CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/527,482, filed June 30, 2017, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[002] The field of the invention relates to methods and processes that make the production of desirable steviol glycosides more efficient and less costly. More specifically, the present disclosure relates to the use of hydrolysis by beta-glucosidase to drive the production of steviol glycosides of interest via bioconversion.

BACKGROUND OF THE INVENTION

[003] The present disclosure is focused on the conversion of identified substrates to steviol glycosides of interest. In particular, the present disclosure relates, in part, to the more efficient production of rebaudioside M ("Reb M") through the use of beta-glucosidase ("B- glul") to hydrolyze specific substrates present in disrupted recombinant cells such as recombinant microbial cells.

[004] Steviol glycosides are natural products isolated from Stevia rebaudiana leaves, and are widely used as high intensity, low-caloric sweeteners in food, feed and beverages. Naturally occurring steviol glycosides have the same base diterpene backbone structure (steviol) but differ in the number and structure of carbohydrate residue modifications (e.g. glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the steviol backbone. Steviol glycosides 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 utilization rebaudioside M itself has been generally regarded as safe ('GRAS' status) but is extremely difficult to source from extraction processes and requires significant microbial modifications to produce through microbial bioconversion alone.

[005] On 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 in wild type Stevia leaves, respectively, while the other steviol glucosides, such as Reb M are present in significantly lower amounts. It is known to use recombinant microbial cells to produce steviol glycosides of interest, however, given the range of biochemical activity of specific enzymes it can occur that a variety of steviol glycosides can be produced from such microbial strain. In these situations, the disrupted cells of these strains may contain high amounts of stevioside (CAS no. 57817-89-7), rebaudioside A (CAS no. 58543-16-1) or other steviol glycosides where Reb M is the desired product. The amounts of these compounds range from about 10-20% for stevioside and about 5-10% for rebaudioside A with other minor constituents being present in the mixture.

[006] As natural sweeteners, different steviol glycosides have different degrees of sweetness, 'mouth feel' and specific after-tastes associated with each rebaudioside species tested. 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 taste tests, while rebaudiosides A and E are 250-450 times sweeter than sucrose and the after-taste is much better than stevioside, however, a noticeable aftertaste is still present. Accordingly, the taste profiles of any stevia extracts are profoundly affected by the relative content of the steviol glycosides in the extract, which in turn may are 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 the stevia extracts, such that the taste profile varies strongly among different batches of extraction products. The taste profile of stevia extracts 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 undesirable for the use of the stevia extract as a sweetener in consumer products.

[007] In addition, the cost of isolating individual or specific combinations of steviol glycosides that are not abundant in stevia extracts is cost and resource prohibitive. The non- biological production of steviol glycosides from stevioside and Reb A is difficult. Acid hydrolysis of stevioside is troublesome because under acidic conditions the steviol that is produced rearranges into isosteviol. 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, Mao et al., US Patent Application Publication Number

US2016/0207954, A Non-Caloric Sweetener) via a fermentation pathway with modified microbes. Alternatively, other non-biologic synthetic means can be used to develop steviol glycosides of interest.

[008] From a biological perspective, all steviol glycosides are formed by a series of glycosylation reactions of steviol, which typically are catalyzed by UDP-glycosyltransferase (UGT) enzymes using uridine 5'-diphosphoglucose (UDP-glucose) as a donor of the sugar moiety. In plants, UGTs are a very divergent group of enzymes that transfer a glucose residue from UDP-glucose to steviol. In these reactions stevioside is often an intermediate in the biosynthesis of various rebaudioside compounds. For example, glycosylation of stevioside at the C-3' at the C-13-O-glucose of stevioside yields rebaudioside A; while glycosylation at the C-2' at the 19-0-glucose position of stevioside yields rebaudioside E.

[009] According to the current disclosure, a practical approach to improve the taste quality of stevia extracts is to increase the yield of those rebaudioside compounds that have more desirable taste characteristics in general and to do this via a more productive synthetic pathway and associated processes. Of those steviol glycosides tested many believe that Reb M has the most desirable taste and chemical characteristics for use in food and beverages. As stated above, however, the plant has vanishingly small amounts of this compound present in its leaves and therefore an alternative processes is needed to enable and assist in the large-scale production of this glycoside as well as to provide alternate sweeteners to the food and beverage industry.

[0010] 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 molecule, so that such production of desirable glycosides can be commercially as cost effective as possible. The present disclosure provides a method of enhancing the production of the desired steviol glycosides.

[0011] New production methods are also needed to reduce costs of steviol glycoside production and lessen the environmental impact of large scale cultivation and processing (Yao et al., 1994). One such potential solution is the use of fermentation bio-conversion technology that allows the production in certain microbial species or other recombinant cells that increases the selectivity, abundance and purity of desired steviol glycosides available for commerce and enhancing the presence of a desired rebaudioside using a hydrolytic enzyme to drive such production in cellular lysate from modified microbial cultures or other modified cell cultures. SUMMARY OF THE INVENTION

[0012] The present disclosure encompasses, in part, a method of producing enhanced amounts of rebaudioside M (Reb M) from various steviol glycosides utilizing the hydrolytic activity of beta-glucosidase. More specifically, the present disclosure provides an improved process to produce desired steviol glycosides from disrupted recombinant cells (e.g., recombinant microbial cells) containing substrates of interest. This is where such cells (e.g., microbes) have been modified to carry genes capable of generating steviol glycosides of interest including rebaudioside A (Reb A), rebaudioside E (Reb E) and/or stevioside. In this embodiment, the process comprises the steps of obtaining the material from disrupted cells (e.g., microbial cells) and carrying out enzymatic hydrolysis on the steviosides present in said waste product to enhance the production of Reb M.

[0013] 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 in dietary supplements. Products containing steviol glycosides of interest can include aerosols, liquids, or granular formulations.

[0014] As for the cellular system of the current disclosure, 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. coli are used to produce the desired steviol glycoside compounds later exposed to beta-glucosidase hydrolysis.

[0015] Beta-glucosidases are constitutive enzymes often present in the lower gastrointestinal tracts of animals and are useful as an aid in digestion and absorption of food material. According to the current disclosure B-glul is used to hydrolyze stevioside, rebaudioside E (Reb E), rebaudioside A (Reb A), rebaudioside I (Reb I), rebaudioside D (Reb D), rebaudioside G (Reb G) and rubusoside. Hydrolysis proceeds via initial formation of steviolbioside with steviol as the final product of hydrolysis.

[0016] Accordingly, aspects of the disclosure provide methods of altering the glycosylation of a steviol glycoside comprising a) providing a recombinant cell (e.g., a microbe, alga or plant cell) modified to produce a first substrate; b) disrupting said

recombinant cell to release its cellular cytosol where such cytosol contains said first substrate; c) obtaining the cytosol from said recombinant cell; d) exposing said cytosol to a beta- glucosidase where such beta-glucosidase has hydrolytic activity for sufficient time to generate a second substrate of interest through the removal of at least one glucosyl group from said first substrate; and, e) collecting said second substrate of interest.

[0017] In some embodiments, said first substrate is a steviol glycoside. In some embodiments, said second substrate of interest is Reb M. In some embodiments, said second substrate of interest is Reb B. In some embodiments, said second substrate of interest is Reb A. In some embodiments, the first and second substrate are those described in Table 1 or in a Figure herein (e.g., FIG. 9).

[0018] In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position of said steviol glycoside. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C13 position of said steviol glycoside. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from either the C19 position of said steviol glycoside or the C13 position of said steviol glycoside. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from both the C19 position of said steviol glycoside and the C13 position of said substrate.

[0019] In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position of rubusoside to produce steviol- 13-gluoside. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position of stevioside to produce steviolbioside. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position of Reb E to produce

steviolbioside. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position of Reb I to produce Reb A. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position of Reb A to produce Reb B.

