HK1230659A1 - Recombinant production of steviol glycosides - Google Patents

Description

Recombinant preparation of steviol glycosides

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 61/898,571 filed on 1/11/2013, the entire contents of which are incorporated herein by reference.

The sequence listing includes

Provided herein are paper copies of the sequence Listing and sequences in computer readable form containing a file named 32559-17_ ST25.txt, the file size being 46,751 bytes (in Microsoft WindowsExplorer) andincorporated herein by reference. The sequence table is composed of SEQ ID NO 1-12.

Background

The present disclosure relates generally to the biosynthesis of steviol glycosides. In particular, the present disclosure relates to recombinant polypeptides that catalyze the preparation of steviol glycosides, such as rebaudioside D, rebaudioside E and a novel rebaudioside (rebaudioside Z).

Steviol glycosides are natural products isolated from the leaves of Stevia rebaudiana (Stevia rebaudiana), and are widely used as high-intensity, low-calorie sweeteners. Naturally occurring steviol glycosides have the same basic structure (steviol) and differ in the content of carbohydrate residues (e.g., glucose, rhamnose, and xylose residues) at the C13 and C19 positions. Steviol glycosides having known structures include stevioside (stevia), rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F and dulcoside (dulcoside) a.

On a dry weight basis, stevioside, rebaudioside a, rebaudioside C, and dulcoside a account for 9.1, 3.8, 0.6, and 0.3, respectively, of the total weight of steviol glycosides in the leaves, while other steviol glycosides were present in much lower amounts. Extracts from the stevia (stevia rebaudiana) plant are commercially available, which usually contain stevioside and rebaudioside a as the main compounds. Other steviol glycosides are typically present in stevia extracts as minor components. For example, the amount of rebaudioside a in the commercial product may vary from about 20% to more than 90% of the total steviol glycoside content, while the amount of rebaudioside B may be about 1-2% of the total steviol glycosides, the amount of rebaudioside C may be about 7-15% of the total steviol glycosides, and the amount of rebaudioside D may be about 2% of the total steviol glycosides.

As a natural sweetener, different steviol glycosides have different sweetness and aftertaste. The sweetness of steviol glycosides is significantly higher than that of sucrose. For example, stevioside is 100-times more sweet than sucrose and has a bitter aftertaste, while rebaudioside A and E are 450-times more sweet than sucrose and has a much better aftertaste than stevioside. Accordingly, the taste profile of any stevia extract is greatly influenced by the relative content of steviol glycosides in the extract, which in turn can be influenced by the source of the plant, environmental factors (such as mud content and climate), and the extraction process. In particular, variations in extraction conditions may lead to inconsistent composition of steviol glycosides in stevia extracts, such that taste characteristics vary between different batches of extracted products. The taste characteristics of stevia extracts can also be affected by plant-derived contaminants (such as pigments, lipids, proteins, phenols and carbohydrates) remaining in the product after the extraction process. These contaminants often have undesirable off-flavors for use of stevia extract as a sweetener.

Most steviol glycosides are formed by several glycosylation reactions of steviol, which are typically catalyzed by a UDP-glycosyltransferase (UGT) that uses uridine-5' -diphosphoglucose (UDP-glucose) as the donor of the sugar moiety. In plants, UGT is a very different group of enzymes that transfer glucose residues from UDP-glucose to steviol. Stevioside is an intermediate in the biosynthesis of rebaudioside compounds. For example, glycosylation of C-3' of C-13-O-glucose of stevioside produces rebaudioside A; glycosylation of stevia sugar at C-2' of 19-O-glucose produces rebaudioside E. Further glycosylation on rebaudioside A (at 19-O-glucose) or rebaudioside E (at C-13-O-glucose) produces rebaudioside D. (FIGS. 1A-1C).

A practical way to improve the taste quality of stevia extracts is to increase the yield of rebaudioside compounds by further glycosylation of the stevia sugar. UGT having 1, 2-19-O-glucosylation activity is an important enzyme for the preparation of rebaudioside D and E.

Sucrose synthase (SUS) catalyzes the conversion of UDP to UDP-glucose in the presence of sucrose. Thus, for glycosylation reactions utilizing UDP-glucose (e.g., those catalyzed by UGT), SUS can be used to regenerate UDP-glucose from UDP, increasing the efficiency of such reactions (fig. 2).

Accordingly, there is a need for steviol glycosides that have consistent taste characteristics and less off-taste than current commercial products. As described herein, the present disclosure provides recombinant polypeptides for the preparation of steviol glycosides, e.g., rebaudioside D and rebaudioside E. The present disclosure also provides methods of using such recombinant polypeptides to prepare steviol glycoside (rebaudioside Z) compositions.

Disclosure of Invention

The subject technology relates generally to recombinant polypeptides having UDP-glycosyltransferase activity. Specifically, the present invention provides polypeptides having 1, 2-19-O-glucose glycosylation activity for steviol glycoside compounds. In one aspect, the subject technology relates to a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6. In an exemplary embodiment, the amino acid sequence of a recombinant polypeptide described herein has 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 even 100% identity to SEQ ID No. 6.

In another aspect, the subject technology relates to an isolated nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide described herein. In another aspect, the subject technology relates to vectors comprising the nucleic acids described herein, and host cells comprising the vectors described herein. In an exemplary embodiment, the host cell of the present technology of interest is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria (cyanobacteria algae), and plant cells.

In another aspect, the subject technology also relates to a method of making a steviol glycoside composition, the method comprising incubating a substrate (e.g., stevioside, rebaudioside a, rebaudioside E or a combination thereof) with a recombinant polypeptide comprising an amino acid sequence at least 80% identical to SEQ ID NO: 6. In an exemplary embodiment, the amino acid sequence of a recombinant polypeptide used in the methods described herein is 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 even 100% identical to SEQ ID No. 6.

In another aspect, the subject technology also relates to a method of making a steviol glycoside composition, the method comprising incubating a substrate (e.g., stevioside and rebaudioside E) with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 11. In an exemplary embodiment, the amino acid sequence of a recombinant polypeptide used in the methods described herein is 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 even 100% identical to SEQ ID No. 11.

In one embodiment, the method further comprises incubating a recombinant sucrose synthase (e.g., a recombinant sucrose synthase having an amino acid sequence at least 80% identical to the amino acid sequence of AtSUS1 set forth in SEQ ID NO:9) with a substrate and a recombinant polypeptide as described herein. In another embodiment, the method further comprises incubating the recombinant UDP-glycosyltransferase with the recombinant sucrose synthase, substrate, and recombinant polypeptide described herein (e.g., a recombinant UDP-glycosyltransferase having an amino acid sequence at least 80% identical to the amino acid sequence of UGT76G1 shown in SEQ ID NO: 11). In another embodiment, the methods described herein comprise incubating the substrate with a host cell expressing the recombinant polypeptide.

The present technology of interest also relates to a novel steviol glycoside, called rebaudioside Z, which is characterized by a retention time on HPLC of about 6.68 minutes under the conditions described herein. The subject technology also relates to a method of making rebaudioside Z described herein, comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6. As used herein, the term "rebaudioside Z" or "Reb Z" refers to a mixture of compounds, particularly to a mixture of rebaudioside Z1 ("Reb Z1") and rebaudioside Z2 ("Reb Z2").

