阅读说明：本技术 甜菊糖苷莱苞迪苷j和莱苞迪苷n的生物合成生产 (Biosynthetic production of steviol glycosides rebaudioside J and rebaudioside N ) 是由 毛国红 M·巴腾 P·宏特 余晓丹 于 2019-03-12 设计创作，主要内容包括：本公开涉及通过使用莱苞迪苷A作为底物和涉及各种1,2RhaT-鼠李糖基转移酶的生物合成途径来产生甜菊糖苷莱苞迪苷J和莱苞迪苷N。(The present disclosure relates to the production of steviol glycosides rebaudioside J and rebaudioside N by using rebaudioside a as a substrate and biosynthetic pathways involving various 1,2 RhaT-rhamnosyltransferases.)

1. A biosynthetic method of making rebaudioside N, the method comprising:

reacting a steviol glycoside composition with a rhamnose donor moiety in the presence of a first recombinant polypeptide having 1, 2-rhamnosyltransferase activity; wherein the first recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO 3, SEQ ID NO 9 or SEQ ID NO 19.

2. The method of claim 1, wherein the first recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 3.

3. The method of claim 1, wherein the first recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 9.

4. The method of claim 1, wherein the first recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 19.

5. The method of any one of claims 1-4, comprising expressing the first recombinant polypeptide in a transformed cell system.

6. The method of claim 5, wherein the transformed cell system is selected from the group consisting of: yeast, non-steviol glycoside producing plants, algae, fungi and bacteria.

7. The method of claim 6, wherein the transformed cell system is a bacterium or yeast selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, and Clostridium.

8. The method of any one of claims 5-7, wherein the reacting step is performed in the transformed cell system.

9. The method of any one of claims 5-7, comprising isolating the first recombinant polypeptide from the transformed cell system, and the reacting step is performed in vitro.

10. The method of any one of claims 1-9, wherein the rhamnose donor moiety is UDP-L-rhamnose.

11. The method of any one of claims 1-10, wherein the steviol glycoside composition comprises rebaudioside a and the reacting step results in the production of rebaudioside J.

12. The method of claim 11, comprising reacting the rebaudioside J with a glucose donor moiety in the presence of a second recombinant polypeptide having glucosyltransferase activity.

13. The method of claim 12, wherein the glucose donor moiety is generated in situ.

14. The method of claim 12 or 13, wherein the second recombinant polypeptide has both glucosyltransferase activity and sucrose synthase activity.

15. The method of claim 12 or 13, wherein the second recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13, or SEQ ID No. 15.

16. The method of claim 12 or 13, comprising reacting the rebaudioside J with a glucose donor moiety in the presence of a third recombinant polypeptide having sucrose synthase activity.

17. The method of any of claims 1-10, wherein the steviol glycoside composition comprises rebaudioside I.

18. The method of claim 17, wherein the rebaudioside I is prepared by reacting a steviol glycoside composition comprising rebaudioside a with a glucose donor moiety in the presence of a second recombinant polypeptide having glucosyltransferase activity.

19. The method of claim 18, wherein the glucose donor moiety is generated in situ.

20. The method of claim 18 or 19, wherein the second recombinant polypeptide has both glucosyltransferase activity and sucrose synthase activity.

21. The method of claim 18 or 19, wherein the second recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13, or SEQ ID No. 15.

22. The method of claim 18 or 19, comprising reacting the rebaudioside a comprising steviol glycoside composition with a glucose donor moiety in the presence of a third recombinant polypeptide having sucrose synthase activity.

23. A biosynthetic method of making rebaudioside J, the method comprising:

reacting a steviol glycoside composition comprising rebaudioside a with a rhamnose donor moiety in the presence of a recombinant polypeptide having 1, 2-rhamnosyltransferase activity; wherein the recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO 3, SEQ ID NO 9 or SEQ ID NO 19.

24. The method of claim 23, wherein the recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 3.

25. The method of claim 23, wherein the recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 9.

26. The method of claim 23, wherein the recombinant polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID No. 19.

27. The method of any one of claims 23-26, comprising expressing the recombinant polypeptide in a transformed cell system.

28. The method of claim 27, wherein the transformed cell system is selected from the group consisting of: yeast, non-steviol glycoside producing plants, algae, fungi and bacteria.

29. The method of claim 28, wherein the transformed cell system is a bacterium or yeast selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, and Clostridium.

30. The method of any one of claims 27-29, wherein the reacting step is performed in the transformed cell system.

31. The method of any one of claims 27-29, comprising isolating the recombinant polypeptide from the transformed cell system, and the reacting step is performed in vitro.

32. The method of any one of claims 23-31, wherein the rhamnose donor moiety is UDP-L-rhamnose.

33. A rebaudioside obtainable by the method of any one of claims 1-32.

34. A nucleic acid comprising a sequence encoding a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 19.

35. A cell comprising the nucleic acid of claim 34.

36. A composition comprising at least one polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 19.

37. A cell comprising at least one polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID No. 3, SEQ ID No. 9 or SEQ ID No. 19.

38. The cell of claim 35 or 37, wherein the cell is a yeast cell, a plant cell that does not produce steviol glycosides, an algal cell, a fungal cell, or a bacterial cell.

Technical Field

The present disclosure relates generally to the biosynthesis of steviol glycosides. More specifically, the disclosure relates to biocatalytic methods for making compositions comprising rebaudioside J ("Reb J") and/or rebaudioside N ("Reb N"), as well as recombinant polypeptides having enzymatic activities useful in related biosynthetic pathways for producing Reb J and/or Reb N.

Background

Steviol glycosides are a class of compounds found in the leaves of the Stevia (Stevia rebaudiana) plant that can be used as high intensity, low calorie sweeteners. These naturally occurring steviol glycosides share the same basic diterpene structure (steviol backbone), but differ in the number and type of sugar residues (e.g., glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the steviol backbone. Interestingly, these changes in the sugar "decoration" of the basic steviol structure often significantly and unpredictably affect the properties of the resulting steviol glycoside. The affected characteristics may include, but are not limited to, overall mouthfeel characteristics, the presence and extent of any off-flavors, crystallization point, "mouthfeel," solubility, and perceived sweetness, among other differences. Steviol glycosides of known structure include stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside J, rebaudioside N, and dulcoside a.

Stevioside, rebaudioside a, rebaudioside C, and dulcoside a comprise about 9.1%, 3.8%, 0.6%, and 0.3% of the total weight of all steviol glycosides found in wild-type stevia leaves, respectively, on a dry weight basis. Other steviol glycosides such as RebJ and Reb N are present at significantly lower levels. Extracts from the stevia plant are commercially available. In such extracts, stevioside and rebaudioside a are typically the major components, while other known steviol glycosides are present as trace or trace components. The actual content levels of the various steviol glycosides in any given stevia extract may vary depending on, for example, the climate and soil in which the stevia plant is growing, the conditions under which the stevia leaves are harvested, and the method used to extract the desired steviol glycosides. To illustrate, the amount of rebaudioside a in commercial preparations can vary from about 20% to greater than about 90% by weight of the total steviol glycoside content, while the amount of rebaudioside B, rebaudioside C, and rebaudioside D can be about 1% to 2%, about 7% to 15%, and about 2% by weight of the total steviol glycoside content, respectively. In such extracts, rebaudioside J and rebaudioside N each typically account for less than 0.5% by weight of the total steviol glycoside content.