[0020] In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C13 position of steviol- 13-gluoside to produce steviol. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C13 position of Reb D to produce Reb B or Reb A. In some embodiments, said hydrolytic activity functions to remove a glucosyl group from the C19 position and from the C13 position of Reb G to produce steviol- 13-glucoside.

[0021] Methods provided herein, in some aspects, further comprise the use of beta- galactosidase or pectinase enzymes to increase the speed of enzymatic hydrolysis. In some embodiments, methods provided herein further comprise expressing a steviol glycoside in a transformed cellular system; growing the cellular system in a medium; and, producing said second substrate of interest. [0022] In some embodiments, said time period for beta-glucosidase exposure to said first substrate is at least 6 hours.

[0023] In some embodiments, said time period for beta-glucosidase exposure to said first substrate is at least 12 hours. In some embodiments, said time period for beta-glucosidase exposure to said first substrate is at least 18 hours. In some embodiments, said time period for beta-glucosidase exposure to said first substrate is at least 24 hours. In some embodiments, said second substrate of interest is a steviol glycoside.

[0024] In some embodiments, the beta-glucosidase has an amino acid sequence that has 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: l. In some embodiments, the beta-glucosidase has an amino acid sequence that is at least 95% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:3.

[0025] In some embodiments, the recombinant cell is selected from the group consisting of: a bacterium, a yeast, a filamentous fungus, a cyanobacteria alga and a plant cell.

In some embodiments, the recombinant cell (e.g., microbe) is selected from the group consisting of: Escherichia; 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; Yarrowia; Klebsiella; Pantoea;

Salmonella Corynebacterium; Clostridium; and, Clostridium acetobutylicum.

[0026] In some embodiments, said second substrate of interest is a steviol glycoside.

In some embodiments, the second substrate of interest is Reb E. In some embodiments, said second substrate is a mixture of Reb E, Reb D4 and Reb M. In some embodiments, the second substrate of interest is Reb D4. In some embodiments, said second substrate of interest is a steviol glycoside mixture and the steviol glycoside content of this mixture combined is greater than any other components of the cytosol derived concentrate by weight on a dry basis.

[0027] In some embodiments, the second substrate of interest in step e) is a crude product and step e) further comprises i) purifying said crude product; and, ii) removing solvents under vacuum to provide a concentrated product.

[0028] In some embodiments, said crude product is purified by column

chromatography. In some embodiments, said crude product is purified by acid-base extraction.

In some embodiments, said crude product is purified by vacuum distillation. [0029] Methods provided herein, in some embodiments, further comprise purifying the concentrated product using a semi-preparative HPLC.

[0030] 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.

[0031] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 shows that rubusoside is hydrolyzed by B-glul enzyme and disrupted

Pichia cells. A: HPLC profiles of the rubusoside hydrolysis products, a: standard of steviol ("S"); b: standard of rubusoside ("Rub"); c-d: Rubusoside was cleaved by recombinant B-glul enzyme at 1 hour (c) and 3 hours (d); e-f: Rubusoside was hydrolyzed by disrupted Pichia cells at 1 hour (e) and 6 hours (f). B: Rubusoside hydrolysis pathway catalyzed by B-glul.

Rubusoside was hydrolyzed by recombinant B-glul enzyme and disrupted Pichia cells to produce steviol- 13-glucoside ("S-13-G") and steviol.

[0033] FIG. 2 shows that stevioside is hydrolyzed by B-glul enzyme. A: HPLC profiles of the stevioside hydrolysis products, a: standard of stevioside ("ST"); b: standard of steviolbioside ("SB"); c-e: Stevioside was hydrolyzed by recombinant B-glul enzyme at 1 hour (c), 6 hours (d) and 24 hours (e). B: Stevioside hydrolysis pathway catalyzed by B-glul. Stevioside is hydrolyzed by recombinant B-glul enzyme and disrupted pichia cells to produce steviolbioside.

[0034] FIG. 3 shows that rebaudioside E is hydrolyzed by B-glul enzyme. A: HPLC profiles of the rebaudioside E hydrolysis products, a: standard of steviolbioside ("SB"); b: standard of rebaudioside E ("E"); c-e: Rebaudioside E was hydrolyzed by recombinant B-glul enzyme at 1 hour (c), 6 hours (d) and 24 hours (e). B: Rebaudioside E hydrolysis pathway catalyzed by B-glul. Rebaudioside E is hydrolyzed by recombinant B-glul enzyme and disrupted pichia cells to produce steviolbioside. [0035] FIG. 4 shows that rebaudioside A is hydrolyzed by B-glul enzyme. A: HPLC profiles of the rebaudioside A hydrolysis products, a: standard of rebaudioside A ("Reb A"); b: standard of rebaudioside B ("Reb B"); c-d: Rebaudioside A was hydrolyzed by recombinant B- glul enzyme at 1 hour (c) and 6 hours (d). B: Rebaudioside A hydrolysis pathway catalyzed by B-glul. Rebaudioside A is hydrolyzed by recombinant B-glul enzyme and disrupted pichia cells to produce Rebaudioside B.

[0036] FIG. 5 shows that rebaudioside I is hydrolyzed by B-glul enzyme. A: HPLC profiles of the rebaudioside I hydrolysis products, a: standard of rebaudioside A ("Reb A"); b: standard of rebaudioside B ("Reb B"); c: standard of rebaudioside I ("Reb I"), d-f:

Rebaudioside I was hydrolyzed by recombinant B-glul enzyme at 1 hour (d), 6 hours (e) and 24 hours(f). B: Rebaudioside I hydrolysis pathway catalyzed by B-glul. Rebaudioside I is hydrolyzed by recombinant B-glul enzyme to produce rebaudioside A and rebaudioside B.

[0037] FIG. 6 shows that rebaudioside D is hydrolyzed by B-glul enzyme. A: HPLC profiles of the rebaudioside D hydrolysis products, a: standard of rebaudioside B ("Reb B"); b: standard of rebaudioside D ("Reb D"); c-e: Rebaudioside D was hydrolyzed by recombinant B- glul enzyme at 1 hour (c), 6 hours (d) and 24 hours(e). B: Rebaudioside D hydrolysis pathway catalyzed by B-glul. Rebaudioside D is hydrolyzed by recombinant B-glul enzyme to produce rebaudioside A and rebaudioside B.

[0038] FIG. 7 shows that rebaudioside G is hydrolyzed by B-glul enzyme and disrupted pichia cells. A: HPLC profiles of the rebaudioside G hydrolysis products, a: standard of steviol ("Steviol"), steviol-13-glucoside ("S-13-G"); b: standard of rebaudioside G ("G"); b- d: Rebaudioside G was hydrolyzed by recombinant B-glul enzyme at 0.5 hour (c), 1 hour (d) and 6 hours (e); f: Rebaudioside G was hydrolyzed by disrupted pichia cells at 24 hours (f). B: Rebaudioside G hydrolysis pathway catalyzed by B-glul. Rebaudioside G was hydrolyzed by recombinant B-glul enzyme and disrupted pichia cells to produce steviol- 13-glucoside ("S-13- G") and steviol.

[0039] FIG. 8 shows that steviol glycosides are hydrolyzed by disrupted pichia cells, a: standard of steviolbioside ("SB"); b: standard of rebaudioside B ("B"); c: Stevioside was hydrolyzed by disrupted pichia cells at 24 hours; d: Rebaudioside E ("E") was hydrolyzed by disrupted pichia cells at 24 hours; e: Rebaudioside A ("A") was hydrolyzed by disrupted pichia cells at 24 hours; f: Rebaudioside I ("I") was hydrolyzed by disrupted pichia cells at 24 hours; g: Rebaudioside D ("D") was hydrolyzed by disrupted pichia cells at 24 hours.