In one embodiment, the present disclosure further relates to a method for synthesizing rebaudioside Z from rebaudioside E. The method comprises the following steps: 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 HV1 and a sucrose synthase, incubating said reaction mixture for a sufficient time to prepare rebaudioside Z, wherein one glucose is covalently linked to rebaudioside E to prepare rebaudioside Z, one glucose is covalently linked to C2 '-13-O-glucose of rebaudioside E to prepare rebaudioside Z1, and one glucose is covalently linked to C2' -19-O-glucose of rebaudioside E to prepare rebaudioside Z2.

In one embodiment, the rebaudioside Z compound is rebaudioside Z1(Reb Z1) having the structure:

in one embodiment, the rebaudioside Z compound is rebaudioside Z2(Reb Z2) having the structure:

as described herein, the recombinant polypeptides of the present technology are biosynthetic methods for developing steviol glycosides, which are typically low abundant in natural sources, such as rebaudioside D and rebaudioside E. Accordingly, the present technology also provides steviol glycoside compositions prepared by the biosynthetic methods described herein. Such compositions may comprise steviol glycoside compounds selected from the group consisting of rebaudioside D, rebaudioside E, neorebaudioside (referred to herein as "rebaudioside Z" and "Reb Z"), and combinations thereof. Further, the present invention also provides sweeteners comprising the steviol glycoside compositions described herein.

In one embodiment, the present disclosure relates to a sweetener comprising a compound having the chemical structure:

in another embodiment, the sweetener comprises a compound having the chemical structure:

the present disclosure further relates to the use of sweeteners in consumable products such as beverages, desserts, baked goods, cookies and chewing gum.

Drawings

The present disclosure may be better understood, and features, aspects, and advantages other than those shown above may become apparent when consideration is given to the following detailed description thereof. This detailed description makes reference to the following drawings, in which:

FIGS. 1A-1C depict protocols demonstrating the pathway for the biosynthesis of steviol glycosides from stevioside. As described herein, a recombinant HV1 polypeptide ("HV 1") contains 1, 2-19-O-glucose glycosylation activity, transferring the second sugar moiety to C-2' of the 19-O-glucose of stevioside to make rebaudioside E ("Reb E"), or similarly to make rebaudioside D ("Reb D") from rebaudioside a ("Reb a"). Figures 1A-1C also show that recombinant UGT76G1 enzyme ("UGT 76G 1", distinct from recombinant HV1 polypeptide) catalyzes the reaction of the sugar moiety to C-3' of the C-13-O-glucose of stevioside to prepare rebaudioside a, or similarly to make rebaudioside D from rebaudioside E.

FIG. 2 shows an exemplary scheme of a coupling reaction system of UDP-glycosyltransferase ("UGT") and sucrose synthase ("SUS"). Reaction 1 shows a UGT catalyzed reaction that converts rebaudioside a ("Reb a") to rebaudioside D ("Reb D"), which uses UDP-glucose as the glucose donor and causes the production of UDP. Reaction 2 shows an SUS-catalyzed reaction to convert UDP to UDP-glucose, which uses sucrose as a glucose donor. Reaction 2 also shows that the SUS catalyzed reaction can be coupled with the UGT catalyzed reaction.

FIG. 3 shows the in vitro preparation of rebaudioside D ("Reb D") from rebaudioside A ("Reb A") catalyzed by recombinant HV1 polypeptide (SEQ ID NO:6) and recombinant AtSUS1(SEQ ID NO:9) in the HV1-AtSUS1 coupling reaction system as described herein. Figure 3A shows standards for stevioside ("Ste"), rebaudioside a ("Reb a"), and rebaudioside D ("Reb D"). 6. The results for 9, 12 and 24 hours are shown in FIGS. 3B-E, respectively. The results of the reaction without recombinant AtSUS1 (i.e., the non-coupling reaction) at 12 and 24 hours are shown in fig. 3F and 3G, respectively.

FIG. 4 shows the in vitro preparation of rebaudioside E ("Reb E") from stevioside catalyzed by recombinant HV1 polypeptide (SEQ ID NO:6) and recombinant AtSUS1(SEQ ID NO:9) in the HV1-AtSUS1 coupling reaction system as described herein. Figure 4A shows standards for stevioside ("Ste"), rebaudioside a ("Reb a"), and rebaudioside D ("Reb D"). The results at 20 hours are shown in fig. 4B, which includes the rebaudioside Z compound ("Reb Z").

FIG. 5 shows the in vitro preparation of rebaudioside D ("Reb D") from stevioside catalyzed by a combination of recombinant HV1 polypeptide (SEQ ID NO:6), recombinant UGT76G1(SEQ ID NO:11), and recombinant AtSUS1(SEQ ID NO: 9). Figure 5A shows standards for stevioside ("Ste"), rebaudioside a ("Reb a"), and rebaudioside D ("Reb D"). 6. The results for 9, 12 and 24 hours are shown in FIGS. 5B-E, respectively.

Figure 6 shows an SDS-PAGE analysis of the purified recombinant HV1 polypeptide.

FIG. 7 shows SDS-PAGE analysis of purified recombinant AtSUS1 polypeptide.

FIG. 8 shows the in vitro preparation of rebaudioside Z ("Reb Z") from rebaudioside E ("Reb E") catalyzed by recombinant HV1 polypeptide (SEQ ID NO:6) and recombinant AtSUS1(SEQ ID NO:9) in the HV1-AtSUS1 coupling reaction system as described herein. Fig. 8A shows a standard of rebaudioside E ("Reb E"). The results at 24 hours are shown in fig. 8B, which includes the rebaudioside Z compound ("Reb Z").

FIG. 9 shows an SDS-PAGE analysis of the purified recombinant UGT76G1 polypeptide.

FIG. 10: rebaudioside Z (including Reb Z1 and Reb Z2) and rebaudioside E.

FIG. 11: key TOCSY and HMBC relationships for Reb Z1 and Reb Z2.

FIG. 12: shows the in vitro preparation of rebaudioside D ("Reb D") from rebaudioside E ("Reb E") catalyzed by recombinant UGT76G1(SEQ ID NO: 11). Fig. 12A-12B show standards of rebaudioside E ("Reb E"), rebaudioside D ("Reb D"). Results of the reaction without recombinant AtSUS1 (i.e., non-coupling reaction) (FIG. 12C) and the reaction with recombinant AtSUS1 (i.e., UGT-SUS coupling reaction) (FIG. 12D) at 6 hours are shown, respectively.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

Detailed Description

The present object technology provides recombinant polypeptides having UDP-glycosyltransferase activity, such as 1, 2-19-O-glucose glycosylation activity and 1, 2-13-O-glucose glycosylation activity for the synthesis of steviol glycosides. The recombinant polypeptides of the present technology of interest (which may also be referred to hereinafter as "recombinant HV1 polypeptides") are useful for the biosynthesis of steviol glycoside compounds. In the context of the present invention, UDP-glycosyltransferase (UGT) refers to an enzyme that transfers a sugar residue from an activated donor molecule, typically UDP-glucose, to an acceptor molecule. 1, 2-19-O-glucose glycosylation activity refers to the enzyme activity of transferring a sugar moiety to C-2' of the 19-O-glucose moiety of stevioside, rebaudioside A or rebaudioside E (FIGS. 1A-1C and 10). 1, 2-13-O-glucose glycosylation activity refers to the enzyme activity that transfers the sugar moiety to C-2' of the 13-O-glucose moiety of rebaudioside E (FIG. 10).