As a natural sweetener, different steviol glycosides have different degrees of sweetness, mouthfeel, and aftertaste. The sweetness of steviol glycosides is significantly higher than that of table sugar (i.e., sucrose). For example, stevioside itself is 100-150 times more sweet than sucrose, but has a bitter aftertaste, as noted in many taste tests, while rebaudioside a and E are 250-450 times more sweet than sucrose, and the aftertaste profile is much better than stevioside. However, these steviol glycosides themselves still retain a noticeable aftertaste. Thus, the overall taste profile of any stevia extract is profoundly influenced by the relative amounts of the various steviol glycosides in the extract, which in turn may be influenced by plant sources, environmental factors (such as soil content and climate) and the extraction process. In particular, variations in extraction conditions can lead to inconsistent composition of steviol glycosides in stevia extracts, such that taste characteristics vary among different batches of extracted products. The taste characteristics of stevia extracts may also be affected by plant-derived or environmental-derived contaminants (such as pigments, lipids, proteins, phenols and sugars) that remain in the product after the extraction process. These contaminants often have undesirable off-flavors that are undesirable for use of stevia extracts as sweeteners. Furthermore, methods of isolating steviol glycosides, alone or in specific combinations, that are not abundant in stevia extracts may be cost and resource prohibitive.

In addition, the method of extraction from plants generally employs a solid-liquid extraction technique using solvents such as hexane, chloroform and ethanol. Solvent extraction is an energy intensive process and can lead to problems associated with toxic waste disposal. Therefore, new production methods are needed to reduce the cost of steviol glycoside production and mitigate the environmental impact of large scale cultivation and processing.

Thus, there is a need in the art for a new method of preparing steviol glycosides, particularly minor amounts of steviol glycosides such as Reb J and Reb N, which can result in products with better and more consistent taste profiles. More preferably, such preparation methods may utilize more abundant steviol glycosides such as Reb a to reduce production costs.

Disclosure of Invention

In some embodiments, the disclosure encompasses methods of making Reb J from Reb a and methods of making Reb N from Reb a through intermediates of Reb J. In some embodiments, the present disclosure provides methods of making Reb N from Reb I, and methods of making Reb N from Reb a through intermediates of Reb I.

In one embodiment, the present disclosure provides the production of the steviol glycoside rebaudioside J "Reb J" or 13- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) oxy ] ent-kauri-16-ene-19-carboxylic acid- [ (2-O- α -L-rhamnopyranosyl- β -D-glucopyranosyl) ester ] from Reb a by the various 1, 2-rhamnosyltransferases described herein. FIG. 1 shows the chemical structure of Reb J.

In another embodiment, the present disclosure provides for the production of the steviol glycoside rebaudioside N "Reb N" or 13- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) oxy ] ent-kauri-16-ene-19-carboxylic acid- [ (2-O- α -L-rhamnopyranosyl-3-O- β -D-glucopyranosyl) oxy ] from RebJ by the various UDP-glycosyltransferases described herein. Fig. 2 shows the chemical structure of Reb N.

The present method provides a method for synthesizing specific steviol glycosides using certain specific synthetic pathways.

In terms of product/commercial utility, there are tens of steviol glycoside-containing products on the U.S. market and can be used in anything from foods, beverages and dietary supplements to analgesics and insect repellents. The steviol glycoside-containing product may be a liquid, a granular formulation, a gel or an aerosol.

Provided herein, inter alia, are biosynthetic methods of making a rebaudioside, such as rebaudioside N, the methods comprising reacting a steviol glycoside composition with a rhamnose donor moiety in the presence of a first recombinant polypeptide having 1, 2-rhamnosyltransferase activity; wherein the first recombinant polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO 3, SEQ ID NO 9, or SEQ ID NO 19.

In some embodiments, the first recombinant polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID No. 3.

In some embodiments, the first recombinant polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID No. 9.

In some embodiments, the first recombinant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 19.

In some embodiments, the biosynthetic methods provided herein include expressing the first recombinant polypeptide in a transformed cell system. In some embodiments, the transformed cell system is selected from the group consisting of: yeast, non-steviol glycoside producing plants, algae, fungi and bacteria. In some embodiments, the bacteria or yeast are selected from the group consisting of: escherichia (Escherichia), Salmonella (Salmonella), Bacillus (Bacillus), Acinetobacter (Acinetobacter), Streptomyces (Streptomyces), Corynebacterium (Corynebacterium), Methylosinus (Methysinus), Methylomonas (Methylomonas), Rhodococcus (Rhodococcus), Pseudomonas (Pseudomonas), Rhodobacterium (Rhodobacter), Synechocystis (Synechocystis), Saccharomyces (Saccharomyces), Zygosaccharomyces, Kluyveromyces (Kluyveromyces), candida (Candida), Hansenula (Hansenula), Debaryomyces (Debaryomyces), Mucor (Mucor), Pichia (Pichia), Torulopsis (Torulopsis), Aspergillus (Aspergillus), Arthrobotrys (Arthrobotrys), Brevibacterium (Brevibacterium), Microbacterium (Microbacterium), Arthrobacter (Arthrobacter), Citrobacter (Citrobacter), Klebsiella (Klebsiella), Pantoea (Pantoea) and Clostridium (Clostridium).

In some embodiments, the biosynthetic methods provided herein include a reaction step performed in a transformed cell system. In other embodiments, the reacting step may be performed in vitro. In some embodiments, the biosynthetic method includes isolating the first recombinant polypeptide from the transformed cell system, and the reacting step can be performed in vitro.

In some embodiments, the rhamnose donor is rhamnose. In some embodiments, the rhamnose donor is L-rhamnose. In some embodiments, the rhamnose donor moiety is UDP-L-rhamnose.

In some embodiments, the steviol glycoside composition comprises rebaudioside a and the reacting step results in the production of rebaudioside J.

In some embodiments, the biosynthetic methods provided herein further comprise reacting rebaudioside J with a glucose donor moiety in the presence of a second recombinant polypeptide having glucosyltransferase activity. In some embodiments, the glucose donor moiety is generated in situ.

In some embodiments, the second recombinant polypeptide has both glucosyltransferase activity and sucrose synthase activity. In some embodiments, the second recombinant polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13, or SEQ ID No. 15.

In some embodiments, the second recombinant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 7.

In some embodiments, the second recombinant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 11.

In some embodiments, the second recombinant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 13.

In some embodiments, the second recombinant polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 15.

In some embodiments, the biosynthetic methods provided herein further comprise reacting rebaudioside J with a glucose donor moiety in the presence of a third recombinant polypeptide having sucrose synthase activity.

In some embodiments, the steviol glycoside composition may include rebaudioside I. In some embodiments, rebaudioside I can be prepared by reacting a steviol glycoside composition comprising rebaudioside a with a glucose donor moiety in the presence of a second recombinant polypeptide having glucosyltransferase activity.

In some embodiments, the biosynthetic methods provided herein further comprise reacting a steviol glycoside composition comprising rebaudioside a with a glucose donor moiety in the presence of a third recombinant polypeptide having sucrose synthase activity.