[0040] FIG. 9 shows that B-Glul can hydrolyze different steviol glycoside substrates. Black line: glycosylation; Dotted line: hydrolysis. [0041] FIG. 10 shows a synthetic pathway for producing Reb M and Reb WB2.

DETAILED DESCRIPTION

Explanation of Terms Used Herein:

[0042] Steviol Glycosides are a class of chemical compounds responsible for the sweet taste of the leaves of the South American plant Stevia rebaudiana (Asteraceae), and can be used as sweeteners in food, feed and beverages.

Definitions:

[0043] Cellular system is any cells that provide for the expression of ectopic proteins. It included bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes the in vitro expression of proteins based on cellular components, such as ribosomes.

[0044] Coding sequence is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence.

[0045] Growing the Cellular System. Growing includes providing an appropriate medium that would allow cells to multiply and divide. It also includes providing resources so that cells or cellular components can translate and make recombinant proteins.

[0046] Protein Expression. Protein production can occur after gene expression. It consists of the stages after DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which are ultimately folded into proteins. DNA is present in the cells through transfection - a process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus -mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection for this application.

[0047] Yeast. According to the current disclosure a yeast as claimed herein are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Yeasts are unicellular organisms which evolved from multicellular ancestors but with some species useful for the current disclosure being those that have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae.

[0048] The names of the UGT enzymes used in the present disclosure for the production of various steviol glycosides are consistent with the nomenclature system adopted by the UGT Nomenclature Committee (Mackenzie et al., "The UDP glycosyltransferase gene super family: recommended nomenclature updated based on evolutionary divergence ," PHARMACOGENETICS, 1997, vol. 7, pp. 255-269), which classifies the UGT genes by the combination of a family number, a letter denoting a subfamily, and a number for an individual gene. For example, the name "UGT76G1" refers to a UGT enzyme encoded by a gene belonging to UGT family number 76 (which is of plant origin), subfamily G, and gene number 1.

Structural Terms:

[0049] As used herein, the singular forms "a, an" and "the" include plural references unless the content clearly dictates otherwise.

[0050] To the extent that the term "include," "have," or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term "comprise" as "comprise" is interpreted when employed as a transitional word in a claim.

[0051] 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.

[0052] The term "complementary" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to describe the relationship between nucleotide bases that are capable to hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the subjection technology also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences

[0053] The terms "nucleic acid" and "nucleotide" are to be given their respective ordinary and customary meanings to a person 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 analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

[0054] The term "isolated" is to 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 an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.

[0055] The terms "incubating" and "incubation" as used herein means a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing a steviol glycoside composition.

[0056] The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can 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 deoxy inosine residues. A nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.

[0057] The terms "polypeptide," "protein," and "peptide" are to be given their respective ordinary' and customary meanings to a person of ordinary skill in the art; the three terms are sometimes used interchangeably, and are used without limitation to refer to a polymer of amino acids, or amino acid analogs, regardless of its size or function. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "polypeptide" as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to a polynucleotide product. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.

[0058] The terms "polypeptide fragment" and "fragment," when used in reference to a reference polypeptide, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.

[0059] The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein, and has substantially the same biological activity, or carries out substantially the same function as the full-length polypeptide or protein (e.g., carrying out the same enzymatic reaction).

[0060] The terms "variant polypeptide," "modified amino acid sequence" or "modified polypeptide," which are used interchangeably, refer to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., by one or more amino acid substitutions, deletions, and/or additions. In an aspect, a variant is a "functional variant" which retains some or all of the ability of the reference polypeptide.

[0061] The term "functional variant" further 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 maintains 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 non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one charged or polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine or arginine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan for another. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "conservatively substituted variant" also includes peptides wherein a residue is replaced with a chemically-derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.

[0062] The term "variant," in connection with the polypeptides of the subject technology, further includes a functionally active polypeptide having an amino acid sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical to the amino acid sequence of a reference polypeptide.

[0063] The term "homologous" in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or polypeptides that possess a "common evolutionary origin," including polynucleotides or polypeptides from super families and homologous polynucleotides or proteins from different species (Reeck et al., CELL 50:667, 1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous polypeptides can have amino acid sequences that are 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.

[0064] "Suitable regulatory sequences" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is 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 influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0065] "Promoter" is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a 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 is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.

Promoters, which cause a gene to be expressed in most cell types at most times, are commonly referred to as "constitutive promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. [0066] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the

transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0067] The term "expression" as used herein, is to be given its ordinary and customary meaning to a person 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 fragment of the subject technology. "Over-expression" refers to the production of a gene product in transgenic or recombinant organisms that exceeds levels of production in normal or non-transformed organisms.

[0068] "Transformation" is to be given its ordinary and customary meaning to a person of original Y skill in the aft, and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or

[0069] The terms "transformed," "transgenic," and "recombinant," when used herein in connection with host cells, are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.

[0070] The terms "recombinant," "heterologous," and "exogenous," when used herein in connection with polynucleotides, are to be given their ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found.

[0071] Similarly, the terms "recombinant," "heterologous," and "exogenous," when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.

[0072] The terms "plasmid," "vector," and "cassette" are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and 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 a single- or double- stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0073] "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell.

[0074] "Expression cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.

[0075] The present disclosure relates to the production of a steviol glycoside of interest by B-glul enzyme.

Synthetic Biology

[0076] Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described, for example, by Sambrook, J., Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., IN CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, published by Greene Publishing and Wiley-Interscience, 1987; (the entirety of each of which is hereby incorporated herein by reference).

[0077] 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 the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

[0078] Glycosylation is often considered a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of transferases in most plant species that have been studied to date. These glycosyltransferases (GTs) have been classified into over 60 families. Of these, the family of GT enzymes, also known as the UDP glycosyltransferases (UGTs), transfer UDP-activated sugar moieties to specific acceptor molecules. These are the molecules that transfer such sugar moieties in the steviol glycosides to create various rebaudiosides. Each of these UGTs have their own activity profile and preferred structure locations where they transfer their activated sugar moieties.

Production Systems

[0079] Expression of proteins in prokaryotes is most often carried out in a bacterial host cell with 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 a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase 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. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such vectors are within the scope of the present disclosure.

[0080] In an embodiment, the expression vector includes those genetic elements for expression of the recombinant polypeptide in bacterial cells. The elements for transcription and translation in the bacterial cell can include a promoter, a coding region for the protein complex, and a transcriptional terminator.

[0081] A person of ordinary skill in the art will be aware of the molecular biology techniques available for the preparation of expression vectors. The polynucleotide used for incorporation into the expression vector of the subject technology, as described above, can be prepared by routine techniques such as polymerase chain reaction (PCR).

[0082] A number of molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homo-polymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0083] In an alternative embodiment, synthetic linkers containing one or more restriction sites provide are used to operably link the polynucleotide of the subject technology to the expression vector. In an embodiment, the polynucleotide is generated by restriction endonuclease digestion. In an embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a polynucleotide carrying polymeric linker sequences at its ends. These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the

polynucleotide.

[0084] Alternatively, a vector having ligation-independent cloning (LIC) sites can be employed. The required PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, NUCL. ACID. RES. 18 6069-74, (1990), Haun, et al, BIOTECHNIQUES 13, 515-18 (1992), which is incorporated herein by reference to the extent it is consistent herewith).

[0085] In an embodiment, in order to isolate and/or modify the polynucleotide of interest for insertion into the chosen plasmid, it is suitable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.

[0086] In an embodiment, a polynucleotide for incorporation into an expression vector of the subject technology is prepared by the use of PCR using appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product. In an embodiment, the amplification primers contain restriction endonuclease recognition sites, which allow the amplified sequence product to be cloned into an appropriate vector.