The names of UGT enzymes used in The context of The present invention are consistent with The nomenclature system adopted by The UGT nomenclature Committee (Mackenzie et al, "The UDP glycosylation transfer enzyme gene super family: semantic expressed on evolution direction," pharmaceuticals, 1997, Vol.7, p.255) which classifies UGT genes by a combination of family number, letter representing a subfamily, and number representing an individual gene. For example, the designation "UGT 76G 1" refers to a UGT enzyme encoded by a gene belonging to UGT family number 76 (which is the origin of a plant), subfamily G, and gene number 1.

There is a large family of UGT genes in plants. However, the biological function of most of these UGTs remains unknown.

Definition of

As used herein, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

To the extent that the terms "includes," "including," "has," "having," or the like are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising" as "comprising" is interpreted when employed as a transitional word in a claim.

The term "exemplary" is used herein to mean "serving as an example, instance, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

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

The terms "nucleic acid" and "nucleotide" are given their respective ordinary and customary meanings to those of ordinary skill in the art, and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

The term "isolated" is given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that exists by the human hand apart from its original environment and is therefore not a natural product. An isolated nucleic acid or polypeptide may be present in a purified form or may be present in a non-native environment, e.g., in a transgenic host cell.

The term "incubating" is used herein to refer to mixing and contacting two or more chemical or biological entities (e.g., chemical compounds and enzymes) with one another under conditions conducive to the preparation of a steviol glycoside composition.

The term "degenerate variant" refers to a nucleic acid sequence having a sequence of residues that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. The nucleic acid sequence and all degenerate variants thereof will express the same amino acid or polypeptide.

The terms "polypeptide", "protein" and "peptide" are given their respective ordinary and customary meanings to one of ordinary skill in the art; these three terms are sometimes used interchangeably and are used without limitation to refer to a polymer of amino acids or amino acid analogs, regardless of size or function. Although "protein" is often used to refer to relatively large polypeptides, and "polypeptide" is often used to refer to small polypeptides, the use of these terms in the art overlaps and varies. The term "polypeptide" is used herein to refer 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.

The terms "polypeptide fragment" and "fragment" when used in reference to a reference polypeptide give their ordinary and customary meaning to those of ordinary skill in the art, and are used without limitation to refer to polypeptides in which amino acid residues are deleted compared to the reference polypeptide itself, with the remaining amino acid sequence generally being identical to the corresponding position on the reference polypeptide. Such deletion may occur at the amino terminus or the carboxy terminus, or alternatively both, of the reference polypeptide.

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

The terms "variant polypeptide," "modified amino acid sequence," or "modified polypeptide," used interchangeably, refer to an amino acid sequence that differs from a reference polypeptide by one or more amino acids (e.g., one or more amino acid substitutions, deletions, and/or additions). In one aspect, a variant is a "functional variant" that retains some or all of the capabilities of a reference polypeptide.

The term "functional variant" further includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from the reference polypeptide by one or more conservative amino acid substitutions and that retains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; substitution of one with a charged or polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; substitution of one basic residue, such as lysine or arginine, for the other; or substitution of one acidic residue, such as aspartic acid or glutamic acid, for the other; or by 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 in which a residue is replaced with a chemically derivatized residue, provided that the resulting peptide retains some or all of the activity of the reference peptide as described herein.

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

The term "homologous" for all grammatical forms and spelling variants refers to the relationship between polynucleotides or polypeptides having a "common evolutionary origin", including polynucleotides or polypeptides from the superfamily 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 particular amino acids or motifs at conserved positions. For example, two homologous polypeptides may have an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 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.

"percent (%) amino acid sequence identity" with respect to a variant polypeptide sequence of the present subject technology is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues of a reference polypeptide (e.g., such as SEQ ID NO:6) after aligning the sequences and, if necessary, introducing a gap to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity.

Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, such as, for example, using publicly available computer software, e.g., BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. For example,% amino acid sequence identity can be determined using the sequence comparison program NCBI-BLAST 2. NCBI-BLAST2 sequence comparison programs are available from NCBI. NCBI BLAST2 employs several search parameters, all of which are set to default values, including, for example, unmask yes, strand all, expected occurrencies 10, minimum low complexity length 15/5, multi-pass-value 0.01, constant for multi-pass 25, drop for final matched alignment 25, and ordering matrix BLOSUM 62. When using NCBI-BLAST2 for amino acid sequence comparisons, the% amino acid sequence identity of a given amino acid sequence a to, with or to a given amino acid sequence B (which may alternatively be stated as having or comprising a given amino acid sequence a with, with or to a given amino acid sequence B) is calculated as follows: 100 times the score X/Y, where X is the number of amino acid residues scored as identity matches by the sequence alignment program NCBI-BLAST2 in an alignment of the programs for A and B, and Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A.

In this sense, techniques for determining amino acid sequence "similarity" are well known in the art. In general, "similarity" means an exact amino acid-to-amino acid comparison of two or more polypeptides at the appropriate position, where the amino acids are identical or have similar chemical and/or physical properties, such as charge or hydrophobicity. This is referred to as "percent similarity" which can then be determined between the polypeptide sequences being compared. Techniques for determining nucleic acid and amino acid sequence identity are also well known in the art and include determining the nucleotide sequence of the mRNA for the gene (usually via a cDNA intermediate) and determining the amino acid sequence encoding it, and comparing it to a second amino acid sequence. In general, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by determining their "percent identity," e.g., can be two or more amino acid sequences. The programs available in the Wisconsin Sequence Analysis Package, 8 th edition (available from Genetics Computer Group, Madison, Wis.) (e.g., the GAP program) enable the calculation of identity between two polynucleotides and identity and similarity, respectively, between two polypeptide sequences. Other procedures for calculating identity or similarity between sequences are known to those skilled in the art.

An amino acid position "relative to" a reference position is a position that is aligned with a reference sequence, as identified by aligning the amino acid sequences. Such alignment can be performed manually or using well known sequence alignment programs such as ClustalW2, Blast2, and the like.

Unless otherwise indicated, percent identity of two polypeptide or polynucleotide sequences refers to the percentage of identical amino acid residues or nucleotides over the entire length of the shorter of the two sequences.

"coding sequence" is given its ordinary and customary meaning to a person of ordinary skill in the art, and is used without limitation to refer to a DNA sequence that encodes a particular amino acid sequence.

