FIELD OF THE INVENTION

The field of the invention relates to methods and processes useful in the production of a specific steviol glycoside via a biosynthetic pathway engineered into selected microorganisms. More specifically, the present disclosure relates to the production of a previously unknown rebaudioside, rebaudioside D4 (“Reb D4”) that can be synthesized from Rebaudioside E (“Reb E”) via enzymatic conversion.

BACKGROUND OF THE INVENTION

Several steviol glycosides were found as compounds inStevia rebaudianaleaves, and have been widely used as high intensity, low-calorie sweeteners in food, feed and beverages. These naturally occurring steviol glycosides have the same basic diterpene structure (steviol backbone) but differ in the number and structure of their carbohydrate residue modifications (e.g. glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the steviol backbone. Interestingly, these changes in sugar ‘ornamentation’ of the base steviol structure dramatically and unpredictably affect the properties of the individual steviol glycosides themselves. These traits include, without limitation, taste profile, crystallization point, solubility and perceived sweetness among other differences. Steviol glycosides with known structures include stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M and dulcoside A. In terms of commercial use rebaudioside M has become generally regarded as safe (that is, it has ‘GRAS’ status) and is being studied for a wide range of uses in the food and beverage markets.

On a dry weight basis, stevioside, rebaudioside A, rebaudioside C, and dulcoside A, account for 9.1, 3.8, 0.6, and 0.30 percent of the total weight of the steviol glycosides found in wild typeStevialeaves, respectively, while the other steviol glycosides, such as Reb E are present in significantly lower amounts, with Reb D4 not being present at all. Extracts fromStevia rebaudianaplant are commercially available and in such extracts stevioside and rebaudioside A are most often the primary components. Comparatively, the other known steviol glycosides are typically present in thesteviaextract as minor or trace components. For example, the amount of rebaudioside A in typical commercial preparations can vary from about 20% to more than 90% of the total steviol glycoside content, with the amount of rebaudioside B at about 1-2%, the amount of rebaudioside C about 7-15%, and the amount of rebaudioside D can be about 2% of the total steviol glycosides. In such extracts rebaudioside M is present only in vanishingly small amounts. Interestingly, Rebaudioside E is also one of the least abundant steviol glycosides present inStevia rebaudianaplant varieties, accounting for less than 0.5% of total glycoside present.

As natural sweeteners, and as mentioned above, each of the different steviol glycosides have different degrees of sweetness, ‘mouth feel’ and specific after-tastes associated with them. Relative to table sugar (i.e., “sucrose”) the sweetness of steviol glycosides is significantly higher. For example, stevioside is 100-150 times sweeter than sucrose but has a bitter after-taste as noted in numerous taste tests, while rebaudiosides A and E are 250-450 times sweeter than sucrose and the after-taste profile is much better than stevioside. However, these steviol glycosides themselves still retain a noticeable aftertaste. Accordingly, it is generally known that the taste profiles ofsteviaextracts are profoundly affected by the relative content of the various steviol glycosides in the extract, which in turn may be affected by the environmental conditions experienced by the underlying plants and the extraction process used. These variations in plant production, weather conditions and extraction conditions can lead to inconsistent compositions of the steviol glycosides in thesteviaextracts, such that the taste profile varies strongly among different batches of extraction products. The taste profile ofsteviaextracts also can be affected by plant-derived or environment-derived contaminants (such as pigments, lipids, proteins, phenolics and saccharides) that remain in the product after the extractions process. These contaminants typically have their own off-flavors, making the resultant extract undesirable for use in consumer products. In addition, the cost of isolating individual or specific combinations of steviol rebaudiosides that are not abundant insteviaextracts is cost and resource prohibitive. Given that there is a limited quality and availability of some specific steviol glycosides, commercial supply can be better addressed by bio-conversion, where natural enzymes, or specific microbes can be modified to carry needed enzymes and use commercially significant fermentation processes to specifically increase the production of glycosides of interest. For example, bio-conversion of Stevioside to Reb E has been reported previously (see, Yu et al., U.S. application Ser. No. 15/016,589, published as US Patent Application Publication Number US2016/0207954) via a fermentation pathway with modified microbes. Alternatively, other non-biologic synthetic means can be used to develop steviol glycosides of interest.

