stevioside at C-3 'of stevioside's C-13-O-glucose produces rebaudioside A; while glycosylation at the C-2' of the 19-0-glucose position of stevioside produces rebaudioside E.
In accordance with the present disclosure, a practical method of improving the taste quality of stevia extracts is to improve the yield of rebaudioside compounds that generally have more desirable taste characteristics, and to achieve this by more efficient synthetic routes and related methods. Among those steviol glycosides tested, Reb M is considered by many to have the most desirable taste and chemical characteristics for use in foods and beverages. However, as mentioned above, the plant contains very small amounts of this compound in its leaves, and therefore alternative methods are needed to achieve and assist in the large scale production of the glycoside and to provide alternative sweeteners for the food and beverage industry.
Thus, there is a need for steviol glycosides to be developed as commercial products with better and more consistent taste profiles and for such steviol glycosides to utilize relatively common starting substrates (e.g., more abundant steviol glycosides) as starting molecules, so that producing the desired glycoside can be as cost-effective as commercially possible. The present disclosure provides methods of enhancing the production of desirable steviol glycosides.
There is also a need for new production methods to reduce the cost of steviol glycoside production and to reduce the environmental impact of large scale cultivation and processing (Yao et al, 1994). One such potential solution is the use of fermentative bioconversion technology, which allows production in certain microbial species or other recombinant cells, which increases the selectivity, abundance, and purity of the desired steviol glycosides that are commercially available; and enhancing the presence of the desired rebaudioside with a hydrolase to drive such production in a cell lysate from a modified microorganism culture or other modified cell culture.
Disclosure of Invention
This disclosure encompasses, in part, methods of producing enhanced amounts of rebaudioside M (Reb M) from a variety of steviol glycosides using the hydrolytic activity of β -glucosidase more particularly, the disclosure provides improved methods of producing desired steviol glycosides from disrupted recombinant cells (e.g., recombinant microbial cells) containing a substrate of interest.
In terms of product/commercial utility, there are several tens of steviol glycoside-containing products on the U.S. market and all products ranging from analgesics to anthelmintics and in food products and dietary supplements. Products containing the steviol glycoside of interest may include aerosol, liquid, or particulate formulations.
As to the cell system of the present disclosure, it is selected from the group consisting of bacteria, yeast and combinations thereof, or any cell system that allows genetic transformation with selected genes and subsequent biosynthetic production of the desired steviol glycoside from steviol.
β -glucosidase is a constitutive enzyme that is normally present in the lower gastrointestinal tract of animals and can be used as an aid to the digestion and absorption of food materials according to the present disclosure, B-glu1 is used to hydrolyze stevioside, rebaudioside E (Reb E), rebaudioside A (Reb A), rebaudioside I (Reb I), rebaudioside D (Reb D), rebaudioside G (Reb G), and rubusoside hydrolysis is performed by the initial formation of steviol biosides with steviol as the final product of hydrolysis.
Accordingly, aspects of the disclosure provide methods of altering glycosylation of steviol glycosides, the methods comprising a) providing a recombinant cell (e.g., a microbial, algal, or plant cell) that is modified to produce a first substrate, b) disrupting the recombinant cell to release its cell cytosol, wherein such cytosol contains the first substrate, c) obtaining cytosol from the recombinant cell, d) exposing the cytosol to β -glucosidase, wherein such β -glucosidase has hydrolytic activity for a sufficient time to produce a second substrate of interest by removing at least one glucosyl group from the first substrate, and e) collecting the second substrate of interest.
In some embodiments, the first substrate is a steviol glycoside. In some embodiments, the second substrate of interest is Reb M. In some embodiments, the second substrate of interest is Reb B. In some embodiments, the second substrate of interest is Reb a. In some embodiments, the first and second substrates are those described in table 1 and figures herein (e.g., fig. 9).
In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of the steviol glycoside. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C13 position of the steviol glycoside. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of the steviol glycoside or the C13 position of the steviol glycoside. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of the steviol glycoside and the C13 position of the substrate.
In some embodiments, the hydrolysis activity acts to remove glucosyl groups from the C19 position of rubusoside to produce steviol-13-glucoside. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of stevioside to produce a steviol bioside. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of Reb E to produce steviol glycosides. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of Reb I to produce Reb a. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 position of Reb a to produce Reb B.
In some embodiments, the hydrolysis activity acts to remove glucosyl groups from the C13 position of steviol-13-glucoside to produce steviol. In some embodiments, the hydrolytic activity acts to remove a glucosyl group from the C13 position of Reb D to produce Reb B or Reb a. In some embodiments, the hydrolytic activity acts to remove glucosyl groups from the C19 and C13 positions of Reb G to produce steviol-13-glucoside.
In some aspects, the methods provided herein further comprise using β -galactosidase or pectinase to increase the rate of enzymatic hydrolysis.
In some embodiments, the period of time that β -glucosidase is exposed to the first substrate is at least 6 hours.
In some embodiments, the period of time during which β -glucosidase is exposed to the first substrate is at least 12 hours, in some embodiments, the period of time during which β -glucosidase is exposed to the first substrate is at least 18 hours, in some embodiments, the period of time during which β -glucosidase is exposed to the first substrate is at least 24 hours.
In some embodiments, β -glucosidase has an amino acid sequence that is at least 90% (e.g., 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%, or 100%) identical to SEQ ID NO. 1 in some embodiments, β -glucosidase has an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO. 3.
In some embodiments, the recombinant cell is selected from the group consisting of: bacteria, yeast, filamentous fungi, cyanobacterial algae, and plant cells. In some embodiments, the recombinant cell (e.g., microorganism) is selected from the group consisting of: escherichia (Escherichia); salmonella (Salmonella); bacillus (Bacillus); acinetobacter (Acinetobacter); streptomyces (Streptomyces); corynebacterium (Corynebacterium); campylobacter methylotrophus (Methylosinus); methylomonas (Methylomonas); rhodococcus (Rhodococcus); pseudomonas (Pseudomonas); rhodobacter (Rhodobacter); synechocystis (Synechocystis); saccharomyces (Saccharomyces); zygosaccharomyces (Zygosaccharomyces); kluyveromyces (Kluyveromyces); candida genus (Candida); hansenula (Hansenula); debaryomyces (Debaryomyces); mucor (Mucor); pichia (Pichia); torulopsis (Torulopsis); aspergillus (Aspergillus); arthrobotlys; brevibacterium (brevibacterium); microbacterium (Microbacterium); arthrobacter (Arthrobacter); citrobacter (Citrobacter); escherichia (Escherichia); klebsiella (Klebsiella); pantoea (Pantoea); corynebacterium salmonella (salmonella corynebacterium); clostridium (Clostridium); and clostridium acetobutylicum (clostridium acetobutylicum).