Also provided herein, inter alia, are biosynthetic methods of making rebaudioside, such as rebaudioside J, comprising reacting a steviol glycoside composition comprising rebaudioside a with a rhamnose donor moiety in the presence of a recombinant polypeptide having 1, 2-rhamnosyltransferase activity; wherein the recombinant polypeptide comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO 3, SEQ ID NO 9, or SEQ ID NO 19.

In some embodiments, a recombinant polypeptide having 1, 2-rhamnosyltransferase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 3.

In some embodiments, a recombinant polypeptide having 1, 2-rhamnosyltransferase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 9.

In some embodiments, a recombinant polypeptide having 1, 2-rhamnosyltransferase activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID No. 19.

Aspects of the disclosure also provide rebaudioside obtainable by or produced by any of the biosynthetic methods described herein (including any of the embodiments above).

Aspects of the disclosure also provide nucleic acids encoding polypeptides as described herein. In some embodiments, the nucleic acid comprises a sequence encoding a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID No. 3, SEQ ID No. 9, or SEQ ID No. 19. In some embodiments, the nucleic acid comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID No. 4, SEQ ID No. 10, or SEQ ID No. 20. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO 4. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO 10. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO 20. In some embodiments, the nucleic acid is a plasmid or other vector.

Aspects of the disclosure also provide a cell comprising a nucleic acid described herein (including any of the above embodiments).

Other aspects of the disclosure provide compositions comprising at least one polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID No. 3, SEQ ID No. 9, or SEQ ID No. 19. In some embodiments, the composition comprises at least one polypeptide comprising the sequence of SEQ ID No. 3. In some embodiments, the composition comprises at least one polypeptide comprising the sequence of SEQ ID No. 9. In some embodiments, the composition comprises at least one polypeptide comprising the sequence of SEQ ID No. 19. In some embodiments, the composition is an in vitro reaction mixture, e.g., comprising a rhamnose donor moiety as described herein and a steviol glycoside composition as described herein.

Aspects of the disclosure provide a cell comprising at least one polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID No. 3, SEQ ID No. 9, or SEQ ID No. 19. In some embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO. 3. In some embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ id No. 9. In some embodiments, the cell comprises at least one polypeptide comprising the sequence of SEQ ID NO 19. In some embodiments, the cell is a yeast cell, a plant cell that does not produce steviol glycosides, an algal cell, a fungal cell, or a bacterial cell. In some embodiments, the bacteria or yeast are selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, and Clostridium. In some embodiments, the cell further comprises a second polypeptide having glucosyltransferase activity or glucosyltransferase activity and sucrose synthase activity as described herein, such as a second polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID No. 7, SEQ ID No. 11, SEQ ID No. 13, or SEQ ID No. 15.

For the cell system in this embodiment, it may be selected from the group consisting of: one or more bacteria, one or more yeasts, and combinations thereof, or any cellular system that allows genetic transformation with a selected gene and subsequent biosynthesis to produce a desired steviol glycoside. In the most preferred microbial system, e.coli (e.coli) is used to produce the desired steviol glycoside compound.

In some embodiments, the present disclosure provides a mutant of the EU11 enzyme comprising the amino acid sequence of SEQ ID NO 3 and identified as EUCP 1. In some embodiments, the present disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 3.

In some embodiments, the present disclosure provides a DNA molecule having a sequence corresponding to EUCP1 and comprising SEQ ID No. 4. In some embodiments, the present disclosure provides nucleic acid molecules having a sequence at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 4.

In some embodiments, the present disclosure provides mutants of the EU11 enzyme comprising the amino acid sequence of SEQ ID NO. 1. In some embodiments, the present disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 1.

In some embodiments, the present disclosure provides a DNA molecule having a sequence corresponding to EU11 and comprising SEQ ID No. 2. In some embodiments, the present disclosure provides nucleic acid molecules having a sequence at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 2.

In some embodiments, the present disclosure provides an enzyme referred to herein as UGT2E-B comprising the amino acid sequence of SEQ ID No. 9. In some embodiments, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 9.

In some embodiments, the present disclosure provides a DNA molecule having a sequence corresponding to UGT2E-B and comprising SEQ id no: 10. In some embodiments, the present disclosure provides nucleic acid molecules having a sequence at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 10.

In some embodiments, the present disclosure provides an enzyme referred to herein as NX114 comprising the amino acid sequence of SEQ ID NO 19. In some embodiments, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 19.

In some embodiments, the present disclosure provides a DNA molecule having a sequence corresponding to NX114 and comprising SEQ ID NO: 20. In some embodiments, the present disclosure provides nucleic acid molecules having a sequence at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 20.

In some embodiments, the present disclosure provides a microbial host cell comprising a vector capable of producing one or more enzymes described herein. In certain embodiments, the enzyme may be selected from the group consisting of: EUCP1[ SEQ ID No.3], UGT2E-B [ SEQ ID No.9] and NX114[ SEQ ID No.19 ]. In some embodiments, the enzyme may be selected from the group consisting of: CP1[ SEQ ID NO.11] and CP2[ SEQ ID NO.13 ]. In some embodiments, the enzyme may be a fusion enzyme referred to herein as GS [ SEQ ID No.15 ]. In some embodiments, the host cell is selected from the group consisting of: bacteria, yeast, filamentous fungi, cyanobacterial algae, and plant cells. In some embodiments, the host cell is selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, Corynebacterium, Clostridium (e.g., Clostridium acetobutylicum). In some embodiments, the host cell is a cell isolated from a plant selected from the group consisting of: soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oat, sorghum, rice, broccoli, cauliflower, cabbage, parsnip, melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, grass seed crops, sugar beet, sugarcane, beans, peas, rye, flax, hardwood, cork, pasture grass, arabidopsis, rice (rice), barley, switchgrass (Panicum virgratum), brachypodium, brassica, and crambe.

In some embodiments, the disclosure provides methods of producing rebaudioside J comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 3, SEQ ID No. 9, or SEQ ID No. 19. In some embodiments, the recombinant polypeptide is a 1,2 rhamnosyltransferase having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 3, SEQ ID No. 9, or SEQ ID No. 19. In some embodiments, the substrate is selected from the group consisting of: rubusoside, stevioside, or rebaudioside A, and combinations thereof.

In some embodiments, the present disclosure provides a sweetener comprising RebJ produced by any of the embodiments of the methods described above.

In some embodiments, the disclosure provides methods of producing rebaudioside N comprising incubating a substrate with a first recombinant polypeptide comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 3, SEQ ID No. 9, or SEQ ID No. 19. In some embodiments, the substrate is selected from the group consisting of: reb J, Reb I, Reb a, stevioside, or rubusoside, and combinations thereof. In some embodiments, the substrate comprises Reb J. In other embodiments, the substrate comprises Reb I. In some embodiments, the method further comprises incubating the second recombinant polypeptide having glucosyltransferase activity and optionally the recombinant sucrose synthase with the substrate and the first recombinant polypeptide.

In some embodiments, the present disclosure provides a sweetener comprising RebN produced by any of the embodiments of the methods described above.