[0087] The expression vectors can be introduced into a recombinant host cell (e.g., plant or microbial host cells) by conventional transformation or transfection techniques.

Transformation of appropriate cells with an expression vector of the subject technology is accomplished by methods known in the art and typically depends on both the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemoporation or electroporation.

[0088] Successfully transformed cells, that is, those cells containing the 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 polypeptides described herein. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.

[0089] The host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector,

[0090] 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 cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.

[0091] Microbial host cell and other host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins is well known to those skilled in the art. Any of these could be used to construct vectors for expression of the recombinant polypeptide of the subjection technology in a microbial host cell. These vectors could then be introduced into appropriate microorganisms via

transformation to allow for high level expression of the recombinant polypeptide of the subject technology.

[0092] Vectors or cassettes useful for the transformation of suitable microbial host cells and other host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.

[0093] Initiation control regions or promoters, which are useful to drive expression of the recombinant polypeptide in the desired microbial host cell or other host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the subject technology including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOXI (useful for expression in Pichia); and lac, trp, JPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli).

[0094] Termination control regions may also be derived from various genes native to the microbial hosts. A termination site optionally may be included for the microbial hosts described herein.

[0095] In plant cells, the expression vectors of the subject technology can include a coding region operably linked to promoters capable of directing expression of the recombinant polypeptide of the subject technology in the desired tissues at the desired stage of

development. For reasons of convenience, the polynucleotides to be expressed may comprise promoter sequences and translation leader sequences derived from the same polynucleotide. 3' non-coding sequences encoding transcription termination signals should also be present. The expression vectors may also comprise one or more introns in order to facilitate polynucleotide expression.

[0096] For plant host cells, any combination of any promoter and any terminator capable of inducing expression of a coding region may be used in the vector sequences of the subject technology. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes. One type of efficient plant promoter that may be used is a high-level plant promoter. Such promoters, in operable linkage with an expression vector of the subject technology should be capable of promoting the expression of the vector. High level plant promoters that may be used in the subject technology include the promoter of the small subunit (ss) of the ribulose-1,5- bisphosphate carboxylase for example from soybean (Berry-Lowe et al., J. MOLECULAR AND APP. GEN., 1:483 498 (1982), the entirety of which is hereby incorporated herein to the extent it is consistent herewith), and the promoter of the chlorophyll alb binding protein. These two promoters are known to be light-induced in plant cells (see, for example, GENETIC

ENGINEERING OF PLANTS, AN AGRICULTURAL PERSPECTIVE, A. Cashmore, Plenum, N.Y. (1983), pages 29 38; Coruzzi, G. et al., The Journal of Biological CHEMISTRY, 258: 1399 (1983), and Dunsmuir, P. et al., JOURNAL OF MOLECULAR AND APPLIED GENETICS, 2:285 (1983), each of which is hereby incorporated herein by reference to the extent they are consistent herewith).

Cell Disruption Techniques

[0097] Cell disruption is a collection of techniques used for releasing biomolecules of interest from inside the cell. Many biotechnologically produced compounds are intracellular and must be released from cells before recovery. The efficient recovery of products requires cell disruption, which can be achieved by using different methods and technologies, either mechanical or non-mechanical methods depending upon the biomolecule of interest and the cellular system being used. It should be noted that all disruption methods will release molecules of interest, here steviol glycosides, and other molecules that can cause the breakdown of proteins of interest within the lysate. Considerations need to be made to prevent this if protein activity is important to the downstream work. Lysing samples in highly denaturing solutions or high pH minimizes enzymatic activity. Performing the disruption on ice or lower temperatures will also help minimize degradation of samples. Thus, one technique to inhibit protease activity is to have a general phosphatase inhibitor cocktail available for the lysate once disrupted.

Known Disruption Techniques that can be Used According to the Current Disclosure

[0098] Standard disruption consists of mechanical homogenization, French press, sonication, bead homogenization, grinding, freeze-thaw lysis, detergent lysis, enzymatic lysis and osmotic lysis.

[0099] Mechanical homogenization includes using a hand-held device, like a Dounce homogenizer, or something like a blender to homogenize the tissue. This method is useful for non-seed plant type material or soft tissues (i.e. Liver tissue.)

[00100] French press uses shear force to homogenize the tissue. An example of this would be taking a cell suspension and forcing it through a narrow gauge syringe using a syringe barrel and plunger. This works well with bacteria, yeast, fungi, algae and mammalian cell culture but is difficult to scale-up.

[00101] Sonication uses short bursts of ultrasonic waves to disrupt the tissue. This method generates a lot of heat and will typically have to be performed on ice to maintain the protein. This method is also effective for bacteria, yeast, fungi, algae and mammalian cell culture and is the preferred method for the current disclosure. [00102] Bead homogenization involves using glass or metal beads to apply gentle abrasion while vortexing them with the tissue or cell suspension. Grinding involves using a mortar and pestle to homogenize the tissue sample. The most common method is to freeze and grind the sample using liquid nitrogen. Freeze-thaw lysis is pretty much as it sounds, using liquid nitrogen or a freezer to freeze the cells and then allow them to thaw.

When cells are frozen the water inside the cells expands as it freezes causing the cells to burst open. This method is effective for mammalian cells.

[00103] Enzymatic lysis consists of suspending the cells in iso-osmotic buffers containing enzymes that can digest the cell wall (i.e. Zymolyase for yeast cells). This lysis method is often used in conjunction with another disruption technique (usually sonication) to ensure complete lysis of the sample. This technique is effective with bacteria, yeast, fungi, algae, non-seed plant material and mammalian cell culture and is a technique that is also embodied in the current disclosure.

[00104] Detergent lysis involves suspending the cells in a detergent solution to solubilize the cell membrane, releasing the cell contents. This method also generally uses another lysis method, such as sonication to ensure complete lysis. This method is effective with mammalian cell culture.

[00105] Osmotic lysis involves suspending cells in hypotonic (low salt) solution.

This causes the cells to swell and eventually burst releasing the contents of the cells for further use.

[00106] Typically, in the in vitro method of the subject technology, the weight ratio of the recombinant polypeptide to the substrate, on a dry weight basis, 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.

[00107] Typically, the reaction temperature of the in vitro method is from about

20°C to about 40°C, suitably from 25° C to about 37° C, more suitably from 28° C to about 32° C.

[00108] One with skill in the art will recognize that the steviol glycoside composition produced by the method described herein can be further purified and mixed with other steviol glycosides, flavors, or sweeteners to obtain a desired flavor or sweetener composition. For example, a composition enriched with rebaudioside D4 or rebaudioside M produced as described herein can be mixed with a natural stevia extract containing

rebaudioside A as the predominant steviol glycoside, or with other synthetic or natural steviol glycoside products to make a desired sweetener composition. Alternatively, a substantially purified steviol glycoside (e.g., rebaudioside D4 or rebaudioside M) obtained from the steviol glycoside composition described herein can be combined with other sweeteners, such as sucrose, maltodextrin, aspartame, sucralose, neotame, acesulfame potassium, and saccharin. The amount of steviol glycoside relative to other sweeteners can be adjusted to obtain a desired taste, as known in the art. The steviol glycoside composition described herein (including rebaudioside D, rebaudioside E, rebaudioside D4, rebaudioside M or a combination thereof) can be included in food products (such as beverages, soft drinks, ice cream, dairy products, confectioneries, cereals, chewing gum, baked goods, etc.), dietary supplements, medical nutrition, as well as pharmaceutical products.

Analysis of Sequence Similarity Using Identity Scoring

[00109] As used herein "sequence identity" refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.