In some embodiments, the second substrate of interest is a steviol glycoside. In some embodiments, the second substrate of interest is Reb E. In some embodiments, the second substrate is a mixture of Reb E, Reb D4, and Reb M. In some embodiments, the second substrate of interest is Reb D4. In some embodiments, the second substrate of interest is a steviol glycoside mixture, and the mixture has a higher steviol glycoside content than any other component of the cytosol-derived concentrate by weight on a dry weight basis.
In some embodiments, the second substrate of interest in step e) is a crude product, and step e) further comprises i) purifying the crude product; and ii) removing the solvent under vacuum to provide a concentrated product.
In some embodiments, the crude product is purified by column chromatography. In some embodiments, the crude product is purified by acid-base extraction. In some embodiments, the crude product is purified by vacuum distillation.
In some embodiments, the methods provided herein further comprise purifying the concentrated product using semi-preparative HPLC.
While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiment disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.
Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments of the invention, which proceeds with reference to the accompanying drawings.
Drawings
FIG. 1 shows that rubusoside is hydrolyzed by the B-glu1 enzyme and disrupted Pichia cells. A: HPLC profile of rubusoside hydrolysate. a: a standard for steviol ("S"); b: standards of rubusoside ("Rub"); c-d: at 1 hour (c) and 3 hours (d), rubusoside was cleaved by the recombinant B-glu1 enzyme; e-f: at 1 hour (e) and 6 hours (f), rubusoside was hydrolyzed by disrupted pichia cells. B: the rubusoside hydrolysis pathway is catalyzed by B-
FIG. 2 shows that stevioside is enzymatically hydrolyzed by B-
FIG. 3 shows that rebaudioside E is enzymatically hydrolyzed by B-
FIG. 4 shows that rebaudioside A is enzymatically hydrolyzed by B-
FIG. 5 shows that rebaudioside I is enzymatically hydrolyzed by B-
FIG. 6 shows that rebaudioside D is enzymatically hydrolyzed by B-
FIG. 7 shows rebaudioside G was hydrolyzed by B-glu1 enzyme and disrupted Pichia cells. A: HPLC spectra of rebaudioside G hydrolysate. a: steviol ("Steviol"), a standard of Steviol-13-glucoside ("S-13-G"); b: a standard of rebaudioside G ("G"); b-d: at 0.5 hours (c), 1 hour (d), and 6 hours (e), rebaudioside G was enzymatically hydrolyzed by recombinant B-
FIG. 8 shows the hydrolysis of steviol glycosides by disrupted Pichia cells. a: a standard of steviol glycosides ("SB"); b: a standard of rebaudioside B ("B"); c: at 24 hours, stevioside was hydrolyzed by disrupted pichia cells; d: at 24 hours, rebaudioside E ("E") was hydrolyzed by disrupted pichia cells; e: at 24 hours, rebaudioside a ("a") was hydrolyzed by disrupted pichia cells; f: at 24 hours, rebaudioside I ("I") was hydrolyzed by disrupted pichia cells; g: at 24 hours, rebaudioside D ("D") was hydrolyzed by disrupted pichia cells.
FIG. 9 shows that B-Glu1 can hydrolyze different steviol glycoside substrates. Black lines: glycosylation; dotted line: and (4) hydrolyzing.
Fig. 10 shows the synthetic pathway for the production of Reb M and
Detailed Description
Interpretation of terms used herein：
Steviol glycosides are a class of chemical compounds that are responsible for the sweetness of the leaves of the stevia rebaudiana (Compositae) plant and are useful as sweeteners in foods, feeds and beverages.
Defining:
cell systemIs any cell that provides for ectopic protein expression. It includes bacteria, yeast, plant cells and animal cells. It includes prokaryotic cells and eukaryotic cells. It also includes in vitro expression of proteins based on cellular components such as ribosomes.
Coding sequenceIt is given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to a DNA sequence encoding a particular amino acid sequence.
Culturing the cell system.Culturing includes providing an appropriate medium that allows the cells to multiply and divide. It also includes providing resources to allow translation and production of recombinant proteins by the cells or cell components.
And (4) protein expression.Protein production may occur after gene expression. It consists of the stage after transcription of DNA into messenger rna (mrna). The mRNA is then translated into polypeptide chains, which ultimately fold into proteins. DNA is present in cells by transfection, a deliberate attempt to introduceThe process of introducing nucleic acid into a cell. The term is commonly used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, but other terms are preferred: "transformation" is more commonly used to describe non-viral DNA transfer in bacterial, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term, as transformation is also used to refer to the development of a cancerous state (carcinogenesis) in these cells. Transduction is commonly used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection in the present application.
A yeast.According to the present disclosure, the yeast claimed herein are eukaryotic, unicellular microorganisms classified as members of the kingdom fungi. Yeast is a unicellular organism evolved from a multicellular ancestor, but species are available for the present disclosure, those capable of developing multicellular characteristics by forming strings of linked germ cells called pseudohyphae or pseudohyphae.
The names of The UGT enzymes used in this disclosure for The production of various steviol glycosides are consistent with The nomenclature system adopted by The UGT nomenclature Committee (Mackenzie et al, "The UDP glycosylation transfer enzyme gene super family: recommended nomenclature based on evolutionary divergence update" pharmaceuticals [ genetics ],1997, Vol.7, p.255-269) which classifies UGT genes by a combination of family number, letter representing The subfamily, and number representing The individual gene. For example, the designation "UGT 76 Gl" refers to a UGT enzyme encoded by a gene belonging to UGT family number 76 (which is the origin of a plant), subfamily G, and gene number l.