Accordingly, there is a need for steviol glycosides with better and more consistent taste profiles to be developed as commercial products and for such steviol glycosides to utilize a relative common starting substrate, such as more abundant steviol glycosides as starting molecules, so that such production of desirable glycosides can be commercially as cost effective as possible.

SUMMARY OF THE INVENTION

The present disclosure encompasses a method of producing Reb D4 from Reb E, such as in a cellular system.

In particular, the current disclosure provides for the production of steviol glycoside rebaudioside D4 “Reb D4” which is identified as (13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester]) by a specific UDP-glycosyltransferase from Reb E.

The current methods provide an approach for the synthesis of specific steviol glycosides using a specific synthetic pathway.

In some aspects, the disclosure provides a mutant CP1 enzyme comprising the amino acid sequence of SEQ ID NO: 7. In some aspects, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 7. In some embodiments, the recombinant polypeptide has the same or substantially the same activity as a mutant CP1 enzyme as described herein, e.g., a mutant CP1 enzyme comprising the amino acid sequence of SEQ ID NO: 7.

In some aspects, the disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 8. In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encodes a mutant CP1 enzyme as described herein, e.g., a mutant CP1 enzyme comprising the amino acid sequence of SEQ ID NO: 7.

In some aspects, the disclosure provides a mutant CP1 enzyme comprising the amino acid sequence of SEQ ID NO: 9. In some aspects, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 9. In some embodiments, the recombinant polypeptide has the same or substantially the same activity as a mutant CP1 enzyme as described herein, e.g., a mutant CP1 enzyme comprising the amino acid sequence of SEQ ID NO: 9.

In some aspects, the disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 10. In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 10. In some embodiments, the nucleotide sequence encodes a mutant CP1 enzyme as described herein, e.g., a mutant CP1 enzyme comprising the amino acid sequence of SEQ ID NO: 9.

In some aspects, the disclosure provides a mutant HV1 enzyme comprising the amino acid sequence of SEQ ID NO: 11. In some aspects, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 11. In some embodiments, the recombinant polypeptide has the same or substantially the same activity as a mutant HV1 enzyme as described herein, e.g., a mutant HV1 enzyme comprising the amino acid sequence of SEQ ID NO: 11.

In some aspects, the disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 12. In some aspects, the disclosure provides a nucleic acid comprising a nucleotide sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 12. In some embodiments, the nucleotide sequence encodes a mutant HV1 enzyme as described herein, e.g., a mutant HV1 enzyme comprising the amino acid sequence of SEQ ID NO: 11.

In some aspects, the disclosure provides a host cell comprising a vector capable of producing a C1m2 enzyme having the amino acid sequence of SEQ ID NO: 7. In some aspects, the disclosure provides a host cell comprising a vector capable of producing a C1m3 enzyme having the amino acid sequence of SEQ ID NO: 9. In some aspects, the disclosure provides a host cell comprising a vector capable of producing a mutant HV1 enzyme having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the host cell is selected from the group consisting of a bacteria, a yeast, a filamentous fungi, a cyanobacteria algae and a plant cell. In some embodiments, the host cell is selected from the group consisting ofEscherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Escherichia; Klebsiella; Pantoea; Salmonella Corynebacterium; Clostridium; andClostridium acetobutylicum. In some embodiments, the host cell is a cell isolated from plants selected from the group consisting of soybean; rapeseed; sunflower; cotton; corn; tobacco; alfalfa; wheat; barley; oats; sorghum; rice; broccoli; cauliflower; cabbage; parsnips; melons; carrots; celery; parsley; tomatoes; potatoes; strawberries; peanuts; grapes; grass seed crops; sugar beets; sugar cane; beans; peas; rye; flax; hardwood trees; softwood trees; forage grasses;Arabidopsis thaliana; rice (Oryza sativa);Hordeum yulgare; switchgrass (Panicum vigratum);Brachypodiumspp.;Brassicaspp.; andCrambe abyssinica.

In some aspects, the disclosure provides a method of producing a steviol glycoside composition, the method 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 7. In some embodiments, the recombinant polypeptide is a UDP-glycosyltransferase having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 7. In some embodiments, the substrate is selected from the group consisting of stevioside or rebaudioside E and combinations thereof. In some embodiments, the steviol glycoside compound produced by the method is rebaudioside D4 such that the steviol glycoside composition comprises rebaudioside D4.