[00110] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) as compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, MA). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this disclosure "percent identity" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[00111] The percent of sequence identity is preferably determined using the "Best Fit" or "Gap" program of the Sequence Analysis Software Package™ (Version 10;

Genetics Computer Group, Inc., Madison, WI). "Gap" utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, JOURNAL OF MOLECULAR BIOLOGY 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit" performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, ADVANCES IN APPLIED MATHEMATICS, 2:482-489, 1981, Smith et al., NUCLEIC ACIDS RESEARCH 11:2205-2220, 1983). The percent identity is most preferably determined using the "Best Fit" program.

[00112] Useful methods for determining sequence identity are also disclosed in the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al., J. MOL. BlOL. 215:403-410 (1990); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.

[00113] As used herein, the term "substantial percent sequence identity" refers to a percent sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity. Thus, one embodiment of the disclosure is a polynucleotide molecule that has 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 with a polynucleotide sequence described herein. Polynucleotide molecules that have the activity of the beta-glucosidase genes of the current disclosure are capable of directing the production of a variety of steviol glycosides and have a substantial percent sequence identity to the polynucleotide sequences provided herein and are

encompassed within the scope of this disclosure. Identity and Similarity

[00114] Identity is the fraction of amino acids that are the same between a pair of sequences after an alignment of the sequences (which can be done using only sequence information or structural information or some other information, but usually it is based on sequence information alone), and similarity is the score assigned based on an alignment using some similarity matrix. The similarity index can be any one of the following BLOSUM62, PAM250, or GONNET, or any matrix used by one skilled in the art for the sequence alignment of proteins.

[00115] Identity is the degree of correspondence between two sub-sequences (no gaps between the sequences). An identity of 25% or higher implies similarity of function, while 18- 25% implies similarity of structure or function. Keep in mind that two completely unrelated or random sequences (that are greater than 100 residues) can have higher than 20% identity. Similarity is the degree of resemblance between two sequences when they are compared. This is dependent on their identity.

[00116] As is evident from the foregoing description, certain aspects of the present disclosure are not limited by the particular details of the examples illustrated herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. It is accordingly intended that the claims shall cover all such modifications and applications that do not depart from the spirit and scope of the present disclosure.

[00117] Moreover, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to or those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described above.

[00118] 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. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

[00119] According to the current disclosure beta-glucosidase ("B-glul") can be used to hydrolyze steviol glycosides and reduce the production of unwanted steviol glycosides in a novel way. The presence of undesirable steviol glycosides in stevia extract or

fermentation productions affect the overall taste profile and usefulness of steviol glycosides. By reducing production steps the current disclosure will reduce both the cost of purification and the production of desired steviol glycosides in stevia crude extract.

[00120] The disclosure will be more fully understood upon consideration of the following non-limiting Examples. It should be understood that the Examples below, 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 of the subject technology to adapt it to various uses and conditions.

Example 1; Identification of beta-glycosidase in Pichi Pastoris

[00121] According to the current disclosure Pichia pastoris genome analysis was completed and several beta-glycosidase candidate genes were identified. Full length DNA fragments of all candidate beta-glucosidase genes were commercially synthesized. Almost all codons of the cDNA were changed to those preferred for E. coli (Genscript, NJ). The synthesized DNA was cloned into a bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen). These same experiments could be performed in Yarrowia lipolytica with sequences optimized for such use.

[00122] Each expression construct was transformed into E. coli BL21 (DE3), which was subsequently grown in LB media containing 50 μg/mL kanamycin at 37 °C until reaching an OD600 of 0.8-1.0. Protein expression was induced by addition of 1 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) and the culture was further grown at 16 °C for 22 hr. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellets were collected and were either used immediately or stored at -80 °C.

[00123] The cell pellets typically were re-suspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25ug/ml lysozyme, 5ug/ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells were disrupted by sonication under 4° C, and the cell debris was clarified by centrifugation (18,000 x g; 30 min).

Supernatant was loaded to an equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading of protein sample, the column was washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged beta-glucosidase recombinant polypeptides were eluted by equilibration buffer containing 250mM imidazole. [00124] The purified candidate beta-glucosidase recombinant polypeptides were assayed for de-glycosylation activity by using various steviol glycosides as substrate.

Typically, the recombinant polypeptide (10 μg) was tested in a 300 μΐ in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer, pH 7.2, 1 mg/ml steviol glycoside. The reaction was performed at 30-37 °C and 50ul reaction was terminated by adding 200 μΐ _^ 1-butanol at various time points. The samples were extracted three times with 200 μΐ _^ 1-butanol. The pooled fraction was dried and dissolved in 100 μΐ _^ 80% methanol for high-performance liquid chromatography (HPLC) analysis.

[00125] Pichia cells were suspended in extraction buffer (50mM potassium phosphate buffer, pH 7.2; 150mM NaCl). After sonication, the supernatant (crude extract) was collected by centrifuge set at 12,000g at 4□ . The resulting crude protein (50ug) was tested in a 300ul in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer, pH 7.2, 1 mg/ml steviol glycoside. The reaction was performed at 30-37 °C and 50ul reaction was terminated by adding 200 μΐ _^ 1-butanol at various time points. The samples were extracted three times with 200 μΐ _^ 1-butanol. The pooled fraction was dried and dissolved in 100 μΐ _^ 80% methanol for high-performance liquid chromatography (HPLC) analysis.

[00126] HPLC analysis was then performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA), including a quaternary pump, a temperature controlled column compartment, an auto sampler and a UV absorbance detector. A Synergi Hydro-RP column (Phenomenex) with guard column was used for the characterization of steviol glycosides in the pooled samples. Acetonitrile in water was used mobile phase in the HPLC analysis. The detection wavelength used in the HPLC analysis was 210nm. After activity screening, we found Beta-glucosidase (B-glul, SEQ: 1) has strong activity to cleave related steviol glycosides and is therefore a useful tool in the production of steviol glycosides of interest.

Example 2: Hydrolysis of rubusoside by use of the recombinant B-glul and disrupted Pichia cells

[00127] Rubusoside can be hydrolyzed by recombinant B-glul enzyme and disrupted Pichia cells to produce steviol- 13-glucoside. The produced steviol- 13-glucoside can be subsequently hydrolyzed to produce steviol (FIG. 1 B). The reactions were setup as described in Example 1. lg/L rubusoside was added in the reaction as substrate. As shown in FIG. 1A, B-glul can remove a glucosyl group from C19 position of rubusoside to produce steviol- 13-gluoside (FIG. 1A - c). The produced steviol-13-gluocside (S- 13-G) will be converted to steviol at later time point (FIG. 1A - d). B-glul remove another glucosyl group from C13 position of steviol-13-glucoside. Since B-glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze rubusoside to steviol-13-glucoside and continually to produce steviol (FIG.1A - e and f).

Example 3: Hydrolysis of stevioside by use of the recombinant B-glul and disrupted Pichia cells

[00128] Stevioside can be hydrolyzed by recombinant B-glul enzyme and disrupted Pichia cells to produce steviolbioside (FIG. 2B). The reactions were setup as described in Example 1. lg/L stevioside was added in the reaction as substrate. As shown in FIG. 2, B-glul can remove a glucosyl group from C19 position of stevioside to produce steviolbioside (FIG. 2A c and d). Since B-glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze stevioside to steviolbioside (FIG. 8 C).

Example 4: Hydrolysis of rebaudioside E by use of the recombinant B-glul and disrupted Pichia cells

[00129] Rebaudioside E can be hydrolyzed by recombinant B-glul enzyme to produce steviolbioside (FIG. 3B). The reactions were setup as described in Example 1. lg/L rebaudioside E was added in the reaction as substrate. As shown in FIG. 3, B-glul can remove the glucosyl group from C19 position of rebaudioside E to produce stevioside and

steviolbioside (FIG. 3 A c-e). Since B-glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze rebaudioside E to steviolbioside (FIG. 8 D).