"suitable regulatory sequences" are given their ordinary and customary meaning to those of ordinary skill in the art, and are used without limitation to refer to nucleotide sequences located upstream (5 '-non-coding sequences), within, or downstream (3' -non-coding sequences) of a coding sequence, and which affect transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

"promoter" is given its ordinary and customary meaning to those of ordinary skill in the art and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. Promoters may be derived in their entirety from the original gene, or consist of different elements derived from different promoters found in nature, or even comprise synthetic DNA fragments. 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 gene expression in most cell types most of the time are commonly referred to as "constitutive promoters". It is further recognized that since the exact boundaries of regulatory sequences are not fully defined in most cases, DNA fragments of different lengths may have the same promoter activity.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression" as used herein gives 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 fragments of the present subject technology. "overexpression" refers to the production of a gene product in a transgenic or recombinant organism, above the level produced in a normal or non-transformed organism.

"transformation" is given its ordinary and customary meaning to one of ordinary skill in the art, and is used without limitation to refer to the transfer of a polynucleotide to a target cell. The transferred polynucleotide may be incorporated into the genomic or chromosomal DNA of the target cell, resulting in genetically stable inheritance, or it may replicate independently of the host chromosome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The terms "transformed", "transgenic" and "recombinant" when used herein in connection with a host cell, have their respective ordinary and customary meanings to those of ordinary skill in the art, and are used without limitation to refer to a cell, e.g., a plant or microbial cell, of a host organism into which an exogenous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the genome of the host cell, or the nucleic acid molecule may be present as an extrachromosomal molecule. Such extrachromosomal molecules may be autonomously replicating. Transformed cells, tissues or subjects are understood to include not only the end product of the transformation procedure but also transgenic progeny thereof.

The terms "recombinant," "heterologous," and "exogenous" when used herein in connection with a polynucleotide are given their ordinary and customary meaning to those of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or gene) that is derived from a source that is foreign to the particular host cell or, if 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, for example, by using site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term relates to a portion of DNA that is foreign or heterologous to the cell, or homologous to the cell, but in a position or form where the element is not normally found in the host cell.

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

The terms "plasmid", "vector" and "cassette" are given their ordinary and customary meanings to those of ordinary skill in the art, and are used without limitation to refer to extra-chromosomal elements that often carry genes that are not part of the central metabolism of the cell, and are usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences of any origin, genome integrating sequences, bacteriophage or nucleotide sequences, linear or circular single-or double-stranded DNA or RNA, wherein a plurality of nucleotide sequences have been ligated or recombined into a single construct capable of introducing into a cell a promoter fragment and a DNA sequence for a selected gene product, together with appropriate 3' untranslated sequence. "transformation cassette" refers to a particular vector that contains an exogenous gene and has elements in addition to the exogenous gene that facilitate transformation of a particular host cell. An "expression cassette" refers to a specific vector that contains a foreign gene and has elements other than the foreign gene that enhance expression of the gene in a foreign host.

Standard recombinant DNA and molecular Cloning techniques used herein are well known in the art and are described, for example, by Sambrook, j., Fritsch, e.f. and manitis, t.molecular Cloning: a Laboratory Manual, 2 nd edition; cold Spring Harbor Laboratory, N.Y.,1989 (hereinafter "Maniatis"); and Silhavy, t.j., Bennan, m.l. and Enquist, l.w. experiments with Gene Fusions; cold spring Harbor, N.Y., 1984; and Ausubel, F.M. et al, In Current protocols In Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987 (the entire contents of each of which are hereby incorporated by reference).

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

The present disclosure will be more fully understood when considered in view of the following non-limiting examples. While preferred embodiments of the subject technology are shown, it should be understood that these examples are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of this subject technology to adapt it to various usages and conditions.

Recombinant polypeptides

In one aspect, the present disclosure relates to recombinant polypeptides having an amino acid sequence that is at least 70%, at least 75%, 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%, and even 100% identical to the amino acid sequence set forth in SEQ ID No. 6. Suitably, the amino acid sequence of the recombinant polypeptide has at least 80% identity with SEQ ID No 6. More suitably, the amino acid sequence of the recombinant polypeptide has 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% and even 100% identity to SEQ ID No. 6. In an exemplary embodiment, the amino acid sequence of the recombinant polypeptide consists of SEQ ID NO 6. Accordingly, recombinant polypeptides described herein include functional fragments of SEQ ID NO. 6, functional variants of SEQ ID NO. 6 and other homologous polypeptides having, for example, 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% and even 100% sequence identity to SEQ ID NO. 6.

In another aspect, the present disclosure relates to an isolated nucleic acid having a nucleotide sequence encoding a recombinant polypeptide described herein. For example, the isolated nucleic acid may include a nucleotide sequence encoding a polypeptide having an amino acid sequence at least 70%, at least 75%, 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%, and even 100% identical to the amino acid sequence set forth in SEQ ID NO 6. Suitably, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence with at least 80% identity to the amino acid sequence set forth in SEQ ID NO. 6. More suitably, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide having an amino acid sequence with 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% and even 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 6. Thus, the isolated nucleic acids include those encoding a functional fragment of SEQ ID NO. 6, a functional variant of SEQ ID NO. 6, or other homologous polypeptides having, for example, 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%, and even 100% sequence identity to SEQ ID NO. 6.

In one embodiment, the present disclosure relates to an isolated nucleic acid having a nucleotide sequence that is at least 70%, at least 75%, 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%, and even 100% identical to the nucleotide sequence set forth in SEQ ID No. 7. Suitably, the isolated nucleic acid comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO. 7. More suitably, the isolated nucleic acid comprises a nucleotide sequence having 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% and even 100% identity to the nucleic acid sequence set forth in SEQ ID No. 7.

In another aspect, the subject technology relates to vectors having the nucleic acids described herein, and host cells having the vectors described herein. In some embodiments, the disclosure relates to an expression vector comprising at least one polynucleotide of the present technology of interest and wherein the expression vector upon transfection into a host cell is capable of expressing at least one recombinant HV1 polypeptide described herein. In one embodiment, the expression vector comprises the nucleotide sequence set forth in SEQ ID NO. 7 or a variant thereof.

The design of the expression vector will depend on such factors as the choice of host cell to be transformed, the level of protein expression desired, and the like. The expression vector may be introduced into a host cell to produce a recombinant polypeptide of the present technology of interest, such as a recombinant HV1 polypeptide having the amino acid sequence of SEQ ID NO. 6 or a variant thereof.

Expression of proteins in prokaryotes is most often accomplished in bacterial host cells with vectors containing constitutive or inducible promoters directing expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, often to the amino terminus of the recombinant protein. Such fusion vectors are commonly used for three purposes: 1) increasing expression of the recombinant protein; 2) increasing the solubility of the recombinant protein; and 3) to aid in the purification of recombinant proteins by acting as a ligand in affinity purification. Often, a protein cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to separate the recombinant protein from the fusion moiety after purification of the fusion protein. Such vectors are within the scope of the present disclosure.

In one embodiment, the expression vector includes those genetic elements used for expression of a recombinant polypeptide in a bacterial cell. Elements for transcription and translation in bacterial cells may include a promoter, a coding region for a protein complex, and a transcription terminator.

In one embodiment, the expression vector of the present technology of interest comprises a bacterial expression vector (e.g., recombinant phage DNA, plasmid DNA, or cosmid DNA), a yeast expression vector (e.g., a recombinant yeast expression vector), a vector for expression in insect cells (e.g., a recombinant viral expression vector such as baculovirus), or a vector for expression in plant cells (e.g., a recombinant viral expression vector such as cauliflower mosaic virus (CaMV), Tobamovirus (TMV), or a recombinant plasmid expression vector (e.g., Ti plasmid)).