In some aspects, the disclosure provides a method of producing rebaudioside D4, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 7. In some embodiments, the substrate is selected from the group consisting of Reb E, stevioside, and combinations thereof. In some embodiments, the method further comprises incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide.

In some aspects, the disclosure provides a method for synthesizing rebaudioside D4 from rebaudioside E, the method comprising (a) preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate glucose (UDP-glucose), and C1m2, and (b) incubating the reaction mixture for a sufficient time to produce rebaudioside D4, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside D4. 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 anArabidopsissucrose synthase 1, anArabidopsissucrose synthase 3 and aVigna radiatesucrose synthase. In some embodiments, the sucrose synthase is anArabidopsis thalianasucrose synthase 1. In some embodiments, the Reb D4 produced is greater than 70% (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or 100%) pure. In some embodiments, the method further comprises adding a HV1 enzyme to the reaction mixture.

In some aspects, the disclosure provides a method for synthesizing rebaudioside D4 from rebaudioside E, the method comprising (a) preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate glucose (UDP-glucose), and C1m3, and (b) incubating the reaction mixture for a sufficient time to produce rebaudioside D4, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside D4. 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 anArabidopsissucrose synthase 1, anArabidopsissucrose synthase 3 and aVigna radiatesucrose synthase. In some embodiments, the sucrose synthase is anArabidopsis thalianasucrose synthase 1. In some embodiments, the Reb D4 produced is greater than 70% (e.g., greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or 100%) pure. In some embodiments, the method further comprises adding a HV1 enzyme to the reaction mixture.

In some aspects, the disclosure provides a sweetener comprising a steviol glycoside composition or steviol glycoside produced by a method as described above or as otherwise described herein.

In some aspects, the disclosure provides a GXT6 enzyme comprising the amino acid sequence of SEQ ID NO: 5. In some aspects, the disclosure provides a recombinant polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 5. In some embodiments, the recombinant polypeptide has the same or substantially the same activity as a GXT6 enzyme as described herein, e.g., a GXT6 enzyme comprising the amino acid sequence of SEQ ID NO: 5. In some aspects, the disclosure provides a host cell comprising a vector capable of producing a GXT6 enzyme having the amino acid sequence of SEQ ID NO: 5.

In some aspects, the disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6.

In some aspects, the disclosure provides a CP1 enzyme comprising SEQ ID NO: 3. In some aspects, the disclosure provides a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4.

In some aspects, the disclosure provides a method of producing Reb M using the enzymes and substrates described inFIG. 9, or a subset thereof (e.g., starting with Stevioside, Reb E, or Reb D4 and/or utilizing HV1, C1m2, C1m3, UGT76G1, CP1 and/or CR1). In some embodiments, the Reb M is produced using an in vitro reaction mixture containing the enzymes and substrates described inFIG. 9, or a subset thereof (e.g., starting with Stevioside, Reb E, or Reb D4 and/or utilizing HV1, C1m2, C1m3, UGT76G1, CP1 and/or CR1). In some embodiments, the Reb M is produced in vivo in a cell that expresses the enzymes described inFIG. 9, or a subset thereof (e.g., HV1, C1m2, C1m3, UGT76G1, CP1 and/or CR1), wherein the cell is incubated with a substrate described inFIG. 14(e.g., Stevioside, Reb E, or Reb D4). In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a plant cell.

In terms of product/commercial utility there are several dozen products containing steviol glycosides on the market in the United States and can be used in everything from analgesics to pest repellents as well as in foods and as a dietary supplement. Products containing steviol glycosides can be aerosols, liquids, gels or granular formulations.

As for the cellular system, in some embodiments, it is selected from the group consisting of bacteria, yeast, and a combination thereof, or any cellular system that would allow the genetic transformation with the selected genes and thereafter the biosynthetic production of the desired steviol glycosides from steviol. In a most preferred microbial system,E. coliare used to produce the desired steviol glycoside compounds.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawing 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 present disclosure as defined by the appended claims.

Other features and advantages of this invention will become apparent in the following detailed description of preferred embodiments of this invention, taken with reference to the accompanying drawings.