Example 5: Hydrolysis of rebaudioside A by use of the recombinant B-glul and disrupted Pichia cells

[00130] Rebaudioside A can be hydrolyzed by recombinant B-glul enzyme to produce rebaudioside B (FIG. 4B). The reactions were setup as described in Example 1. lg/L rebaudioside A was added in the reaction as substrate. As shown in FIG. 4, B-glul can remove the glucosyl groups from C19 position of rebaudioside A to produce rebaudioside B (FIG. 4A c-d). Since B-glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze rebaudioside A to

rebaudioside B (FIG. 8e). Example 6: Hydrolysis of rebaudioside I by use of the recombinant B-glul and disrupted Pichia cells

[00131] Rebaudioside I can be hydrolyzed by recombinant B-glul enzyme to produce rebaudioside A and the produced A can be hydrolyzed to produce rebaudioside B (FIG. 5B). The reactions were setup as described in Example 1. lg/L rebaudioside I was added in the reaction as substrate. As shown in FIG. 5, B-glul can remove a glucosyl group from C19 position of rebaudioside I to produce rebaudioside A and subsequently remove another glucosyl group at C19 position of rebaudioside A to produce rebaudioside B (FIG. 5A d-f). Rebaudioside I can be converted to rebaudioside A completely at 24hrs (FIG.5A f). Since B- glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze rebaudioside I to rebaudioside B (FIG. 8 f).

Example 7: Hydrolysis of rebaudioside D by use of the recombinant B-glul and disrupted Pichia cells

[00132] Rebaudioside D can be hydrolyzed by recombinant B-glul enzyme to produce rebaudioside B (FIG. 6B) and Reb A. The reactions were setup as described in Example 1. lg/L rebaudioside D was added in the reaction as substrate. As shown in FIG. 6, B- glul can remove the glucosyl groups from C 19 position of rebaudioside D to produce rebaudioside (FIG. 6A c-e). Since B-glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze rebaudioside D to rebaudioside B (FIG. 8g).

Example 8: Hydrolysis of rebaudioside G by use of the recombinant B-glul and disrupted Pichia cells

[00133] Rebaudioside G can be hydrolyzed by recombinant B-glul enzyme and disrupted Pichia cells to produce steviol-13-glucoside. The produced steviol-13-glucoside can be continually hydrolyzed to produce steviol (FIG. 7 B). The reactions were setup as described in Example 1. lg/L rebaudioside G was added in the reaction as substrate. As shown in FIG. 7A, B-glul can remove the glucosyl groups from C13 and C19 position of rebaudioside G to produce steviol- 13-gluoside (FIG. 7A b-c). The produced steviol- 13-gluocside (S-13-G) will be converted to steviol (FIG. 7A). The produced steviol- 13-glucoside will be converted to steviol completely at 24hrs (FIG. 7A d). B-glul remove another glucosyl group from C13 position of steviol- 13-glucoside. Since B-glul is an intercellular enzyme in Pichia cells, the disrupted Pichia cells release B-glul enzyme, which has same enzymatic activity to hydrolyze rebaudioside G to steviol- 13-glucoside and continually to produce steviol (FIG.7A e).

[00134] According to the current disclosure we identified and quantified the enzymatic activity of beta-glucosidase on a Pichia pastoris cell lysate. This enzyme has specific activity that allows it to hydrolyze specific steviol glycosides (Table 1). Referring to FIG. 7, Reb G is hydrolyzed by B-glul enzyme and disrupted Pichia cells. The HPLC shows the products of Reb G hydrolysis. In this effort steviol ("S"); steviol-13-glucoside ("S-13-G"); rubusoside ("Rub") were produced. The time course was a series of time points over 24 hours. Steviol-13-glucoside ("S-13-G") and steviol were produced by recombinant Pichia pastoris cultures.

[00135] Using this technique we have hydrolyzed Reb M to remove Reb A, Reb

D3, Reb D4, Reb W, Reb V, Reb E, Reb I and Reb D to increase purification efficacy (FIG. 9). This technique involves the use of stevioside and Reb A to produce steviol, steviol- 13-0- glucoside, steviolbioside and Reb B through beta-glucosidase bioconversion. Referring to FIG. 8, steviol glycosides were hydrolyzed by disrupted Pichia pastoris cells. The samples included a steviolbioside ("SB") and rebaudioside B ("B") standard. In our experiments stevioside was hydrolyzed by disrupted Pichia cells at 24 hours; Reb E and Reb A were hydrolyzed by disrupted Pichia cells at 24 hours. Likewise, Reb I and Reb D were hydrolyzed by disrupted Pichia cells at 24 hours (FIG. 8).

[00136] In another embodiment of the current disclosure we can use beta- glucosidase to control steviol glycoside production pathways. (Ex: Reb M from Reb W) (FIG. 10).

[00137] According to preferred embodiments of the current disclosure the beta- glucosidase can be used to hydrolyze additional steviol glycosides including Reb V, Reb W, Reb Zl, Reb Z2, Reb D3 and Reb D4 to drive them to steviol glycosides of interest (FIG. 9). Thereafter we can separate, collect and concentrate the hydrolyzed products. In another embodiment of the current disclosure we use a beta-glucosidase deficient strain, which will reduce side production in our bioconversion process. Table 1: Summary of hydrolysis of steviol glycosides by B-glul.

STATEMENT OF INDUSTRIAL APPLICABILITY / TECHNICAL FIELD

[00138] This disclosure has applicability in the food, feed, beverage, and pharmacological industries. This disclosure relates generally to a method for the steviol glycosides biosynthesis via hydrolysis of beta glucosidase.

Literature Cited and Incorporated by Reference:

1. Brandle, J. E. et al., (1998). Stevia Rebaudiana: Its Agricultural, Biological, and Chemical Properties, CANADIAN J. PLANT SCIENCE. 78 (4): 527-36.

2. Ceunen, S., and J.M. C. Geuns, Steviol Glycosides: Chemical Diversity, Metabolism, and Function, J. NAT. PROD., 2013, 76 (6), pp 1201-28 (2013).

3. Du J et al., (2011), Engineering microbial factories for synthesis of value-added products, J IND MICROBIOL BIOTECHNOL. 38: 873-90.

4. GRAS Notices, USA Food and Drug Administration, United States Health & Human

Services. (2016) (relevant to steviol glycosides & polyglycosides). 5. Hausler A, and Miinch T., (1997), Microbial production of natural flavors, ASM NEWS

63:551-59.

6. Prakash I., et al.; Isolation and Characterization of a Novel Rebaudioside M Isomer from a Bioconversion Reaction of Rebaudioside A and NMR Comparison Studies of Rebaudioside M Isolated from Stevia rebaudiana Bertoni and Stevia rebaudiana Morita, BlOMOLECULES,

2014 Jun; 4(2): 374-89. (Published online 2014 Mar 31. 2014).

7. Prakash I., et al., Development of Next Generation Stevia Sweetener: Rebaudioside M, FOODS, 2014, 3: 162-175.

8. Shockey JM. Et a., (2003), Arabidopsis contains a large superfamily of acyl-activating enzymes: phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme A synthetases. PLANT PHYSIOL 132 1065-76. Sequences of Interest:

Sequences:

B-glul:

B-glul Amino Acid: (SEQ ID NO: 1 ) Pichia pastoris sequence (GS 115)