In some embodiments, the present disclosure provides a method of synthesizing rebaudioside N from rebaudioside J, the method comprising: preparing a reaction mixture comprising rebaudioside J, a glucose donor moiety selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate glucose (UDP-glucose), and UGT enzyme, incubating the reaction mixture for a sufficient time to produce rebaudioside N, wherein glucose is covalently coupled to rebaudioside J to produce rebaudioside N. In various embodiments, the UGT enzyme can be a polypeptide comprising an amino acid sequence having at least 80% sequence identity to UGT76G1[ SEQ ID No.7], CP1[ SEQ ID No.11], CP2[ SEQ ID No.13] or GS [ SEQ ID No.15 ]. In some embodiments, the method further comprises adding a sucrose synthase to the reaction mixture. In some embodiments, the sucrose synthase is selected from the group consisting of: arabidopsis sucrose synthase 1, arabidopsis sucrose synthase 3 and mungbean sucrose synthase. In some embodiments, the sucrose synthase is Arabidopsis sucrose synthase 1(SEQ ID NO: 17).

In some embodiments, the Reb N is greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%) pure.

In some embodiments, the present disclosure provides a host cell comprising a vector capable of producing an enzyme wherein the amino acid sequence corresponds to SEQ ID NO 3, SEQ ID NO 9 or SEQ ID NO 19. In some embodiments, the host cell is selected from the group consisting of: bacteria, yeast, filamentous fungi, cyanobacterial algae, and plant cells. In some embodiments, the host cell is selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, Corynebacterium, Clostridium. In some embodiments, the host cell is a cell isolated from a plant selected from the group consisting of: soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oat, sorghum, rice, broccoli, cauliflower, cabbage, parsnip, melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, grass seed crops, sugar beet, sugarcane, beans, peas, rye, flax, hardwood, cork, pasture grass, arabidopsis, rice (rice), barley, switchgrass (Panicum virgratum), brachypodium, brassica, and crambe.

In some embodiments, the present disclosure provides a beverage product comprising: up to about 125ppm rebaudioside N; and at least one non-nutritive sweetener selected from the group consisting of: reb J, Reb W, Reb V, Reb D4, Reb E, and Reb M, and combinations thereof, wherein the at least one non-nutritive sweetener is present at a concentration from about 30ppm to about 600 ppm. In some embodiments, the at least one non-nutritive sweetener is selected from the group consisting of: reb J, Reb W, Reb V, Reb D4, Reb E, and Reb M, and combinations thereof, and wherein the Reb N and the at least one non-nutritive sweetener are present in a weight ratio of about 1: 5.

In some embodiments, the present disclosure provides a GS fusion enzyme comprising a first domain having an amino acid sequence at least 90% identical to UGT76G1 and a second domain having an amino acid sequence at least 90% identical to AtSUS 1. The GS fusion enzyme may have an amino acid sequence comprising SEQ ID NO: 15. In some embodiments, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 15.

In some embodiments, the present disclosure provides a DNA molecule having a sequence corresponding to GS and comprising SEQ ID NO: 16. In some embodiments, the present disclosure provides nucleic acid molecules having a sequence at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 16.

In some embodiments, the present disclosure provides a UDP-glycosyltransferase comprising a UGT2E-B enzyme and having an amino acid sequence of SEQ ID No. 9. In some embodiments, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID No. 9.

In some embodiments, the present disclosure provides a DNA molecule having a sequence corresponding to UGT2E-B and comprising SEQ id no: 10. In some embodiments, the present disclosure provides nucleic acid molecules having a sequence at least 90% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID No. 10.

In some embodiments, the present disclosure provides a microbial host cell comprising a vector capable of producing a UGT2E-B enzyme. In some embodiments, the host cell is selected from the group consisting of: bacteria, yeast, filamentous fungi, cyanobacterial algae, and plant cells. In some embodiments, the host cell is selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, Corynebacterium, Clostridium. In some embodiments, the host cell is a cell isolated from a plant selected from the group consisting of: soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oat, sorghum, rice, broccoli, cauliflower, cabbage, parsnip, melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, grass seed crops, sugar beet, sugarcane, beans, peas, rye, flax, hardwood, cork tree, pasture grass, arabidopsis, rice (rice), barley, switchgrass (panicum virgratum), brachypodium, brassica, and crambe.

In some embodiments, the disclosure provides a method of producing rebaudioside N comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO: 15. In some embodiments, the substrate is selected from the group consisting of: reb J, Reb a, stevioside, or rubusoside, and combinations thereof. In some embodiments, the method further comprises incubating the recombinant sucrose synthase with the substrate and the recombinant polypeptide.

In some embodiments, the present disclosure provides host cells comprising vectors capable of producing enzymes CP1 and CP2 wherein the amino acid sequences correspond to SEQ ID Nos. 11 and 13. In some embodiments, the host cell is selected from the group consisting of: bacteria, yeast, filamentous fungi, cyanobacterial algae, and plant cells. In some embodiments, the host cell is selected from the group consisting of: escherichia, Salmonella, Bacillus, Acinetobacter, Streptomyces, Corynebacterium, methylotrophus, methylomonas, Rhodococcus, Pseudomonas, rhodobacter, Synechocystis, Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Torulopsis, Aspergillus, Arthrobacter, Brevibacterium, Microbacterium, Arthrobacter, Citrobacter, Klebsiella, Pantoea, Corynebacterium, Clostridium. In some embodiments, the host cell is a cell isolated from a plant selected from the group consisting of: soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oat, sorghum, rice, broccoli, cauliflower, cabbage, parsnip, melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, grass seed crops, sugar beet, sugarcane, beans, peas, rye, flax, hardwood, cork, pasture grass, arabidopsis, rice (rice), barley, switchgrass (Panicum virgratum), brachypodium, brassica, and crambe.

In some embodiments, the present disclosure provides a mutant enzyme comprising the amino acid sequence of SEQ ID NO. 11 and identified as CP 1.

In some embodiments, the present disclosure provides a mutant enzyme comprising the amino acid sequence of SEQ ID NO 13 and identified as CP 2.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are 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 the chemical structure of rebaudioside J ("Reb J").

Fig. 2 shows the chemical structure of rebaudioside N ("Reb N").

FIG. 3A shows the biosynthetic pathway for the production of Reb J and Reb N. More specifically, starting from rebaudioside a ("Reb a"), Reb J can be produced using 1,2 rhamnosyltransferase ("1, 2 RhaT") by catalyzing the transfer of a rhamnose moiety from a UDP-rhamnose donor to C-2' of 19-O-glucose of the Reb a acceptor. In a subsequent reaction, UDP-glycosyltransferase ("UGT") may be used to produce Reb N from Reb J by catalyzing the transfer of a glucose moiety from a UDP-glucose donor to C-3' of the 19-O-glucanase of the Reb J acceptor. Fig. 3B shows the biosynthetic pathway for the production of Reb I from Reb a using UGT. Reb I can then be converted to Reb N using 1,2 RhaT.

Figure 4 shows the in vitro production of Reb J catalyzed by selected 1,2 RhaT enzymes from Reb a by HPLC analysis. Panel a shows retention time of Reb a standard. Panel B shows retention time of Reb J standard. Panel C shows the retention time of the product obtained from a reaction system catalyzed by EU11(SEQ ID NO:1) using Reb A as substrate. Panel D shows the retention time of the product obtained from the reaction system catalyzed by EUCP1(SEQ ID NO:3) using Reb A as substrate. Panel E shows the retention time of the product obtained from a reaction system catalyzed by HV1(SEQ ID NO:5) using Reb A as substrate. Arrows indicate the presence of Reb J.