In one embodiment, the vector comprises a bacterial expression vector. In another embodiment, the expression vector comprises a high copy number expression vector; alternatively, the expression vector comprises a low copy number expression vector, e.g., a Mini-F plasmid.

One of ordinary skill in the art will be aware of molecular biology techniques that can be used to prepare expression vectors. As described above, polynucleotides for inclusion in the expression vectors of the subject technology can be prepared by conventional techniques, such as Polymerase Chain Reaction (PCR).

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

In an alternative embodiment, synthetic linkers containing one or more restriction sites are provided for operably linking the polynucleotides of the present technology to the expression vector. In one embodiment, the polynucleotide is produced by restriction endonuclease digestion. In one embodiment, the nucleic acid molecule is treated with bacteriophage T4DNA polymerase or e.coli DNA polymerase I, which removes the protruding 3 '-single stranded ends with their 3' -5 'exonuclease activity and fills the recessed 3' -ends with their polymerizing activity, thereby generating blunt-ended DNA fragments. The blunt-ended fragments are then incubated with a large molar excess of a linker molecule in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as bacteriophage T4DNA ligase. Thus, the reaction product is a polynucleotide carrying a polymeric linker sequence at its terminus. These polynucleotides are then cleaved with appropriate restriction enzymes and ligated into an expression vector that has been cleaved with enzymes to yield termini compatible with those of the polynucleotide.

Alternatively, a vector having a ligation-independent cloning (LIC) site may be used. The claimed PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digestion or ligation (Aslanidis and de Jong, Nucl. acid. Res.18,6069-6074, (1990), Haun, et al, Biotechniques 13, 515-.

In one embodiment, PCR is suitable for the isolation and/or modification of the target polynucleotide for insertion into the selected plasmid. Suitable primers for PCR preparation of the sequences can be designed to isolate the desired coding region of the nucleic acid molecule, to add restriction endonuclease or LIC sites, to place the coding region in the desired reading frame.

In one embodiment, the polynucleotide for inclusion in an expression vector of the subject technology is prepared using PCR using appropriate oligonucleotide primers. The coding region is amplified while the primer itself becomes incorporated into the amplified sequence product. In one embodiment, the amplification primers comprise a restriction endonuclease recognition site that allows the amplified sequence product to be cloned into a suitable vector.

In one embodiment, the polynucleotide of SEQ ID NO. 7 or variants thereof are obtained by PCR and introduced into an expression vector using restriction endonuclease digestion and ligation, according to techniques well known in the art.

The present disclosure further relates to host cells comprising the expression vectors described herein. Suitable hosts for the subject technology generally include microbial or plant hosts. For example, the host cell of the present technology of interest is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria and plant cells.

The microbial host can include any organism capable of expressing the polynucleotide (e.g., SEQ ID NO:7) to produce the recombinant HV1 polypeptide described herein. The microorganism used in the present subject technology includes bacteria (e.g., enteric bacteria (e.g., Escherichia and Salmonella) and bacilli, Acinetobacter, Actinomyces (e.g., Streptomyces), Corynebacterium, Methanobacterium (e.g., Methylosinus), Methylomonas, Rhodococcus and Pseudomonas, cyanobacteria (e.g., Rhodobacter and Synechococcus), yeasts (e.g., Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis), and filamentous fungi (e.g., Aspergillus and nematode-trapping), and algae, and Escherichia, Klebsiella, Pantoea, Salmonella, Clostridium (e.g., Clostridium acetobutylicum). preferably, the microbial host is a bacterium (e.g., Escherichia) or a yeast (e.g., Saccharomyces) To produce large, commercial quantities of steviol glycosides.

In one embodiment, the recombinant polypeptide may be expressed in a host cell that is a plant cell. As used herein, the term "plant cell" is understood to mean any cell derived from a monocotyledonous or dicotyledonous plant and capable of constituting an undifferentiated tissue (such as a callus), a differentiated tissue (such as an embryo), a part of a monocotyledonous plant, a monocotyledonous plant or a seed. The term "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, including monocotyledonous and dicotyledonous plants. In some embodiments, the plant cell may be an arabidopsis plant cell, a tobacco plant cell, a soybean plant cell, a morning glory plant cell, or a cell from another oil crop plant (including but not limited to a 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, and a sesame plant cell).

Useful plant hosts may include any plant that supports the production of recombinant polypeptides of the subject technology. Suitable green plants for use as hosts include, but are not limited to, soybean, rapeseed (brassica napus), sunflower (heliothis), cotton (gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), wheat (Triticum sp), barley (Hordeum vulgare), oat (Avena sativa), Sorghum (Sorghum bicolor), rice (Oryza sativa), arabidopsis, cruciferous vegetables (broccoli, cauliflower, cabbage, parsnip, etc.), melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, grass seed crops, sugar beet, sugarcane, bean, pea, rye, flax, broad leaf trees, conifers, and grasses. Algae include, but are not limited to, commercially important hosts such as spirulina, Haemotacoccus, and dunaliella. Suitable plants for use in the methods of the subject technology also include biofuels, biomass, and bioenergy crop plants. Exemplary plants include Arabidopsis, rice (Oryza sativa), barley, switchgrass (Panicum virgatum), brachypodium, Brassica, and crambe.

In some embodiments, the disclosure includes a transgenic host cell or a host that has been transformed with one or more vectors disclosed herein.

Alternatively, the host cells may be those suitable for biosynthetic products, including unicellular organisms, microorganisms, multicellular organisms, plants, fungi, bacteria, algae, cultured crops, non-cultured crops, and/or the like.

The expression vector may be introduced into a plant or microbial host cell by conventional transformation or transfection techniques. Transformation of appropriate cells with the expression vectors of the subject technology is accomplished by methods known in the art and generally depends on the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemical perforation (electroporation) or electroporation.

Successfully transformed cells (i.e., those containing an expression vector) can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce a polypeptide described herein. The cells can be examined for the presence of expression vector DNA by techniques well known in the art.

The host cell may contain a single copy of the expression vector described above, or alternatively, multiple copies of the expression vector.

In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is a plant cell selected from the group consisting of: canola plant cells, rapeseed plant cells, palm plant cells, sunflower plant cells, cotton plant cells, corn plant cells, peanut plant cells, flax plant cells, sesame plant cells, soybean plant cells, and morning glory plant cells.

Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high levels of expression of foreign proteins are well known to those skilled in the art. Any of these can be used to construct vectors for expressing recombinant polypeptides of the present technology of interest in microbial host cells. These vectors can then be introduced into suitable microorganisms by transformation to allow high level expression of the recombinant polypeptides of the present technology of interest.

Vectors or cassettes for transformation of suitable microbial host cells are well known in the art. Typically the vector or cassette contains sequences that direct the transcription and translation of the relevant polynucleotide, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors include a 5 'region of a polynucleotide having transcriptional initiation control and a 3' region of a DNA segment that controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the host cell being transformed, although it will be understood that these control regions need not be derived from the native gene of the particular species chosen as the host.