Structural terms:
as used herein, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.
To the extent that the terms "includes," "including," "has," "having," or the like are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising" as "comprising" is interpreted when employed as a transitional word in a claim.
The term "exemplary" is used herein to mean "serving as an example, instance, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
The term "complementary" is given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to describe the relationship between nucleotide bases capable of hybridizing to one another. For example, in the case of DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the subject technology also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying sequence listing, as well as those substantially similar nucleic acid sequences.
The terms "nucleic acid" and "nucleotide" are given their respective ordinary and customary meanings to those of ordinary skill in the art, and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
The term "isolated" is given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that exists by the human hand free from its original environment and is therefore not a natural product. An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment, such as a transgenic host cell.
As used herein, the term "incubating" means a method of mixing and contacting two or more chemical or biological entities (e.g., chemical compounds and enzymes) with one another under conditions conducive to the production of a steviol glycoside composition.
The term "degenerate variant" refers to a nucleic acid sequence having a sequence of residues that differs from a reference nucleic acid sequence by substitution of one or more degenerate codons. Degenerate codon substitutions may be effected by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues. The nucleic acid sequence and all degenerate variants thereof will express the same amino acid or polypeptide.
The terms "polypeptide", "protein" and "peptide" are given their respective ordinary and customary meanings to one of ordinary skill in the art; these three terms are sometimes used interchangeably and are used without limitation to refer to polymers of amino acids or amino acid analogs, regardless of their size or function. Although "protein" is generally used to refer to relatively large polypeptides, and "polypeptide" is generally used to refer to small polypeptides, the usage of these terms in the art overlaps and varies. As used herein, the term "polypeptide" refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to a polynucleotide product. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins and homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing.
The terms "polypeptide fragment" and "fragment" when used in reference to a reference polypeptide are given their ordinary and customary meaning to those of ordinary skill in the art, and are used without limitation to refer to polypeptides in which amino acid residues are deleted compared to the reference polypeptide itself, with the remaining amino acid sequence generally being identical to the corresponding position on the reference polypeptide. Such deletions may occur at the amino-terminus or the carboxy-terminus of the reference polypeptide, or alternatively at both.
The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of a full-length polypeptide or protein and has substantially the same biological activity, or performs substantially the same function (e.g., performs the same enzymatic reaction), as the full-length polypeptide or protein.
The terms "variant polypeptide", "modified amino acid sequence" or "modified polypeptide" used interchangeably refer to an amino acid sequence that differs from a reference polypeptide by one or more amino acids, e.g., by one or more amino acid substitutions, deletions and/or additions. In one aspect, a variant is a "functional variant" that retains some or all of the ability of a reference polypeptide.
The term "functional variant" further includes variants that are conservatively substituted. The term "conservatively substituted variant" refers to a peptide that has an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions and that retains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include: mutual substitution between nonpolar (hydrophobic) residues such as isoleucine, valine, leucine or methionine; mutual substitution between charged or polar (hydrophilic) residues, such as arginine and lysine, glutamine and asparagine, threonine and serine; mutual substitution between basic residues such as lysine or arginine; or mutual substitution between acidic residues such as aspartic acid or glutamic acid; or mutual substitution between aromatic residues such as phenylalanine, tyrosine or tryptophan. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "conservatively substituted variant" also includes peptides in which a residue is replaced with a residue of chemical origin, provided that the resulting peptide retains some or all of the activity of the reference peptide as described herein.
The term "variant" in connection with a polypeptide of the subject technology further includes functionally active polypeptides having an amino acid sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, and even 100% identical to the amino acid sequence of a reference polypeptide.
The term "homologous" in all its grammatical forms and spelling changes refers to the relationship between polynucleotides or polypeptides having "common evolutionary origin", including polynucleotides or polypeptides from the superfamily as well as homologous polynucleotides or proteins from different species (Reeck et al, Cell [ Cell ]50:667,1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of particular amino acids or motifs at conserved positions. For example, two homologous polypeptides may have an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 900%, 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%, and even 100% identical.
"suitable regulatory sequences" have its ordinary and customary meaning to those skilled in the art, and are used without limitation to refer to nucleotide sequences located upstream (5 '-non-coding sequences), within or downstream (3' -non-coding sequences) of a coding sequence, and which affect transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
"promoter" is given its ordinary and customary meaning to those of ordinary skill in the art and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause the expression of genes in most cell types are most often referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences are not yet fully defined, DNA fragments of different lengths may have identical promoter activity.
The term "operably linked" refers to the association between nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
As used herein, "expression" is given its ordinary and customary meaning to those of ordinary skill in the art, and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragments of the subject technology. "overexpression" refers to the production of a gene product in a transgenic or recombinant organism above the level of production in a normal or non-transformed organism.
"transformation" is given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide may be incorporated into the genomic or chromosomal DNA of the target cell, resulting in genetically stable inheritance, or it may replicate independently of the host chromosome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or.
The terms "transformed", "transgenic" and "recombinant" when used herein in connection with a host cell, give their respective ordinary and customary meaning to a person of ordinary skill in the art, and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the genome of the host cell, or the nucleic acid molecule may exist as an extrachromosomal molecule. Such extrachromosomal molecules may be autonomously replicating. Transformed cells, tissues or subjects should be understood to encompass not only the end product of the transformation process, but also transgenic progeny thereof.
The terms "recombinant," "heterologous," and "exogenous" when used herein in connection with a polynucleotide give their ordinary and customary meaning to those of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or gene) that is foreign to a particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to that particular host cell, but has been modified, for example, by using site-directed mutagenesis or other recombinant techniques. These terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, these terms refer to DNA segments that are foreign or heterologous to the cell, or homologous to the cell but in a position or form in which the element is not normally found in the host cell.
Similarly, the terms "recombinant," "heterologous," and "exogenous" when used herein in connection with a polypeptide or amino acid sequence, refer to a polypeptide or amino acid sequence that originates from an external source, or, if from the same source, is modified in its original form, relative to the particular host cell. Thus, the recombinant DNA segment can be expressed in a host cell to produce a recombinant polypeptide.