FIG. 5 shows in vitro production of Reb J catalyzed by UGT2E-B from RebA by HPLC analysis. Panel a shows retention time of Reb a standard. Panel B shows retention time of Reb J standard. Panel C shows the retention time of the product obtained from the reaction system catalyzed by UGT2E-B (SEQ ID NO:9) using Reb A as a substrate. Arrows indicate the presence of RebJ.

Figure 6 shows the in vitro production of Reb J catalyzed by NX114 from Reb a by HPLC analysis. Panel a shows retention time of Reb J standard. Panel B shows retention time of Reb a standard. Panel C shows the retention time of the product obtained from a reaction system catalyzed by NX114(SEQ ID NO:19) using Reb A as a substrate. Arrows indicate the presence of Reb J.

Figure 7 shows in vitro production of Reb N catalyzed by UGT76G1 from Reb J by HPLC analysis. Panel a shows retention times for various steviol glycoside standards including rebaudioside O, Reb N, rebaudioside C and dulcoside a. Panel B shows the retention time of the product obtained from the reaction system catalyzed by UGT76G1(SEQ ID NO:7) using Reb J obtained from the EU 11-catalyzed reaction. Panel C shows the retention time of the product obtained from the reaction system catalyzed by UGT76G1(SEQ ID NO:7) using Reb J obtained from the EUCP 1-catalyzed reaction. Arrows indicate the presence of Reb N.

Figure 8 shows the in vitro production of Reb N catalyzed by selected UGT enzymes from Reb J by HPLC analysis. Panel a shows retention times for various steviol glycoside standards including rebaudioside O, Reb N, rebaudioside C and dulcoside a. Panel B shows retention time of Reb J standard. Panel B shows the retention time (as indicated by the arrow) of the Reb J intermediate obtained from the EUCP 1-catalyzed reaction. Panel D shows the retention time of the Reb N product obtained from a reaction system catalyzed by UGT76G1(SEQ ID NO:7) using the Reb J intermediate shown in Panel B. Panel E shows the retention time (as indicated by the arrow) of the Reb N product obtained from a reaction system catalyzed by CP1(SEQ ID NO:11) using the RebJ intermediate shown in panel B. Panel F shows the retention time (as indicated by the arrow) of the Reb N product obtained from a reaction system catalyzed by CP2(SEQ ID NO:13) using the Reb J intermediate shown in Panel B. Panel G shows the retention time (as indicated by the arrow) of the Reb N product obtained from the reaction system catalyzed by the fusion enzyme GS (SEQ ID NO:15) using the Reb J intermediate shown in panel B.

Figure 9 shows the in vitro production of Reb N catalyzed by selected UGT enzymes in the presence of UDP and sucrose from Reb J by HPLC analysis. Panel a shows retention time of Reb N standard. Panel B shows retention time of Reb J standard. Panel C shows the retention time of the product obtained from the UGT76G 1-catalyzed reaction. No production of Reb N was observed. Arrows indicate the presence of Reb J. Panel D shows the retention times of the products obtained from the reaction systems catalyzed by both UGT76G1 and AtSUS1(SEQ ID NO: 17). Arrows indicate the presence of Reb N. FIG. E shows the retention time of the product obtained from the reaction system catalyzed by the fusion enzyme GS (SEQ ID NO: 15). Arrows indicate the presence of Reb N.

Detailed Description

Explanations of terms used herein：

Steviol glycosides are a class of compounds responsible for the sweetness of the leaves of the south american plant stevia (Asteraceae) and are useful as sweeteners in food, feed and beverages.

Definition of：

Cell systemIs any cell that provides for ectopic protein expression. It includes bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes in vitro expression of proteins based on cellular components such as ribosomes.

Coding sequenceWill be 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 encoding a particular amino acid sequence.

Growing a cell systemGrowth involves providing an appropriate medium for the cells to multiply and divide. It also includes providing resources such that the cell or cell component can translate and produce a recombinant protein.

Protein expression. Protein production may occur after gene expression. It consists of a stage after the DNA has been transcribed into messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which ultimately fold into proteins. DNA is present in cells by transfection, which is a method of deliberately introducing nucleic acid into cells. This term is commonly used for non-viral methods in eukaryotic cells. It may also relate to other methods and cell types, but other terms are preferred: "transformation" is more generally 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 progression of a cancerous state (carcinogenesis) in these cells. Transduction is commonly used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are encompassed under the definition of transfection in the present application.

Yeast. In accordance with the present disclosure, yeast as claimed herein are classified as trueEukaryotic, unicellular microorganisms that are members of the kingdom fungi. Yeast are unicellular organisms evolved from multicellular progenitors, but among the species useful in the present disclosure are those with the ability to develop multicellular characteristics by forming strings of linked budding cells called pseudohyphae (pseudo-hyphae) or pseudohyphae.

UGT name. The name of the UGT enzyme and the UGT naming Committee used in this disclosure (Mackenzie et al, "the UDP glycosylation transfer enzyme gene super family: registered nomenclature updated on evolution direction," P_{HARMACOGENETICS}1997, volume 7, pages 255-269) using a nomenclature system consistent with that which classifies UGT genes by a combination of family number, letter representing a subfamily, and number of individual genes. For example, the name "UGT 76 Gl" refers to the UGT enzyme encoded by the gene belonging to UGT family number 76 (which is of plant origin), subfamily G, and gene number 1.

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," "has," "having," "has," "with," "having," "has," "having," "contains," "containing," "involving," or any other variation thereof, are used in the specification, it is intended that all such terms be interpreted as including a reference to a common usage of the claims.

The word "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" will be given its ordinary and customary meaning to those 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, for DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the subject technology also includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing, as well as those substantially similar nucleic acid sequences.

The terms "nucleic acid" and "nucleotide" will be 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 natural 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" will be 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 is artificially present outside its natural environment and is therefore not a natural product. An isolated nucleic acid or polypeptide may exist in a purified form, or may exist in a non-natural environment, such as in a transgenic host cell.

As used herein, the term "incubating" means a process that mixes two or more chemical or biological entities (e.g., compounds and enzymes) and allows them to interact under conditions favorable for 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 achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with 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" will be given their respective meanings that are common and customary to those of ordinary skill in the art; these three terms are sometimes used interchangeably and are used to refer to polymers of amino acids or amino acid analogs regardless of their size or function, without limitation. Although "protein" is generally used to refer to relatively large polypeptides and "peptide" is generally used to refer to small polypeptides, the use of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise specified. 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, 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 polypeptide shall be given their ordinary and customary meaning to those of ordinary skill in the art, and are used without limitation to refer to polypeptides lacking amino acid residues as compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally identical to the corresponding position in the reference polypeptide. Such deletions may occur at the amino-terminus or the carboxy-terminus, or both, of the reference polypeptide.

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 as the full-length polypeptide or protein, or performs substantially the same function as the full-length polypeptide or protein (e.g., performs the same enzymatic reaction).

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 capabilities of a reference polypeptide.

The term "functional variant" also includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having 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 the substitution of one nonpolar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; substitution of one charged or polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; substitution of one basic residue such as lysine or arginine for the other; or substitution of one acidic residue such as aspartic acid or glutamic acid for the other; or one aromatic residue such as phenylalanine, tyrosine or tryptophan for the other. Such substitutions are expected to have little 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 chemically derivatized residue, provided that the resulting peptide retains some or all of the activity of the reference peptide as described herein.