Initiation control regions or promoters useful for driving expression of recombinant polypeptides in a desired microbial host cell are numerous and familiar to those skilled in the art. In fact, any promoter capable of driving these genes is suitable for the present technology of interest, including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (for expression in Saccharomyces); AOX1 (for expression in Pichia) (ii) a And lac, trp, IP_L、IP_RT7, tac and trc (for expression in e.

The termination control region may also be derived from various genes native to the microbial host. For the microbial hosts described herein, a termination site may optionally be included.

In plant cells, the expression vector of the present technology of interest may include a coding region operably linked to a promoter capable of directing expression of the recombinant polypeptide of the present technology of interest in a desired tissue at a desired developmental stage. For convenience, the polynucleotide to be expressed may comprise a promoter sequence and a translation leader sequence from the same polynucleotide. A3' -non-coding sequence encoding a transcription termination signal should also be present. The expression vector may also contain one or more introns to facilitate polynucleotide expression.

For plant host cells, any combination of any promoter and any terminator capable of inducing expression of the coding region may be used in the vector sequences of the present technology of interest. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs), and cauliflower mosaic virus (CaMV) genes. One type of efficient plant promoter that can be used is a high level plant promoter. Such a promoter, operably linked to an expression vector of the subject technology, should be capable of promoting expression of the vector. High-level plant promoters that may be used in the present subject technology include promoters of the small subunit (ss) of ribulose-1, 5-bisphosphate carboxylase, such as from soybean (Berry-Lowe et al, j. molecular appl. gen, 1: 483498 (1982), which is hereby incorporated herein in its entirety to the extent consistent therewith), and promoters of chlorophyll a/b binding proteins. Both promoters are known to be photoinduced in plant cells (see, e.g., Genetic Engineering of Plants, an Agricultural chemical Perfect, A. Cashmore, Plenum, N.Y. (1983), p. 2938; 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), The entire contents of each of which are hereby incorporated by reference to The extent they are consistent with each other).

The choice of plasmid vector depends on the method to be used for transforming the host plant. The skilled artisan is familiar with the genetic elements that must be present on a plasmid vector to successfully transform, select and propagate a host cell containing the chimeric polynucleotide. The skilled artisan will also recognize that different independent transformation events will cause different levels and types of expression (Jones et al, EMBO J.4: 24112418 (1985); De Almeida et al, mol. Gen. genetics 218: 7886 (1989), the contents of each of which are hereby incorporated by reference to the extent they are consistent therewith), and that multiple events must therefore be screened to obtain lines exhibiting the desired expression levels and types. Such screening can be accomplished by Southern analysis of the Southern blot, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

Introduction of the expression vector of the present technology of interest into a plant cell can be carried out by various methods known to those skilled in the art, including insertion of the nucleic acid sequence of interest into an agrobacterium rhizogenes Ri or agrobacterium tumefaciens Ti plasmid, microinjection, electroporation or direct precipitation. As an example, in some embodiments, transient expression of a polynucleotide of interest can be performed by Agrobacterium penetration methods. In this regard, a suspension of agrobacterium tumefaciens containing a target polynucleotide can be grown in culture, and then the suspension injected into a plant by placing the tip of a syringe on the back of the leaf while gently applying a counter pressure to the other side of the leaf. The agrobacterium solution is then injected into the interior space of the leaf through the stomata. Once inside the leaves, agrobacterium transforms the gene of interest into a portion of the plant cell where it is then transiently expressed.

As another example, transformation of a target plasmid into a plant cell can be performed by a particle gun bombardment technique (i.e., particle gun method). In this regard, a suspension of plant embryos can be grown in liquid culture and then bombarded with plasmids or polynucleotides bound to gold particles, where the gold particles bound to the target plasmid or nucleic acid can be pushed through the membrane of the plant tissue, such as the embryogenic tissue. After bombardment, the transformed embryos can then be selected with appropriate antibiotics to produce new, clonally propagated, transformed embryogenic suspension cultures.

The host cell may be an unmodified cell or cell line, or a cell line that has been genetically modified. In some embodiments, the host cell is a cell line that has been engineered to grow under desired conditions (e.g., at lower temperatures).

Standard recombinant DNA methods can be used to obtain nucleic acids encoding the recombinant polypeptides described herein, to include the nucleic acids into expression vectors, and to introduce the vectors into host cells, as described in Sambrook, et al (eds), molecular cloning; a Laboratory Manual, third edition, cold spring harbor, (2001); and those described in Ausubel, F.M. et al (eds.) Current Protocols in Molecular Biology, John Wiley & Sons (1995). Nucleic acids encoding the polypeptides may be inserted into expression vectors or vectors such that the nucleic acids are operably linked to transcriptional and translational control sequences (e.g., promoter sequences, transcriptional termination sequences, etc.). The expression vector and expression control sequences are typically selected to be compatible with the expression host cell used.

Expression of the polypeptide in the hosts described herein may be further enhanced by codon optimization. For example, altering less common codons with more common codons can affect the half-life of the mRNA or alter its structure by introducing secondary structures that interfere with translation of the message. All or part of the coding region may be optimized. In some cases the desired modulation of expression is achieved by optimizing substantially all of the genes. In other cases, the desired adjustment will be achieved by optimizing part, but not all, of the gene sequence.

The codon usage of any coding sequence may be adjusted to achieve desired properties, such as high level expression in a particular cell type. The starting point for this optimization may be a coding sequence with 100% common codons, or a coding sequence containing a mixture of common and uncommon codons.

Two or more candidate sequences that differ in their codon usage can be generated and tested to determine whether they have the desired properties. Candidate sequences can be evaluated by using a computer to find the presence of regulatory elements (such as silencers or enhancers), and to find the presence of regions of the coding sequence that can be converted to such regulatory elements by changes in codon usage. Additional criteria may include abundance of a particular nucleotide (e.g., A, C, G or U), codon bias of a particular amino acid, or presence or absence of a particular mRNA secondary or tertiary structure. Adjustments to the candidate sequences may be made based on a number of these criteria.

In some embodiments, a codon optimized nucleic acid sequence may express its protein at a level of about 110%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, or about 500% of that expressed by a nucleic acid sequence whose codons are not optimized.

In addition to the nucleic acid encoding the recombinant polypeptide of the present technology of interest, the expression vector of the present technology of interest may additionally carry regulatory sequences that control the expression of the protein in the host cell, such as promoters, enhancers or other expression control elements that control the transcription or translation of the nucleic acid. Such regulatory sequences are known in the art. One skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. In addition, the recombinant expression vectors of the present technology of interest may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene.

Biosynthesis of steviol glycosides

As described herein, the recombinant polypeptides of the present technology have UDP-glycosyltransferase activity, more specifically including 1, 2-19-O-glucose glycosylation activity, and are useful in developing biosynthetic methods for the preparation of steviol glycosides, which are typically low-abundant in natural sources, such as rebaudioside D and rebaudioside E. The recombinant polypeptide of the present technology has UDP-glycosyltransferase activity and is useful for the development of biosynthetic methods for the preparation of novel steviol glycosides, such as rebaudioside Z1 and rebaudioside Z2.

Accordingly, in one aspect, the subject technology also relates to a method of making a steviol glycoside composition, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6.