The terms "plasmid", "vector" and "cassette" are given their respective ordinary and customary meanings to those skilled in the art and are used without limitation to refer to extra-chromosomal elements that normally carry genes that are not part of the central metabolism of the cell and are usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences of any origin, genome integrating sequences, phage or nucleotide sequences, linear or circular single-or double-stranded DNA or RNA, in which a number of nucleotide sequences have been joined or recombined into a unique configuration which is capable of introducing into a cell a promoter fragment of a selected gene product and a DNA sequence, together with appropriate 3' untranslated sequence.
"transformation cassette" refers to a specific vector that contains a foreign gene and has elements in addition to the foreign gene that facilitate transformation of a particular host cell.
"expression cassette" refers to a specific vector that contains a foreign gene and has elements other than the foreign gene that allow for enhanced expression of the gene in a foreign host.
The disclosure relates to the enzymatic synthesis of steviol glycosides of interest by B-
Synthetic biology
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in: for example, Sambrook, J., Fritsch, E.F. and Maniatis, T.molecular Cloning: A Laboratory Manual [ molecular Cloning: laboratory manual ],2 nd edition; cold Spring Harbor Laboratory Cold Spring Harbor [ Cold Spring Harbor Laboratory: Cold Spring Harbor ], N.Y. [ New York ],1989 (hereinafter "Maniatis"); and Silhavy, t.j., Bennan, m.l. and Enquist, l.w. experiments with Gene Fusions [ Gene fusion experiments ]; cold Spring Harbor Laboratory Cold Spring Harbor [ Cold Spring Harbor Laboratory: Cold Spring Harbor ], N.Y. [ New York ], 1984; and Ausubel, F.M. et al, published by Greene Publishing and Wiley-Interscience [ Green Press and Willi International science publishers ], 1987 in Current Protocols in Molecular Biology [ Current Protocols ]; (each of these documents is hereby incorporated by reference in its entirety).
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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred materials and methods are described below.
Glycosylation is generally considered to be a ubiquitous response that controls the biological activity and storage of plant natural products. In most plant species that have been studied to date, glycosylation of small molecules is catalyzed by the transferase superfamily. These Glycosyltransferases (GT) have been classified into more than 60 families. Among these, the family of GT enzymes (also known as UDP Glycosyltransferases (UGTs)) transfer UDP activated sugar moieties to specific acceptor molecules. These are molecules that transfer such sugar moieties in steviol glycosides to produce rebaudiosides. Each of these UGTs has their own spectrum of activity and preferred structural positions where they transfer their activated sugar moieties.
Production system
Expression of proteins in prokaryotes is most commonly accomplished in bacterial host cells with vectors containing constitutive or inducible promoters directing expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, typically to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: l) increasing the expression of the recombinant protein; 2) increasing the solubility of the recombinant protein; and 3) to aid in the purification of recombinant proteins by acting as a ligand in affinity purification. Typically, a protein cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to separate the recombinant protein from the fusion moiety after purification of the fusion protein. Such vectors are within the scope of the present disclosure.
In embodiments, the expression vector includes those genetic elements used to express the recombinant polypeptide in a bacterial cell. Elements for transcription and translation in bacterial cells may include a promoter, a coding region for a protein complex, and a transcription terminator.
One of ordinary skill in the art will be aware of molecular biology techniques that can be used to prepare expression vectors. As noted above, polynucleotides for incorporation into expression vectors of the subject technology can be prepared by conventional techniques, such as Polymerase Chain Reaction (PCR).
Many molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer segments can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between complementary homopolymer tails to form a recombinant DNA molecule.
In an alternative embodiment, synthetic linkers containing one or more restriction sites are provided for operably linking the polynucleotides of the subject technology to an expression vector. In embodiments, the polynucleotide is produced by restriction endonuclease digestion. In the examples, nucleic acid molecules are treated with bacteriophage T4 DNA polymerase or E.coli DNA polymerase I, which remove protruding 3 '-single stranded ends with their 3' -5 'exonuclease activity and fill recessed 3' -ends with their polymerizing activity, thereby generating blunt-ended DNA fragments. The blunt-ended fragments are then incubated with a large molar excess of linker molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the reaction product is a polynucleotide carrying a polymeric linker sequence at its end. These polynucleotides are then cleaved with appropriate restriction enzymes and ligated into an expression vector that has been cleaved with enzymes to produce termini compatible with those of the polynucleotide.
Alternatively, a vector with a ligation-independent cloning (LIC)) site may be used. The desired PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digestion or ligation (Aslanidis and de Jong, Nucl. acid.Res. [ nucleic acids research ]18,6069-74 (1990); Haun et al, Biotechniques [ Biotechnology ]13,515-18(1992), which are incorporated herein by reference to the extent consistent herewith).
In the examples, PCR is suitably used for the isolation and/or modification of the target polynucleotide for insertion into the selected plasmid. Appropriate primers for use in PCR preparation of the sequences may be designed to isolate the desired coding region of the nucleic acid molecule, to add restriction endonuclease or LIC sites, to place the coding region in the desired reading frame.
In the examples, polynucleotides for incorporation into the expression vectors of the subject technology are prepared by using PCR using appropriate oligonucleotide primers. The coding region is amplified while the primer itself becomes incorporated into the amplified sequence product. In the examples, the amplification primers contain restriction endonuclease recognition sites that allow the amplified sequence product to be cloned into an appropriate vector.
The expression vector may be introduced into a recombinant host cell (e.g., a plant or microbial host cell) by conventional transformation or transfection techniques. Transformation of appropriate cells with the expression vectors of the subject technology is accomplished by methods known in the art and typically depends on the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE dextran-mediated transfection, lipofection, chemical perforation (electroporation), or electroporation.
Successfully transformed cells (i.e., those containing an expression vector) can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce a polypeptide described herein. Cells can be examined for the presence of expression vector DNA by techniques well known in the art.
The host cell may contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.
In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is a plant cell selected from the group consisting of: canola (canola) plant cells, rapeseed plant cells, palm plant cells, sunflower plant cells, cotton plant cells, corn plant cells, peanut plant cells, flax plant cells, sesame plant cells, soybean plant cells, and petunia plant cells.