Mtqldvesliqeltlnekvqllsgsdfwhttpv gipkmrlsdgpngvrgtkffngvptacfpcgtglgatfdkellkeagslmade akakaasvvlgptaniargpnggrgfesfgedpvvnglssaaminglqgkyiaatmkhyv cndlemdrncidaqvshralrevyll pfqiavrdanpraimtaynkangehvsqskflldevlrkewgwdgllmsdwfgvydakss itngldlempgppqcrvhsatdha insgeihindvdervrsllslinychqsgvteedpetsdnntpetieklrkisresivll kdddmrsilplkksdkiavignnakqaayc gggsasvlsyhtttpfdsiksrledsntpaytigadayknlpplgpqmtdsdgkpgfdak ffvgsptskdrklidhfqltnsqvflvdy yneqipenkefyvdvegqfipeedgtynfgltvfgtgrlfvddklvsdssqnqtpgdsff glaaqevigsihlvkgkaykikvlygss vtrtyeiaasvafeggaftfgaakqrnedeeiaraveiakandkvvlciglnqdfesegf drpdikipgatnkmvsavlkanpntviv nqtgtpvempwasdapvilqawfggseagtaiadvlfgdynpsgkltvtfplrfednpay lnfqsnkqacwygedvyvgyryyet idrpvlfpfghglsftefdftdmfvrleeenlevevvvrntgkydgaevvqlyvapvsps lkrpikelkeyakiflasgeaktvhlsvp ikyatsffdeyqkkwcsekgeytillgsssadikvsqsitlekttfwkgl

B-glul DNA: (SEQ ID NO: 2) (codon optimized for E. coli)

ATGACCCAACTGGATGTGGAGAGCCTGATTCAAGAGCTGACCCTGAACGAAAAGG TGCAACTGCTGAGCGGTAGCGACTTCTGGCATACCACCCCGGTTCGTCGTCTGGGC ATCCCGAAGATGCGTCTGAGCGACGGTCCGAACGGCGTTCGTGGTACCAAATTCTT TAACGGTGTTCCGACCGCGTGCTTCCCGTGCGGTACCGGTCTGGGCGCGACCTTTG ACAAGGAACTGCTGAAAGAGGCGGGTAGCCTGATGGCGGATGAAGCGAAAGCGA AAGCGGCGAGCGTGGTTCTGGGTCCGACCGCGAACATTGCGCGTGGTCCGAACGG TGGCCGTGGCTTCGAGAGCTTCGGCGAGGACCCGGTGGTTAACGGTCTGAGCAGC GCGGCGATGATCAACGGCCTGCAGGGCAAGTACATTGCGGCGACCATGAAACACT ATGTTTGCAACGATCTGGAAATGGACCGTAACTGCATTGACGCGCAAGTTAGCCA CCGTGCGCTGCGTGAGGTGTACCTGCTGCCGTTCCAAATCGCGGTGCGTGATGCGA ACCCGCGTGCGATTATGACCGCGTATAACAAGGCGAACGGCGAACACGTTAGCCA GAGCAAATTCCTGCTGGACGAAGTGCTGCGTAAGGAGTGGGGCTGGGATGGTCTG CTGATGAGCGACTGGTTTGGTGTTTACGATGCGAAAAGCAGCATCACCAACGGCC TGGACCTGGAGATGCCGGGTCCGCCGCAGTGCCGTGTGCACAGCGCGACCGATCA CGCGATCAACAGCGGCGAAATCCACATTAACGATGTTGACGAGCGTGTGCGTAGC CTGCTGAGCCTGATTAACTACTGCCACCAAAGCGGTGTTACCGAGGAAGATCCGG AAACCAGCGACAACAACACCCCGGAAACCATCGAGAAGCTGCGTAAAATCAGCC GTGAGAGCATTGTGCTGCTGAAGGACGATGACCGTAACCGTAGCATTCTGCCGCT G A AG A A A AGC G AC A A A ATC GCGGTT ATTGGT A AC A AC GCG A A AC A AGC GGC GT A TTGCGGTGGCGGTAGCGCGAGCGTGCTGAGCTATCACACCACCACCCCGTTCGAC AGCATCAAGAGCCGTCTGGAAGATAGCAACACCCCGGCGTACACCATTGGTGCGG ACGCGTATAAAAACCTGCCGCCGCTGGGTCCGCAAATGACCGATAGCGACGGCAA GCCGGGTTTTGATGCGAAATTCTTTGTTGGCAGCCCGACCAGCAAGGATCGTAAAC TGATCGACCACTTCCAGCTGACCAACAGCCAAGTTTTTCTGGTGGACTACTATAAC GAACAGATCCCGGAAAACAAGGAGTTCTACGTTGACGTGGAGGGTCAATTTATTC CGGAGGAAGATGGCACCTATAACTTCGGTCTGACCGTGTTTGGTACCGGCCGTCTG TTCGTTGATGACAAACTGGTTAGCGACAGCAGCCAGAACCAAACCCCGGGCGATA GCTTCTTTGGTCTGGCGGCGCAGGAAGTGATCGGCAGCATTCACCTGGTGAAGGGT AAAGCGTACAAGATCAAAGTTCTGTATGGCAGCAGCGTGACCCGTACCTACGAAA TTGCGGCGAGCGTTGCGTTTGAGGGCGGTGCGTTCACCTTTGGTGCGGCGAAACAG CGTAACGAAGACGAGGAAATCGCGCGTGCGGTGGAGATTGCGAAGGCGAACGAC AAAGTGGTTCTGTGCATCGGCCTGAACCAAGATTTCGAAAGCGAGGGTTTTGATCG TCCGGACATCAAGATTCCGGGCGCGACCAACAAAATGGTTAGCGCGGTGCTGAAG GCGAACCCGAACACCGTTATTGTGAACCAGACCGGTACCCCGGTTGAGATGCCGT GGGCGAGCGATGCGCCGGTGATCCTGCAAGCGTGGTTTGGCGGTAGCGAGGCGGG TACCGCGATTGCGGATGTTCTGTTTGGCGACTACAACCCGAGCGGCAAGCTGACC GTGACCTTCCCGCTGCGTTTTGAGGATAACCCGGCGTACCTGAACTTCCAGAGCAA CAAACAAGCGTGCTGGTATGGCGAAGACGTTTACGTGGGTTATCGTTACTATGAG ACCATCGATCGTCCGGTGCTGTTCCCGTTTGGTCACGGCCTGAGCTTCACCGAGTT CGATTTTACCGACATGTTTGTTCGTCTGGAGGAAGAGAACCTGGAAGTTGAGGTGG TTGTGCGTAACACCGGCAAGTACGACGGTGCGGAAGTGGTGCAGCTGTATGTTGC GCCGGTTAGCCCGAGCCTGAAACGTCCGATCAAGGAACTGAAAGAGTACGCGAAA ATTTTCCTGGCGAGCGGTGAAGCGAAGACCGTTCACCTGAGCGTGCCGATCAAAT ACGCGACCAGCTTCTTTGATGAGTATCAAAAGAAATGGTGCAGCGAAAAGGGCGA GTATACCATTCTGCTGGGTAGCAGCAGCGCGGACATCAAAGTTAGCCAAAGCATC ACCCTGGAAAAAACCACCTTCTGGAAAGGTCTGTAA

B-glu2 Amino Acid: (SEQ ID NO: 3 ) Pichia pastoris sequence (GS 115)

MKSQLIFMALASLVASAPLEHQQQHHKHEKRAVVTQTVTVAAGQTAAAGSAQAVVT SSAAPASVASSAAASASSSSSSYTSGASGDLSSFKDGTIKCSEFPSGDGVVSVSWLGFG GWSSIMNLQGGTSESCENGYYCSYACEAGYSKTQWPSNQPSDGRSVGGLLCKDGLLY RSNTAFDTLCVPGKGTASVENNVSKGISICRTDYPGSENMCVPTWVDAGNSNTLTVV DEDNYYEWQGLKTSAQYYVNNAGVSVEDGCIWGDESSGVGNWAPLVLGAGSTGGL TYLSLIPNPNNKKAPNFNVKIVATDGSSINGDCKYENGIFVGSSTDGCTVTVTSGSAKL VFY