The term "variant" in relation to a polypeptide of the subject technology also includes functionally active polypeptides having 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 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 the 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 and homologous polynucleotides or proteins from different species (Reeck et al, C_ELL50:667,1987). 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 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" shall be given their ordinary and customary meaning to those of ordinary skill 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 relevant coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"promoter" shall be 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 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 a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is also recognized that since the precise boundaries of regulatory sequences have not been completely defined in most cases, DNA fragments of different lengths may have identical promoter activity.

The term "operably linked" refers to the association of 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 can affect the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in sense or antisense orientation.

As used herein, the term "expression" will be 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 that exceeds the level produced in a normal or non-transformed organism.

"transformation" shall be given its ordinary and customary meaning to those skilled 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 "transformed".

The terms "transformed", "transgenic" and "recombinant" when used herein in connection with a host cell will be given their respective ordinary and customary meanings to those of ordinary skill in the art, and are used without limitation to refer to a cell, such as a plant or microbial cell, of a host organism 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 be present as an extrachromosomal molecule. Such extrachromosomal molecules can replicate themselves. Transformed cells, tissues or subjects are to be understood as encompassing 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 will be given 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 derived from a source that is foreign to the particular host cell or, if derived from the same source, is modified relative to its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example, by using site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to the cell or homologous to the cell but where the element is not normally present 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 is derived from a source that is foreign to the particular host cell or that is modified relative to its original form if derived from the same source. Thus, the recombinant DNA segment can be expressed in a host cell to produce a recombinant polypeptide.

The terms "plasmid", "vector" and "cassette" will be given their respective ordinary and customary meanings to those of ordinary skill 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, genome integrating sequences, phage or nucleotide sequences, linear or circular, of single-or double-stranded DNA or RNA, derived from any source, many of which have been joined or recombined into a unique construct capable of introducing into a cell a promoter fragment and DNA sequence of a selected gene product and appropriate 3' untranslated sequence. "transformation cassette" refers to a particular vector that contains a foreign gene and has, in addition to the foreign gene, elements that facilitate transformation of a particular host cell. "expression cassette" refers to a specific vector that contains an exogenous gene and has elements in addition to the exogenous gene that allow for enhanced expression of the gene in an exogenous host.

In some embodiments, the present disclosure relates to the production of steviol glycoside Reb N of interest from Reb a using at least one novel UDP-rhamnosyltransferase (RhaT) described herein, which can transfer a rhamnose moiety from UDP-L-rhamnose to a steviol glycoside acceptor in a rhamnosylation reaction. Referring to fig. 3, the synthetic pathway may involve rebaudioside J as an intermediate (fig. 3A) or rebaudioside I as an intermediate (fig. 3B). In some embodiments, the subject technology provides recombinant polypeptides having UDP glycosyltransferase activity for use in the synthesis of steviol glycosides. In some embodiments, the recombinant polypeptide may have 1, 2-19-O-rhamnose glycosylation activity. In some embodiments, the recombinant polypeptide may have 1, 3-19-O-glucose glycosylation activity. The recombinant polypeptides of the subject technology can be used for the biosynthesis of steviol glycoside compounds. In the present disclosure, UDP-rhamnosyltransferase (Rha T) refers to an enzyme that transfers a rhamnose moiety from an activated donor molecule (typically UDP-L-rhamnose) to an acceptor steviol glycoside molecule. 1,2 Rha T refers to the enzymatic activity of the C-2' transfer of the rhamnose moiety from UDP-L-rhamnose to 19-O-glucose or 13-O-glucose of steviol glycoside. 1, 2-19-O-rhamnose glycosylation activity refers to the enzymatic activity of transferring a rhamnose moiety to the C-2' of the 19-O glucose moiety of a steviol glycoside such as rebaudioside A (to produce rebaudioside J) or rebaudioside I (to produce rebaudioside N). 1, 3-19-O-glucose glycosylation activity refers to the enzyme activity of transferring a glucose moiety to C-3' of a 19-O glucose moiety of a steviol glycoside such as rebaudioside J (to produce rebaudioside N) or rebaudioside A (to produce rebaudioside I) (FIG. 3).

Synthetic biology

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, in the following references: sambrook, j., Fritsch, e.f. and manitis, t., M_OLECULARC_LONING:A L_ABORATORYM_ANUAL2 nd edition; cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.,1989 (hereinafter referred to as "Maniatis"); and Silhavy, t.j., Bennan, m.l., and Enquist, l.w., E_XPERIMENTS_WITHG_ENEFUSIONS; cold Spring Harbor Laboratory Cold Spring Harbor, N.Y., 1984; and Ausubel, F.M. et al, I_NC_URRENTP_{ROTOCOLS IN}M_OLECULARB_IOLOGYPublished in 1987 by Greene Publishing and Wiley-Interscience; (the entire contents of each of these documents are incorporated herein by reference).

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 below.

The present disclosure will be more fully understood by consideration of the following non-limiting examples. It should be understood that these examples, while indicating preferred embodiments of the subject technology, 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 to the subject technology to adapt it to various uses and technologies.

Glycosylation is generally considered to be a ubiquitous response that controls the biological activity and storage of plant natural products. Glycosylation of small molecules is catalyzed by the transferase superfamily in most plant species that have been studied to date. These Glycosyltransferases (GTs) have been divided into 60 families. Among these, the GT enzyme family (also known as UDP Glycosyltransferase (UGT) and UDP-rhamnosyltransferase) transfers sugar moieties to specific acceptor molecules. These are molecules that transfer such sugar moieties in stevioside to help produce various rebaudioside glycosides. Each of these enzymes has its own activity profile and preferably their structural position to transfer its activated sugar moiety.

Production system

Expression of proteins in prokaryotes is most often carried out in bacterial host cells using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors are commonly used for 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 the recombinant protein by acting as a ligand in affinity purification. Typically, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable the recombinant protein to be separated from the fusion moiety after purification of the fusion protein. Such vectors are within the scope of the present disclosure.

In one embodiment, the expression vector includes those genetic elements used for expression of the recombinant polypeptide in a bacterial cell. Elements used for transcription and translation in bacterial cells may include promoters, coding regions for protein complexes, and transcription terminators.

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 the expression vectors of the subject technology can be prepared by conventional techniques such as Polymerase Chain Reaction (PCR).

Several molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive ends. In one embodiment, complementary homopolymer tracts may be added to the nucleic acid molecules to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

In alternative embodiments, the polynucleotides of the subject technology are operably linked to an expression vector using a synthetic linker containing one or more restriction sites. In one embodiment, the polynucleotide is generated by restriction endonuclease digestion. In one embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E.coli DNA polymerase I, which removes the protruding 3 '-single stranded ends using its 3' -5 '-exonucleolytic activity and fills the recessed 3' -ends using its polymerization 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 that catalyzes ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the reaction product is a polynucleotide carrying a polymer linker sequence at its ends. These polynucleotides are then cleaved with appropriate restriction enzymes and ligated into an expression vector that has been cleaved with an enzyme that produces ends compatible with the ends of the polynucleotides.

Alternatively, a vector having 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, N)_UCL.A_CID.R_ES186069-74, (1990), Haun et al, B_IOTECHNIQUES13,515-18(1992), which is incorporated herein by reference).