Microbial host cells and other host cell expression systems and expression vectors containing regulatory sequences that direct high levels of expression of foreign proteins are well known to those skilled in the art. Any of these can be used to construct vectors for expressing recombinant polypeptides of the subject technology in microbial host cells. These vectors can then be introduced into an appropriate microorganism by transformation to allow for high level expression of the recombinant polypeptides of the subject technology.
Vectors or cassettes that can be used to transform suitable microbial host cells and other host cells are well known in the art. Typically, the vector or cassette contains sequences that direct transcription and translation of the relevant polynucleotide, a selectable marker, and sequences that allow for autonomous replication or chromosomal integration. Suitable vectors comprise a 5 'region of the polynucleotide having transcriptional initiation control and a 3' region of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the host cell being transformed, although it will be understood that these control regions need not be derived from the native genes of the particular species chosen as the host.
Initiation control regions or promoters useful for driving expression of a recombinant polypeptide in a desired microbial or other host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the subject technology, including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in pichia); and lac, trp, JPL, IPR, T7, tac, and trc (useful for expression in E.coli).
Termination control regions may also be derived from various genes native to the microbial host. For the microbial hosts described herein, a termination site may optionally be included.
In plant cells, the expression vectors of the subject technology can include a coding region operably linked to a promoter capable of directing expression of the recombinant polypeptide of the subject technology in a desired tissue at a desired developmental stage. For convenience, the polynucleotide to be expressed may comprise a promoter sequence and a translation leader sequence derived from the same polynucleotide. A3' non-coding sequence encoding a transcription termination signal should also be present. The expression vector may also comprise one or more introns to facilitate polynucleotide expression.
For plant host cells, any combination of any promoter and any terminator capable of inducing expression of the coding region may be used in the vector sequences of the subject technology. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs), and cauliflower mosaic virus (CaMV) genes. One type of efficient plant promoter that can be used is a high level plant promoter. Such promoters, operably linked to the expression vectors of the subject technology, should be capable of promoting expression of the vector. High level plant promoters that may be used in the subject technology include promoters of the small subunit (ss) of ribulose-1, 5-bisphosphate carboxylase, such as promoters from soybean (Berry-Lowe et al, J.molecular randapp. Gen. [ molecular and application genes ],1:483498(1982), the entire contents of which are hereby incorporated by reference) and chlorophyll alb binding proteins. Both promoters are known to be photoinduced in plant cells (see, e.g., Genetic Engineering of Plants, an Agricultural complete [ plant Engineering Agricultural view ], A. Cashmore, Plenum [ Prolan publishing Co., N.Y. [ New York ] (1983), p 2938; Coruzzi, G. et al, The Journal of Biological Chemistry [ J. Biochem., 258:1399 (1983)), and Dunsmuir, P. et al, Journal of Molecular and Applied Genetics [ J. mol. App. J. App., 2:285 (1983)), which are hereby incorporated by reference to The extent consistent herewith).
Cell disruption techniques
Cell disruption is a collection of techniques for releasing biomolecules of interest from within cells. Many biotechnologically produced compounds are intracellular and must be released from the cells before recovery. Efficient recovery of the product requires cell disruption, which can be achieved by using different methods and techniques (mechanical or non-mechanical methods) depending on the biomolecule of interest and the cell system used. It should be noted that all disruption methods will release the molecule of interest (here the steviol glycoside), as well as other molecules that may lead to the breakdown of the protein of interest in the lysate. If protein activity is important for downstream work, consideration is given to preventing this. Lysis of the sample in high denaturing solutions or at high pH minimizes enzymatic activity. Breaking on ice or at lower temperatures also helps to minimize degradation of the sample. Thus, one technique for inhibiting protease activity is to have a mixture of phosphatase inhibitors in general available for the lysate once it is broken.
Known crushing techniques that may be used in accordance with the present disclosure
Standard breakup consisted of: mechanical homogenization, french crush, sonication, bead homogenization, milling, freeze-thaw lysis, detergent lysis, enzymatic lysis, and osmotic lysis.
Mechanical homogenization involves the use of a hand-held device, such as a Dounce homogenizer, or a stirrer-like device, to homogenize the tissue. The method can be used for non-seed plant materials or soft tissues (i.e., liver tissue).
French crush uses shear force to homogenize the tissue. One example of this is to take a cell suspension and use a syringe barrel and plunger to push the cell suspension through a narrow gauge syringe. This applies to bacterial, yeast, fungal, algal and mammalian cell cultures, but is difficult to scale up.
Sonication uses short pulses of ultrasound to destroy tissue. This process generates a lot of heat and will typically have to be carried out on ice to maintain the protein. The method is also effective for bacterial, yeast, fungal, algal and mammalian cell culture and is the presently disclosed preferred method.
Bead homogenization involves the use of glass or metal beads for slight abrasion while vortexing the beads with a tissue or cell suspension. Grinding involves homogenizing the tissue sample using a mortar and pestle. The most common method is to freeze and grind the sample using liquid nitrogen. Freeze-thaw lysis is literally used to freeze cells using liquid nitrogen or a freezer and then to thaw them. When cells are frozen, the water within the cells expands upon freezing, causing the cells to burst. The method is effective on mammalian cells.
Enzymatic lysis involves suspending cells in an isotonic buffer containing an enzyme that digests the cell wall (i.e., the cytolytic enzyme of the yeast cells). This lysis method is usually used in conjunction with another disruption technique (usually sonication) to ensure complete lysis of the sample. This technique is effective for bacteria, yeast, fungi, algae, non-seed plant materials, and mammalian cell culture, and is embodied by the present disclosure.
Detergent lysis involves suspending cells in a detergent solution to lyse the cell membrane, releasing the cell contents. The method also typically uses another lysis method (e.g., sonication) to ensure complete lysis. The method is effective for mammalian cell culture.
Osmotic lysis involves suspending cells in a hypotonic (low salt) solution. This causes the cells to swell and eventually rupture releasing the cell contents for further use.