B-glu2 DNA (SEQ ID NO: 4 ) Pichia pastoris sequence (GS 115)

atgaaaagccagctgatctttatggctttggcctcccttgtagcaagtgcaccgctg gaacaccagcagcagcatcataaacatgagaaa cgcgccgtagttacgcagacagtaactgttgcggcgggccagacagcagcagcgggttcc gcccaggcagttgttacctcaagcgcg gcgccagcatccgttgcttcaagtgcggccgcgtctgctagctcatcttcttccagctat acctctggcgcttcaggcgatcttagtagtttc aaagatggtactattaaatgttcagaattcccatcaggggatggcgtggtgtccgtctct tggttaggcttcggcggctggtctagtattatg aatctgcagggtggtacttcagagagttgtgagaacggctattattgttcatatgcatgt gaagccggttatagcaaaacacagtggccatc taaccagccgtcagatgggagatcagtgggagggttgctgtgtaaagatggcctgttata tcgctccaatacagcgttcgatacattatgtg tgcctggaaaaggtacagcatccgtggagaataatgtgtctaaaggtatttccatttgta gaacggattatccggggtctgaaaacatgtgc gtcccgacgtgggtcgatgccggtaactcaaacaccttgacagtggtagatgaagataat tattatgaatggcagggccttaaaactagtg ctcagtattatgtgaataacgccggtgttagtgttgaagatgggtgcatctggggcgatg agtccagcggcgttggaaactgggcgccgt tggttttgggggccggttccacggggggtctgacctatctgtctctgattccgaatccaa acaacaaaaaagcaccgaattttaacgtaaa aatcgtggccacggatggaagttcaattaacggagattgcaaatatgaaaatgggatctt tgtcggttcttcaaccgatggctgcacggta actgttacctcaggtagtgcaaaactggttttttattaa

B-glu3 Amino Acid (SEQ ID NO: 5 ) Pichia pastoris sequence (GS 115) Mqvksivnlllacslavarpleha hqhdkrgvvvvtktivvdgstveataaaqvqehaetfaestpsavvssssapssassasa pas sgsfsagtkgvtyspyqagggcktaeevasdlsqltgyeiirlygvdcnqvenvfkakap gqklflgiffvdaiesgvsaiasavksy gswddvhtvsvgnelvnngeatvsqigqyvstaksalrsagftgpvlsvdtfiavinnpg lcdfadeyvavnahaffdggiaasgag dwaaeqiqrvssacggkdvlivesgwpskgdtngaavpsksnqqaavqslgqkigsscia fnafndywkadgpfnaekywgil ds

B-glu3 DNA (SEQ ID NO: 6 ) Pichia pastoris sequence (GS 115)

Atgcaggttaaatctattgttaatttactgcttgcctgttccttggctgtggcgcgt ccgttggaacacgctcaccatcagcatgataaacgc ggcgttgtagtagtaacgaaaaccatcgtcgttgatggtagcacagttgaggctaccgcc gctgctcaggtgcaggagcatgcagaaac ctttgcagaatcaaccccgtcagccgtcgtttccagttcatccgccccttcatcagcaag ctcagcttccgctccagctagttcaggttctttt tcagctggtaccaaaggcgtgacatattctccatatcaggccggtggtgggtgtaaaaca gcggaagaagtggcatccgatctgtcaca gcttaccggttatgaaattattcggctttatggcgtagattgcaaccaggttgagaacgt gtttaaagccaaagcccctggccagaaactttt tttgggtatcttttttgtggatgccatcgagtctggcgtatcagctatcgcaagtgccgt taaatcctatggttcttgggatgatgtacacactgt atctgttggcaacgagctggtgaacaatggcgaagccactgttagccagattggacagta tgttagtacggccaaatcagccttacgctct gccggtttcacagggccagtattgtctgttgatacttttattgcagtgattaacaatccg gggctgtgtgatttcgcggatgaatatgttgctgt gaacgcccatgcgttcttcgatgggggtattgctgcctcaggggcgggcgattgggcggc agagcagatccagcgcgtctccagtgcg tgcggcgggaaagatgtcttaattgtagaaagcggttggccgtctaaaggagatacgaac ggcgccgcagtgccgtcaaaatccaatca gcaggctgcagtccagagtcttggccagaaaattgggagctcatgcattgcctttaacgc atttaatgattattggaaagccgatggtccgt tcaacgccgaaaaatattgggggatccttgatagttaa

B-glu4 Amino Acid (SEQ ID NO: 7 ) Pichia pastoris sequence (GS 115)

mlstilnifilllfiqaslqapipvvtkyvtegiavvtetnvrvvtktipivqvlis dgatythtlttvstaeengnfqpitttsivnkevvvpt svtpntqqtrptqvdttqnnadtpaaptpspttssnngvfttysttrsvvtsvvvvgpdg spientgqtanptttapttsttaarttsststtpt asstpggnhprsivyspysdssqckdattietdlefiaskgisavriygndcnyltvvlp kcaslglkvnqgfwigpsgvdsiddavq efiqavngnngfnwdlfelitvgneaisagyvsassliskikevssilssagytgpitta eppnvyedygdlcstdvmsivgvnahsyf ntlfaasdsgsfvksqievvqkacsrsditiietgypsqgatngknvpskenqktaifsi fevvgtdvtilstyddlwkdpgpygieqff gaidlfs

B-glu4 DNA (SEQ ID NO: 8 ) Pichia pastoris sequence (GS 115) atgctgtccacaattctgaatatttttattcttctgttattcatccaggcgtctcttcag gcgcctattccggtggtgaccaaatatgtgaccgaa ggtattgccgttgtgactgaaaccaatgtgcgggttgttactaaaaccattccgattgtg caggtgctgatctccgatggtgcaacctatact cataccctgacgacagtgtcaacggcggaagaaaatggcaacttccagcctattaccacg acatctattgtcaacaaagaagttgtagta ccaacaagcgtaaccccgaatacccagcagacgcgtccgacccaggtagataccacacag aacaatgcggatacaccagcggcgcc tacaccatcacctactactagttcaaacaacggcgtgttcaccacatattccacaacacg tagcgtagtcactagtgtagtcgtagtcggac cggatggaagccctattgaaaatactggacagacagcaaaccctactacaactgccccaa ctacaagcactactgctgcccggaccac aagcagtacgtccaccacacctaccgctagctctacgccaggaggtaatcatccacgtag catcgtctattctccatattccgatagcagt cagtgtaaagatgcgacaacgatcgaaaccgatcttgagttcattgcctctaaaggcatc agcgcggtacgtatttatggcaatgattgtaa ctatcttacagttgttttgcctaaatgtgccagtctgggattaaaagtgaatcagggctt ttggattggtccaagtggagtagatagcatcgat gatgcagtacaggagtttattcaggcagtcaacggcaacaacggctttaattgggattta ttcgaattaattaccgtcggaaacgaagcaat cagtgccggttatgtttcagcgagctccctgatttccaaaattaaagaagtatctagcat tctgagctccgcgggttatactggtccaattacc acagccgaaccgcctaacgtatatgaggattatggcgatctgtgctcaaccgatgtaatg tccatcgtgggtgtaaacgcgcattcctatttt aataccctttttgcggcctccgattcaggttcatttgtgaaatcacagatcgaagtagtc cagaaagcatgctcacgttccgatattactattat tgaaaccgggtatccgtcccagggagctaccaatggaaaaaacgttcctagtaaagagaa tcagaaaacagcgattttttcaatctttgag gtcgttggaacagatgtaactattcttagtacttatgatgatttgtggaaagatcctgga ccgtatgggattgaacagttttttggtgcgatcga tcttttttcttaa