The substrate may be any natural or synthetic compound that can be converted to a steviol glycoside compound in a reaction catalyzed by one or more UDP-glycosyltransferases. For example, the substrate may be a natural stevia extract, steviol-13-O-glycoside, steviol-19-O-glycoside, steviol-1, 2-bioside, rubusoside, stevioside, rebaudioside A, rebaudioside G or rebaudioside E. The substrate may be a pure compound or a mixture of different compounds. Preferably, the substrate comprises a compound selected from the group consisting of rubusoside, stevioside, rebaudioside a, rebaudioside E, and combinations thereof.

The methods described herein also provide coupled reaction systems in which the recombinant peptides described herein are allowed to act in combination with one or more additional enzymes to increase efficiency or modulate the results of the overall biosynthesis of steviol glycoside compounds. For example, additional enzymes may regenerate UDP-glucose required for the glycosylation reaction by converting UDP produced by the glycosylation reaction back to UDP-glucose (e.g., using sucrose as a donor for glucose residues), thus increasing the efficiency of the glycosylation reaction. In another example, a recombinant polypeptide of the present technology of interest can produce an intermediate of a steviol glycoside (e.g., rebaudioside E), which is further converted to another steviol glycoside (e.g., rebaudioside D) in a reaction catalyzed by another UDP-glycosyltransferase (e.g., UGT76G 1). In another example, a recombinant polypeptide of the present technology of interest may produce an intermediate of a steviol glycoside (e.g., rebaudioside E), which is further converted to another steviol glycoside (e.g., rebaudioside Z1 and rebaudioside Z2) in a reaction catalyzed by a UDP-glycosyltransferase (e.g., HV 1).

Accordingly, in one embodiment, the methods of the present technology of interest further comprise incubating a recombinant sucrose synthase (SUS) with a substrate and a recombinant polypeptide as described herein. The recombinant sucrose synthase converts UDP to UDP-glucose using sucrose as a source of glucose. Suitable sucrose synthases include those from the arabidopsis and mungbean SUS genes, or from any gene encoding a functional homolog of a sucrose synthase encoded by the arabidopsis and mungbean SUS1 sequences, or a functional homolog thereof. Suitable sucrose synthases may be, for example, an arabidopsis sucrose synthase 1, an arabidopsis sucrose synthase 3, and a mungbean sucrose synthase. A particularly suitable sucrose synthase may be, for example, arabidopsis sucrose synthase 1. For example, the recombinant SUS includes an amino acid sequence that is at least 70%, at least 75%, 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 even 100% identical to the amino acid sequence of AtSUS1 set forth in SEQ ID No. 9. Preferably, the recombinant SUS of the present subject technology includes an amino acid sequence having at least 80% identity to the amino acid sequence of AtSUS1 shown in SEQ ID NO. 9.

The recombinant sucrose synthase of the present technology of interest can be obtained by expressing a nucleic acid having a nucleotide sequence encoding an amino acid sequence of interest (e.g., an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:9) in a host cell as described above. For example, a vector comprising the nucleotide sequence set forth in SEQ ID NO. 10 can be introduced into a microbial host (e.g., E.coli) by conventional transformation techniques to produce a recombinant sucrose synthase.

In another embodiment, the methods of the present technology of interest further comprise incubating the recombinant UDP-glycosyltransferase with the recombinant sucrose synthase, substrate, and recombinant polypeptide described herein. The recombinant UDP-glycosyltransferase can catalyze a glycosylation reaction that is different from the reaction catalyzed by the recombinant polypeptide of the subject technology. For example, a recombinant UDP-glycosyltransferase can catalyze a reaction that transfers a sugar moiety to C-3 'of C-13-O-glucose of stevioside to produce rebaudioside a (or similarly produce rebaudioside D from rebaudioside E), while a recombinant polypeptide of the present technology of interest transfers a second sugar moiety to C-2' of 19-O-glucose of stevioside to produce rebaudioside E (or similarly produce rebaudioside D from rebaudioside a).

Suitable UDP-glycosyltransferases include any UGT known in the art that catalyzes one or more reactions in the biosynthesis of steviol glycoside compounds, such as UGT85C2, UGT74G1, UGT76G1, or functional homologs thereof. For example, a UDP-glycosyltransferase as described herein can include an amino acid sequence that is at least 70%, at least 75%, 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 even 100% identical to the amino acid sequence of UGT76G1 set forth in SEQ ID NO. 11. Preferably, the UDP-glycosyltransferase comprises an amino acid sequence having at least 80% identity to the amino acid sequence of UGT76G1 as shown in SEQ ID NO: 11.

The recombinant UDP-glycosyltransferase can be obtained by expressing a nucleic acid having a nucleotide sequence encoding an amino acid sequence of interest (e.g., an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:11) in a host cell as described above. For example, a vector comprising the nucleotide sequence shown in SEQ ID NO. 12 can be introduced into a microbial host (e.g., E.coli) by conventional transformation techniques to produce the recombinant UDP-glycosyltransferase of the present subject technology.

Both in vitro and in vivo preparation of steviol glycoside compounds are encompassed by the present technology of interest.

For example, the incubation may be an in vitro method, wherein a substrate is allowed to interact with the recombinant polypeptide of the present technology of interest. Preferably, in the in vitro method, the recombinant polypeptide is purified prior to incubation with said substrate. Conventional polypeptide purification techniques, such as centrifugation, cell lysis and chromatography, are included in the methods of the subject technology. For example, a nucleic acid encoding a recombinant polypeptide of the subject technology can be cloned into an expression vector with a histidine tag, such that the expressed recombinant polypeptide can be purified by affinity column chromatography.

The in vitro methods of the subject technology include any buffer system suitable for steviol glycoside preparation with one or more recombinant polypeptides of the subject technology. Typically, the buffer system is an aqueous solution, such as Tris buffer, HEPES buffer, MOPS buffer, phosphate buffer having a pH of from about 6.0 to about 8.0. More suitably, the pH is from about 6.5 to about 7.5. Even more suitably, the pH is from about 7.0 to about 7.5.

Typically, in the in vitro methods of the present subject technology, the substrate is present in the buffer at a concentration of from about 0.2mg/mL to about 5mg/mL, preferably from about 0.5mg/mL to about 2mg/mL, more preferably from about 0.7mg/mL to about 1.5 mg/mL.

Typically, in the in vitro methods of the present subject technology, UDP-glucose is contained in the buffer at a concentration of from about 0.2mM to about 5mM, preferably from about 0.5mM to about 2mM, more preferably from about 0.7mM to about 1.5 mM. In one embodiment, when the recombinant sucrose synthase is included in the reaction, sucrose is also included in the buffer at a concentration of from about 100mM to about 500mM, preferably from about 200mM to about 400mM, more preferably from about 250mM to about 350 mM.

Typically, in the in vitro methods of the present technology of interest, the weight ratio of the recombinant polypeptide to the substrate is from about 1:100 to about 1:5, preferably from about 1:50 to about 1:10, more preferably from about 1:25 to about 1:15, on a dry weight basis.