Typically, in the in vitro methods of the subject technology, the weight ratio of recombinant polypeptide to substrate is from about 1: 100 to about 1: 5, preferably from about 1: 50 to about 1: 10, more preferably from about 1: 25 to about 1: 15, on a dry weight basis.
Typically, the reaction temperature of the in vitro process is from about 20 ℃ to about 40 ℃, suitably from 25 ℃ to about 37 ℃, more suitably from 28 ℃ to about 32 ℃.
One skilled in the art will recognize that the steviol glycoside compositions produced by the methods described herein may be further purified and mixed with other steviol glycosides, flavors, or sweeteners to obtain the desired flavor or sweetener composition. For example, a composition enriched in rebaudioside D4 or rebaudioside M produced as described herein can be mixed with a natural stevia extract containing rebaudioside a as the primary steviol glycoside, or with other synthetic or natural steviol glycoside products to prepare the desired sweetener composition. Alternatively, the substantially pure steviol glycosides (e.g., rebaudioside D4 or rebaudioside M) obtained from the steviol glycoside compositions described herein may be combined with other sweeteners such as sucrose, maltodextrin, aspartame, sucralose, neotame, potassium acesulfame and saccharin. The amount of steviol glycosides relative to other sweeteners can be adjusted to obtain the desired taste, as is known in the art. The steviol glycoside compositions described herein, including rebaudioside D, rebaudioside E, rebaudioside D4, rebaudioside M, or combinations thereof, can be included in food products (such as beverages, soft drinks, ice creams, dairy products, desserts, cereals, chewing gums, baked goods, and the like), dietary supplements, medical nutraceuticals, and pharmaceuticals.
Sequence similarity analysis Using identity scores
As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire alignment window of components (e.g., nucleotides or amino acids). The "identity score" for an aligned fragment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence fragment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence).
As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) as compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (the appropriate nucleotide insertions, deletions, or gaps collectively being less than 20% of the reference sequence over a comparison window). Optimal alignments of sequences for alignment comparison windows are well known to those skilled in the art and can be performed by tools such as the local homology algorithms of Smith and Waterman, the homology alignment algorithms of Needleman and Wunsch, the search similarity methods of Pearson and Lipman, and preferably by computerized implementation of these algorithms, e.g., GAP, BESTFIT, FASTA and TFASTA, as can
Percentage of Sequence identity is preferably determined using the Sequence Analysis software package (Sequence Analysis software Package)TM) (
Useful methods for determining sequence identity are also disclosed in the Basic Local Alignment Search Tool (BLAST) program, publicly available from the National Center Biotechnology Information (NCBI) of the National Library of Medicine (National Library of Medicine) at the National Institute of health (besida, Md 20894), National Institute of health; see BLAST Manual [ BLAST Manual ], Altschul et al, NCBI, NLM, NIH; altschul et al, J.mol.biol. [ J.M. J.215: 403-; version 2.0 or higher of the BLAST program allows gaps (deletions and insertions) to be introduced in the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; also, for peptide sequences, BLASTN can be used to determine sequence identity.
As used herein, the term "substantial percent sequence identity" refers to a percent sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity (e.g., about 98% or about 99% sequence identity). accordingly, one embodiment of the disclosure is a polynucleotide molecule having at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, e.g., about 98% or about 99% sequence identity, to a polynucleotide sequence described herein. a polynucleotide molecule having the activity of the presently disclosed β -glucosidase gene is capable of directing the production of a variety of steviol glycosides, and having substantial percent sequence identity to a polynucleotide sequence provided herein, and is encompassed by the scope of the disclosure.
Identity and similarity
Identity is the fraction of amino acids that are identical between a pair of sequences after alignment of the sequences (which can be done using only sequence information or structural information or some other information, but is usually based only on sequence information), and similarity is the score assigned based on the alignment using some similarity matrix. The similarity index may be any of BLOSUM62, PAM250, or gon net, or any matrix used by those skilled in the art for protein sequence alignment.
Identity is the degree of correspondence between two subsequences (no gaps between sequences). 25% or greater identity means functional similarity, and 18% -25% means structural or functional similarity. Please remember that two completely unrelated or random sequences (more than 100 residues) can have more than 20% identity. Similarity is the degree of similarity between two sequences when they are compared. Depending on their identity.
It will be apparent from the foregoing that certain aspects of the present disclosure are not limited by the specific details of the examples illustrated herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. It is therefore contemplated that the claims shall cover all such modifications and applications that do not depart from the spirit and scope of the present disclosure.
Moreover, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described above.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Accordingly, the description and examples should not be construed as limiting the scope of the invention, which is set forth in the following claims.
In accordance with the present disclosure, β -glucosidase ("B-
The present disclosure will be more fully understood when considered in view of the following non-limiting examples. While preferred embodiments of the subject technology are indicated, it should be understood that the following examples are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of the subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the subject technology to adapt it to various usages and conditions.
Example 1 identification of β -glycosidase in Pichia pastoris (Pichia pastoris)
According to the present disclosure, pichia pastoris genomic analysis was completed and several β -glycosidase candidate genes were identified, full length DNA fragments of all candidate β -glucosidase genes were commercially synthesized, codons of almost all cdnas were changed to the preferred codons for e.coli (Genscript, NJ), cloning of the synthesized DNA into a bacterial expression vector pETite N-HisSUMO Kan vector (Lucigen), these same experiments can be performed in Yarrowia lipolytica (Yarrowia lipolytica) with sequences optimized for this use.
Each expression construct was transformed into E.coli BL21(DE3) and then grown in LB medium containing 50. mu.g/mL kanamycin at 37 ℃ until OD600 reached 0.8-1.0. protein expression was induced by addition of 1mM isopropyl β -D-1-thiogalactoside (IPTG) and the culture was grown for an additional 22 hours at 16 ℃. cells were harvested by centrifugation (3,000x g; 10 min; 4 ℃). cell pellets were collected and used immediately or stored at-80 ℃.