In one embodiment, PCR is suitably used in order to isolate and/or modify the polynucleotide of interest for insertion into the selected plasmid. Suitable primers for sequence PCR preparation can 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 one embodiment, the polynucleotide for incorporation into the expression vector of the subject technology is prepared using PCR using appropriate oligonucleotide primers. The coding region is amplified while the primer itself is incorporated into the amplified sequence product. In one embodiment, the amplification primers comprise a restriction endonuclease recognition site that allows for cloning of the amplified sequence product into a suitable vector.

The expression vector may be introduced into a plant or microbial host cell by conventional transformation or transfection techniques. Transformation of a suitable cell with the expression vector of the subject technology is accomplished by methods known in the art and generally 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 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. The presence of expression vector DNA in the cell can be checked by techniques well known in the art.

The host cell may contain a single copy of the expression vector as previously described, 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 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 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 suitable microorganisms via transformation to allow for high level expression of the recombinant polypeptides of the subject technology.

Vectors or cassettes for transforming suitable microbial host cells are well known in the art. Typically, the vector or cassette comprises sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors include a 5 'region of a polynucleotide having transcriptional initiation control and a 3' region of a DNA fragment which controls transcriptional termination. Preferably, both control regions are derived from genes homologous to the transformed host cell, although it will be understood that such control regions need not be derived from the native gene 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 host cell are numerous and familiar to those skilled in the art. Indeed, any promoter capable of driving these genes is suitable for use in the subject technology, including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (for expression in saccharomyces); AOXI (for expression in pichia); and lac, trp, JPL, IPR, T7, tac, and trc (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. The 3' non-coding sequence encoding the 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 useful 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 useful in the subject technology include, for example, the promoter of the small subunit (ss) of ribulose-l, 5-bisphosphate carboxylase from soybean (Berry-Lowe et al, J.M) _{OLECULAR AND}A_PP.G_EN1: 483498 (1982), which is hereby incorporated herein in its entirety to the extent consistent herewith) and the promoter of the chlorophyll alb binding protein. Both promoters are known to be light-inducible in plant cells (see, e.g., G)_ENETICE_{NGINEERING OF}P_LANTS,ANA_GRICULTURALP_ERSPECTIVECashmore, Plenum, N.Y. (1983), pages 29-38; coruzzi, G.et al, the journal of Biological C_HEMISTRY258:1399(1983), and Dunsmuir, P. et al, J_{OURNAL OF}M_{OLECULAR AND}A_PPLIEDG_ENETICS2:285(1983), each of which is hereby incorporated by reference to the extent consistent herewith).

Precursor synthesis of Reb J or Reb I

As mentioned previously, steviol glycosides are compounds responsible for the sweet taste of the south american plant stevia (asteraceae) and the plant rubuscarpus rubicuni (Rosaceae). These compounds are glycosylated diterpenes. In particular, their molecules may be considered as steviol molecules, wherein the hydroxyl hydrogen atom thereof is replaced by a glucose molecule to form an ester, and the combination of the hydroxyl hydrogen with glucose and rhamnose forms an acetal.

One method of making compounds of interest in the present disclosure is to employ common or inexpensive precursors such as steviol, stevioside, Reb a or rubusoside, chemically derived or produced via biosynthesis in engineered microorganisms such as bacteria and/or yeast, and synthesize the target steviol glycoside such as Reb J or Reb I by known or inexpensive methods.

Aspects of the present disclosure relate to methods involving recombinant expression of enzymes in a microbial system capable of producing steviol. Typically, such enzymes may include: copalyl diphosphate synthase (CPS), Kaurene Synthase (KS) and geranylgeranyl diphosphate synthase (GGPPS). This should occur in microbial strains expressing an endogenous isoprenoid synthesis pathway such as the non-Mevalonate (MEP) pathway or the mevalonate pathway (MVA)). In some embodiments, the cell is a bacterial cell, including an escherichia coli or yeast cell (such as a saccharomyces cell, a pichia cell, or a Yarrowia cell). In some embodiments, the cell is an algal cell or a plant cell.

Thereafter, the precursor is recovered from the fermentation culture for chemical synthesis. Typically, this is steviol, although it may be kaurene, or a steviol glycoside from cell culture. In some embodiments, steviol, kaurene, and/or steviol glycosides are recovered from the gas phase, while in other embodiments, an organic layer or polymeric resin is added to the cell culture, and then kaurene, steviol, and/or steviol glycosides are recovered from the organic layer or polymeric resin. In some embodiments, the steviol glycoside is selected from rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside N, rebaudioside E, rebaudioside F, rebaudioside J, or dulcoside a. In some embodiments, the terpenoid produced is steviolbioside or stevioside. It is also understood that in some embodiments, at least one enzymatic step, such as one or more glycosylation steps, is performed ex vivo.

Part of the present disclosure is the production of Reb J steviol glycosides, which can be further enzymatically converted to Reb N. In some embodiments, Reb a may be converted to Reb J using a rhamnosyltransferase (RhaT) (e.g., EU11, EUCP1, HV1, UGT2E-B, or NX114) and a rhamnose donor moiety (e.g., UDP-rhamnose) as described herein. In some embodiments, biosynthesis of microbial produced steviol conversion to a desired steviol glycoside (e.g., Reb N) occurs when diterpene steviol is converted to rubusoside and stevioside using multi-step chemical assembly of sugar moieties into the steviol backbone using specifically identified and/or modified enzymes produced by the inventors. In addition to the EU11, EUCP1, HV1, and UGT76G1 enzymes used herein, other enzymes were identified that could also be used to deliver these steviol rebaudioside. For example, other UGT enzymes (CP 1 and CP 2-the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:13, respectively) and UGT76G1-AtSUS1 fusion enzymes have been determined to convert Reb J to Reb N.

Part of the present disclosure is the production of Reb I steviol glycosides from Reb a, which can be further enzymatically converted to Reb N. In some embodiments, the biosynthesis of the conversion of Reb a to Reb I is performed by reacting Reb a with a glucose donor moiety in the presence of a recombinant polypeptide having glucosyltransferase activity. In some embodiments, the glucose donor moiety is generated in situ. In some embodiments, a glucose donor moiety is added to the reaction. In some embodiments, the recombinant polypeptide having glucosyltransferase activity further comprises sucrose synthase activity. For example, in some embodiments, the enzyme identified as UGT76G1(SEQ ID NO:7) can convert Reb A to Reb I. In some embodiments, other UGT enzymes (e.g., CP1 and CP 2-the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:13, respectively) can convert Reb A to Reb I. In some embodiments, the UCT76G1-AtSUS1 fusion enzyme can convert Reb a to Reb I. In some embodiments, Reb I may be converted to Reb N using a rhamnosyltransferase (RhaT) such as EU11, EUCP1 UDP, HV1, UGT2E-B, or NX114, and a rhamnose donor moiety such as UDP-rhamnose, as described herein.

Biosynthesis of steviol glycosides

As described herein, recombinant polypeptides of the present technology have UDP-glycosyltransferase (UDP-glucosyltransferase and/or UDP-rhamnosyltransferase) activity and can be used to develop biosynthetic methods for making steviol glycosides such as rebaudioside J, rebaudioside I and rebaudioside N, respectively, that do not occur in nature or that are typically low abundant in natural sources. The recombinant polypeptide of the present technology has UDP-glycosyltransferase activity, can be used to develop a biosynthetic method for producing steviol glycosides such as rebaudioside J or rebaudioside I and to achieve synthetic production of rebaudioside N.