The cell pellet is typically resuspended in lysis buffer (50mM potassium phosphate buffer, pH 7.2, 25ug/ml lysozyme, 5ug/ml DNase I, 20mM imidazole, 500mM NaCl, 10% glycerol, and 0.4% Triton X-100). cell disruption is achieved by sonication at 4 deg.C and cell debris is clarified by centrifugation (18,000X g; 30 min.) the supernatant is applied to an equilibrated (equilibration buffer: 50mM potassium phosphate buffer, pH 7.2, 20mM imidazole, 500mM NaCl, 10% glycerol) Ni-NTA (Qiagen)) affinity column.
Typically, recombinant polypeptides (10 μ g) were tested in a 300 μ L in vitro reaction system containing 50mM potassium phosphate buffer (pH 7.2), 1mg/ml steviol glycoside the reaction was carried out at 30 ℃ -37 ℃ and 50ul reaction was stopped at various time points by addition of 200 μ L of 1-butanol, the samples were extracted three times with 200 μ L of 1-butanol, the combined fractions were dried and dissolved in 100 μ L of 80% methanol for High Performance Liquid Chromatography (HPLC) analysis.
Pichia cells were suspended in extraction buffer (50mM potassium phosphate buffer (pH 7.2); 150mM NaCl). After sonication, the supernatant (crude extract) was collected by centrifugation at 12,000g set at 4 ℃. The resulting crude protein (50ug) was tested in a 300ul in vitro reaction system. The reaction system contained 50mM potassium phosphate buffer (pH 7.2), 1mg/ml steviol glycoside. The reaction was carried out at 30-37 ℃ and 50ul of the reaction was stopped at various time points by the addition of 200 ul of 1-butanol. The sample was extracted three times with 200. mu.L of 1-butanol. The combined fractions were dried and dissolved in 100 μ L80% methanol for High Performance Liquid Chromatography (HPLC) analysis.
The HPLC analysis was then performed using a Dionex UPLC ultate 3000 system (Sunnyvale, Calif. (CA)) including a quaternary pump, temperature-controlled column chamber, autosampler and UV absorbance detector Synergi Hydro-RP column with guard column (Phenomenex) was used to characterize the steviol glycosides in the combined samples in the HPLC analysis, acetonitrile in water was used as the mobile phase, the detection wavelength used in the HPLC analysis was 210nm after activity screening, we found β -glucosidase (B-glu1, SEQ:1) to have strong activity to cleave the relevant steviol glycosides and thus be a useful tool for producing the steviol glycosides of interest.
Example 2: hydrolysis of rubusoside by Using recombinant B-glu1 and disrupted Pichia cells
Rubusoside can be hydrolyzed by recombinant B-glu1 enzyme and disrupted Pichia cells to produce steviol-13-glucoside. The produced steviol-13-glucoside may then be hydrolyzed to produce steviol (fig. 1B). The reaction was set up as described in example 1. 1g/L rubusoside was added as a substrate in the reaction. As shown in FIG. 1A, B-glu1 can remove glucosyl group from C19 position of rubusoside to produce steviol-13-glucoside (FIGS. 1A-C). At a later point in time, the produced steviol-13-glucoside (S-13-G) will be converted to steviol (FIGS. 1A-d). B-glu1 removed another glucosyl group from the C13 position of steviol-13-glucoside. Since B-glu1 is an intercellular enzyme in Pichia pastoris cells, disrupted Pichia pastoris cells release B-glu1 enzyme with the same enzymatic activity to hydrolyze rubusoside to steviol-13-glucoside and continue hydrolysis to produce steviol (FIGS. 1A-e and f).
Example 3: hydrolysis of stevioside by use of recombinant B-glu1 and disrupted Pichia cells
Stevioside can be hydrolyzed by recombinant B-glu1 enzyme and disrupted Pichia cells to produce steviol bioside (FIG. 2B). The reaction was set up as described in example 1. 1g/L stevioside was added as substrate in the reaction. As shown in FIG. 2, B-glu1 can remove glucosyl groups from the C19 position of stevioside to produce steviol bioside (FIGS. 2A C and d). Since B-glu1 is an intercellular enzyme in pichia cells, the disrupted pichia cells released B-glu1 enzyme, which had the same enzymatic activity to hydrolyze stevioside to steviol bioside (fig. 8C).
Example 4: hydrolysis of rebaudioside E by Using recombinant B-glu1 and disrupted Pichia cells
Rebaudioside E could be hydrolyzed by recombinant B-glu1 enzyme to produce steviol glycosides (fig. 3B). The reaction was set up as described in example 1. 1g/L rebaudioside E was added to the reaction as a substrate. As shown in fig. 3, B-glu1 can remove glucosyl groups from the C19 position of rebaudioside E to produce stevioside and steviol bioside (fig. 3A C-E). Since B-glu1 is an intercellular enzyme in pichia cells, the disrupted pichia cells released B-glu1 enzyme with the same enzymatic activity to hydrolyze rebaudioside E to steviol biosides (fig. 8D).
Example 5: hydrolysis of rebaudioside A by Using recombinant B-glu1 and disrupted Pichia cells
Rebaudioside a could be enzymatically hydrolyzed by recombinant B-glu1 to produce rebaudioside B (fig. 4B). The reaction was set up as described in example 1. 1g/L rebaudioside A was added to the reaction as a substrate. As shown in fig. 4, B-glu1 can remove the glucosyl group from the C19 position of rebaudioside a to produce rebaudioside B (fig. 4A C-d). Since B-glu1 is an intercellular enzyme in pichia cells, the disrupted pichia cells released B-glu1 enzyme, which had the same enzymatic activity to hydrolyze rebaudioside a to rebaudioside B (fig. 8 e).
Example 6: hydrolysis of rebaudioside I by Using recombinant B-glu1 and disrupted Pichia cells
Rebaudioside I could be enzymatically hydrolyzed by recombinant B-glu1 to produce rebaudioside a, and the produced a could be hydrolyzed to produce rebaudioside B (fig. 5B). The reaction was set up as described in example 1. 1g/L rebaudioside I was added to the reaction as a substrate. As shown in fig. 5, B-glu1 can remove a glucosyl group from the C19 position of rebaudioside I to produce rebaudioside a and then remove another glucosyl group from the C19 position of rebaudioside a to produce rebaudioside B (fig. 5A d-f). At 24 hours, rebaudioside I could be completely converted to rebaudioside a (fig. 5A f). Since B-glu1 is an intercellular enzyme in pichia cells, the disrupted pichia cells released B-glu1 enzyme with the same enzymatic activity to hydrolyze rebaudioside I to rebaudioside B (fig. 8 f).