The substrate or starting steviol glycoside composition may be any natural or synthetic compound capable of being converted to a steviol glycoside compound in a reaction catalyzed by one or more UDP-rhamnosyltransferases. For example, the substrate may be natural stevia extract, steviol-13-O-glucoside, steviol-19-O-glucoside, steviol-l, 2-bioside, rubusoside, stevioside, rebaudioside A, rebaudioside B or rebaudioside I. The substrate may be a pure compound or a mixture of different compounds. Preferably, the substrate comprises a compound selected from the group consisting of: rubusoside, stevioside, steviol, rebaudioside A, rebaudioside B, rebaudioside J, and combinations thereof.

The methods described herein also provide coupled reaction systems in which the recombinant peptides described herein can act in combination with one or more additional enzymes to improve the efficiency of or alter the results of the overall biosynthesis of steviol glycoside compounds. For example, the additional enzyme may regenerate UDP-rhamnose required for the rhamnosylation reaction (see, e.g., Pei et al, "structural a novel UDP-rhamnose regeneration system by a two-enzyme reaction system administration in glycosylation of recombinant," biological engineering journal,139:33-42(2018), the entire disclosure of which is incorporated herein by reference), and the additional enzyme may regenerate UDP-glucose required for the glycosylation reaction by converting UDP produced by the glycosylation reaction back to UDP-glucose (e.g., using sucrose as a donor for glucose residues), thereby increasing the efficiency of the glycosylation reaction.

In another embodiment, the methods of the subject technology further comprise incubating a recombinant and novel UDP-rhamnosyltransferase (RhaT) according to the present disclosure with a substrate and one or more additional recombinant polypeptide(s) (e.g., recombinant UGT) described herein. The recombinant UGT can catalyze glycosylation reactions that differ from the glycosylation reactions catalyzed by the recombinant polypeptides of the subject technology, resulting in the production of Reb J and Reb N.

Suitable UDP-glycosyltransferases include any UGT known in the art capable of catalyzing one or more reactions in the biosynthesis of a stevioside compound, such as UGT85C2, UGT74G1, UGT76G1, or functional homologs thereof.

In some embodiments, in the in vitro methods of the subject technology, UDP-glucose and/or UDP-L-rhamnose may be comprised in a buffer at a concentration of about 0.2mM to about 5mM, preferably about 0.5mM to about 2mM, more preferably about 0.7mM to about 1.5 mM. In one embodiment, when a recombinant sucrose synthase is included in the reaction, sucrose is also included in the buffer at a concentration of about 100mM to about 500mM, preferably about 200mM to about 400mM, more preferably about 250mM to about 350 mM.

In some embodiments, 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.

In some embodiments, the reaction temperature of the in vitro method is from about 20 ℃ to about 40 ℃, suitably from 25 ℃ to about 37 ℃.

One skilled in the art will recognize that the steviol glycoside composition produced by the process described herein can 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 Reb J or Reb N 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 produce the desired sweetener composition. Alternatively, the substantially purified steviol glycosides (e.g., rebaudioside J and rebaudioside N) obtained by the methods described herein can be combined with other sweeteners such as sucrose, maltodextrin, aspartame, sucralose, neotame, acesulfame potassium, and saccharin. As is known in the art, the amount of steviol glycosides relative to other sweeteners can be adjusted to achieve a desired taste. Steviol glycosides, including rebaudioside N, rebaudioside I, rebaudioside J, or combinations thereof, as described herein can be included in food products (such as beverages, soft drinks, ice cream, dairy products, candies, cereals, chewing gum, baked goods, and the like), dietary supplements, medical nutraceuticals, and pharmaceutical products.

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" of 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 the 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 (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20% of the reference sequence over the comparison window). Optimal alignments of sequences for alignment comparison windows are well known to those skilled in the art and can be performed by such means 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 computer implementation of these algorithms, such as

Wisconsin

GAP, BESTFIT, FASTA and TFASTA (Accelrys Inc., Burlington, Mass.). The "identity score" of 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. Percent sequence identity is expressed as the identity score multiplied by 100. Comparison of one or more polynucleotide sequences may involve full-length polynucleotide sequences or portions thereof, or longer polynucleotide sequences. For purposes of this disclosure, "percent identity" can also be determined using BLASTX version 2.0 (for translated nucleotide sequences) and BLASTN version 2.0 (for polynucleotide sequences).

Percentage Sequence identity is preferably determined using the Sequence Analysis Software Package^TMIs determined by the "best fit" or "null" program (version 10; Genetics Computer Group, inc., Madison, WI). The "holes" utilize the algorithms of Needleman and Wunsch (Needleman and Wunsch, J)_{OURNAL OF}Molecular Biology 48:443-453, 1970) to find an alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "best fit" uses the local homology algorithm of Smith and Waterman (Smith and Waterman, A) _{DVANCES IN}A_PPLIEDM_ATHEMATICS482-489, 1981, Smith et al, N_UCLEICA_CIDSR_ESEARCH2205-2220, 1983) to perform an optimal alignment for optimal fragment similarity between the two sequences and to insert gaps to maximize the number of matches. The percent identity is most preferably determined using a "best fit" procedure.

Available methods for determining sequence identity are also disclosed in the local alignment search basic tools (BLAST) program, which is publicly available from National Center Biotechnology Information (NCBI) of National Library of Medicine, National Institute of Health, Bethesda, Md.20894; see BLAST Manual, Altschul et al, NCBI, NLM, NIH; altschul et al, J.M_OL.B_IOL215:403-410 (1990); BLAST programs of version 2.0 or higher allow for the introduction of gaps (deletions and insertions)Comparing and centering; for peptide sequences, BLASTX can be used to determine sequence identity; also, for polynucleotide sequences, BLASTN can be used to determine sequence identity.

As used herein, the term "significant 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, such as about 98% or about 99% sequence identity. Accordingly, one embodiment of the present 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, such as about 98% or about 99% sequence identity, to a polynucleotide sequence described herein. Polynucleotide molecules having the activity of the 1,2 RhaT and UGT enzymes of the present disclosure are capable of directing the production of a variety of steviol glycosides and have a significant percentage sequence identity to the polynucleotide sequences provided herein and are included within the scope of the present disclosure.

Identity and similarity

Identity is the fraction of amino acids that are identical between a pair of sequences after aligning the sequences (which may be done using only sequence information or structural information or some other information, but is typically based on only sequence information), while similarity is based on the score assigned for alignment using some similarity matrix. The similarity index may be any of BLOSUM62, PAM250, or gon net below, or any matrix used by one skilled in the art to align protein sequences.

Identity is the degree of identity between two subsequences (no gaps between sequences). Identity of 25% or higher means similarity of function, whereas 18% to 25% means similarity of structure or function. Please remember that two completely unrelated or random sequences (which are more than 100 residues) can have more than 20% identity. Similarity is the degree of similarity between two sequences when the two sequences are compared. Depending on their identity.

As is apparent from the foregoing description, certain aspects of the present disclosure are not limited by the specific details of the embodiments described herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. Therefore, the claims should be intended to cover all such modifications and applications that do not depart from the spirit and scope of the present disclosure.

Furthermore, 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 specification and examples should not be construed as limiting the scope of the invention, which is defined by the appended claims.