Example 7: hydrolysis of rebaudioside D by Using recombinant B-glu1 and disrupted Pichia cells
Rebaudioside D could be enzymatically hydrolyzed by recombinant B-glu1 to produce rebaudioside B (fig. 6B) and Reb a. The reaction was set up as described in example 1. 1g/L rebaudioside D was added to the reaction as a substrate. As shown in fig. 6, B-glu1 can remove the glucosyl group from the C19 position of rebaudioside D to produce rebaudioside (fig. 6A C-e). Since B-glu1 is an intercellular enzyme in pichia cells, the disrupted pichia cells released B-glu1 enzyme with the same enzymatic activity to hydrolyze rebaudioside D to rebaudioside B (fig. 8 g).
Example 8: hydrolysis of rebaudioside G by Using recombinant B-glu1 and disrupted Pichia cells
Rebaudioside G can be hydrolyzed by recombinant B-glu1 enzyme and disrupted pichia cells to produce steviol-13-glucoside. The produced steviol-13-glucoside may be continuously hydrolyzed to produce steviol (fig. 7B). The reaction was set up as described in example 1. 1G/L rebaudioside G was added to the reaction as a substrate. As shown in FIG. 7A, B-glu1 can remove glucosyl groups from the C13 and C19 positions of rebaudioside G to produce steviol-13-glucoside (FIG. 7A B-C). The produced steviol-13-glucoside (S-13-G) was converted into steviol (FIG. 7A). At 24 hours, the produced steviol-13-glucoside was completely converted into steviol (FIG. 7A d). B-glu1 removed another glucosyl group from the C13 position of steviol-13-glucoside. Since B-glu1 is an intercellular enzyme in pichia cells, the disrupted pichia cells released B-glu1 enzyme with the same enzymatic activity to hydrolyze rebaudioside G to steviol-13-glucoside, and continued hydrolysis to produce steviol (fig. 7A e).
According to the present disclosure, we identified and quantified β -glucosidase for enzymatic activity on pichia pastoris cell lysates, the enzyme having a specific activity that allows the enzyme to hydrolyze specific steviol glycosides (table 1.) with reference to fig. 7, RebG is hydrolyzed by B-glu1 enzyme and disrupted pichia pastoris cells HPLC shows the products of Reb G hydrolysis.
Using this technique, we have hydrolyzed Reb M to remove Reb A, Reb D3, Reb D4, Reb W, Reb V, Reb E, Reb I, and Reb D to improve purification efficacy (FIG. 9). The technique involves the use of stevioside and Reb A to produce steviol, steviol-13-O-glucoside, steviol bioside, and Reb B by β -glucosidase bioconversion referring to FIG. 8, steviol glycosides were hydrolyzed by disrupted Pichia pastoris cells.
In another embodiment of the present disclosure, we can use β -glucosidase to control the steviol glycoside production pathway (e.g., from Reb W to Reb M) (fig. 10).
According to a preferred embodiment of the present disclosure, β -glucosidase can be used to hydrolyze additional steviol glycosides (including Reb V, Reb W, Reb Z1, Reb Z2, Reb D3, and Reb D4) to drive these steviol glycosides to the steviol glycosides of interest (fig. 9).
Table 1: summary of hydrolysis of steviol glycosides by B-
Statement of Industrial Applicability/technical field
The present disclosure relates generally to methods for steviol glycoside biosynthesis by hydrolysis of β glucosidase.
Documents cited and incorporated by reference:
1.Brandle,J.E.et al.,(1998).Stevia Rebaudiana:Its Agricultural,Biological,and Chemical Properties,Canadian J.Plant Science.78(4):527-36.
2.Ceunen,S.,and J.M.C.Geuns,Steviol Glycosides:Chemical Diversity,Metabolism,and Function,J.Nat.Prod.,2013,76(6),pp 1201-28(2013).
3.Du J et al.,(2011),Engineering microbial factories for synthesis ofvalue-added products,J Ind Microbiol Biotechnol.38:873-90.
4.GRAS Notices,USA Food and Drug Administration,United States Health&Human Services.(2016)(relevant to steviol glycosides&polyglycosides).
5.A,and Münch T.,(1997),Microbial production of naturalflavors,ASM News 63:551-59.
6.Prakash I.,et al.；Isolation and Characterization of a NovelRebaudioside MIsomer from a Bioconversion Reaction of Rebaudioside A and NMRComparison Studies of Rebaudioside M Isolated from Stevia rebaudiana Bertoniand Stevia rebaudiana Morita,Biomolecules,2014 Jun；4(2):374-89.(Publishedonline 2014 Mar 31.2014).
7.Prakash I.,et al.,Development of Next Generation Stevia Sweetener:Rebaudioside M,Foods,2014,3:162-175.
8.Shockey JM.Et a.,(2003),Arabidopsis contains a large superfamily ofacyl-activating enzymes:phylogenetic and biochemical analysis reveals a newclass of acyl-coenzymeA synthetases.Plant Physiol 132 1065-76.
the target sequence is as follows:
the sequence is as follows:
B-glu1：
b-glu1 amino acid: (SEQ ID NO: 1) Pichia pastoris sequence (GS115)
B-glu1 DNA: (SEQ ID NO: 2) (codon optimization for E.coli)
B-glu2 amino acid: (SEQ ID NO: 3) Pichia pastoris sequence (GS115)
B-glu2 DNA (SEQ ID NO: 4) Pichia pastoris sequence (GS115)
Amino acid B-glu3 (SEQ ID NO: 5) Pichia pastoris sequence (GS115)
B-glu3 DNA (SEQ ID NO: 6) Pichia pastoris sequence (GS115)
Amino acid B-glu4 (SEQ ID NO: 7) Pichia pastoris sequence (GS115)
B-glu4 DNA (SEQ ID NO: 8) Pichia pastoris sequence (GS115)
- 上一篇：一种医用注射器针头装配设备
- 下一篇：食用油中缩水甘油酯含量的减少