Typically, the reaction temperature of the in vitro process is from about 20 ℃ to about 40 ℃, suitably from 25 ℃ to about 37 ℃, more suitably from 28 ℃ to about 32 ℃.

The present disclosure also provides steviol glycoside compositions prepared by the biosynthetic methods described herein. The exact nature of the steviol glycoside compositions prepared by the methods described herein (e.g., the type of molecular species and their percentage in the final product) depends on the substrate used, the incubation conditions, and the enzyme activity contained in the reaction system. For example, when stevioside is used as the substrate, the steviol glycoside composition prepared may include rebaudioside a, rebaudioside D, rebaudioside E, rebaudioside Z1, and rebaudioside Z2, and combinations thereof.

The present object technology provides a method for converting the major steviol glycoside species (i.e., stevioside and rebaudioside a) in a natural stevia extract to rebaudioside D and rebaudioside E, which are low abundant in the natural extract. Accordingly, the present subject technology also provides a method of enriching the content of one or more specific steviol glycosides, such as rebaudioside D and rebaudioside E, the method comprising incubating a substrate (such as a natural stevia extract) with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6. For example, when a natural stevia extract is used as a substrate, the prepared steviol glycoside composition may be enriched with rebaudioside D and/or rebaudioside E, which are low abundant in the natural stevia extract.

One skilled in the art will recognize that the steviol glycoside compositions prepared by the methods described herein may be further purified and mixed with other steviol glycosides, flavors, or sweeteners to obtain the desired flavor or sweetener composition. For example, a rebaudioside D enriched composition prepared as described herein may be mixed with a natural stevia extract containing rebaudioside a as the primary steviol glycoside, or with other synthetic or natural steviol glycoside products to make the desired sweetener composition. Alternatively, the substantially pure steviol glycosides (e.g., rebaudioside D) obtained from the steviol glycoside compositions described herein can be combined with other sweeteners (e.g., sucrose, maltodextrin, aspartame, sucralose, neotame, potassium acesulfame, and saccharin). The amount of steviol glycosides relative to other sweeteners can be adjusted to obtain the desired taste, as is known in the art. The steviol glycoside compositions described herein, including rebaudioside D, rebaudioside E, rebaudioside Z1, rebaudioside Z2 or combinations thereof, may be included in food products (e.g., beverages, soft drinks, ice creams, dairy products, desserts, cereals, chewing gum, baked goods, etc.), dietary supplements, medical nutraceuticals, and pharmaceutical products.

Examples

Example 1: selection of candidate UGT genes

Phylogenetic analysis and protein BLAST analysis were used to identify 7 candidate genes belonging to the UGT91 subfamily for 1, 2-19-O-glucose glycosylation activity (table 1).

Table 1: UGT candidate Gene List

Example 2: enzyme Activity screening of candidate UGT genes

Full-length DNA fragments of all candidate UGT genes were commercially synthesized. Almost all codons of the cDNA were changed to those preferred by e. The synthesized DNA was cloned into the bacterial expression Vector pETite N-HisSUMO Kan Vector (Lucigen).

Each expression construct was transformed into E.coli BL21(DE3), which was subsequently grown in LB medium containing 50. mu.g/mL kanamycin at 37 ℃ until an OD600 of 0.8-1.0 was reached. Protein expression was induced by addition of 1mM isopropyl beta-D-1-thiogalactoside (IPTG) and the culture was further grown for 22 hours at 16 ℃. The cells were collected by centrifugation (3,000x g; 10 min; 4 ℃). The cell pellet was collected and either used immediately or stored at-80 ℃.

The cell pellet is usually resuspended in lysis buffer (50mM potassium phosphate buffer, pH7.2, 25ug/ml lysozyme, 5ug/ml DNase I, 20mM imidazole, 500mM NaCl, 10% glycerol and 0.4% Triton X-100). The cells were lysed by sonication at 4 ℃ and the cell debris was clarified by centrifugation (18,000x g; 30 min). The supernatant was applied to a Ni-NTA (Qiagen) affinity column equilibrated (equilibration buffer: 50mM potassium phosphate buffer, pH7.2, 20mM imidazole, 500mM NaCl, 10% glycerol). After loading the protein sample, the column was washed with equilibration buffer to remove unbound contaminating proteins. The histidine-tagged UGT recombinant polypeptide was eluted with equilibration buffer containing 250mM imidazole.

The 1, 2-19-O-glucose glycosylation activity of the purified candidate UGT recombinant polypeptides was assayed by using stevia or Reb A as a substrate (FIGS. 1A-1C). Typically, recombinant polypeptide (10. mu.g) is detected in a 200. mu.l in vitro reaction system. The reaction system contained 50mM potassium phosphate buffer, pH7.2, 3mM MgCl₂1mg/ml stevioside or rebaudioside A, 1mM UDP-glucose. The reaction was carried out at 30 ℃ and stopped by the addition of 200. mu.L of 1-butanol. The sample was extracted three times with 200. mu.L of 1-butanol. The combined fractions were dried and dissolved in 70 μ L80% methanol for High Performance Liquid Chromatography (HPLC) analysis. Stevia extract (Blue California, CA) containing 95% stevioside was used as the stevioside substrate. Rebaudioside a (99% pure) was also supplied by Blue California.

The UGT-catalyzed glycosylation reaction was coupled to a UDP-glucose generation reaction catalyzed by a sucrose synthase (such as AtSUS1 of SEQ ID NO: 9). In this process, UDP-glucose is produced from sucrose and UDP (fig. 2), so that the addition of additional UDP-glucose can be neglected. The AtSUS1 sequence (Bieniowska et al, Plant J.2007,49:810-828) was synthesized and inserted into a bacterial expression vector. Recombinant AtSUS1 protein was expressed and purified by affinity chromatography. The purified recombinant AtSUS1 polypeptide was analyzed by SDS-PAGE (molecular weight: 106.3kD, FIG. 7).

Accordingly, the activity of the recombinant UGT polypeptide can be coupled without AtSUS1 (50mM potassium phosphate buffer, pH7.2, 3mM MgCl)₂1mg/ml stevioside or rebaudioside A, 1mM UDP) or with an AtSUS coupling (50mM potassium phosphate buffer, pH7.2, 3mM MgCl₂1mg/ml stevioside or rebaudioside A, 1mM UDP and 285mM sucrose). In general, 10. mu.g of AtSUS1 was used for 200. mu.l of in vitro reaction. The in vitro reaction was incubated at 30 ℃ and stopped by extraction with 1-butanol.

HPLC analysis was then performed using a Dionex UPLC ultate 3000 system (Sunnyvale, CA) including a quaternary pump, a temperature-controlled column compartment, an autosampler, and a UV absorption detector. Phenomenex NH2 with guard column was used for the characterization of steviol glycosides. Acetonitrile water was used for isocratic elution in HPLC analysis. The rebaudioside D, rebaudioside E, and rebaudioside Z products were identified by NMR analysis.

The recombinant polypeptide encoded by SEQ ID NO:7 (SEQ ID NO:6) showed 1, 2-19-O-glucose glycosylation activity and was used for additional analysis. This gene is derived from a barley subsp (Hordeum vulgare subsp. vulgare) (abbreviated herein as "HV 1"). The purified recombinant HV1 polypeptide was analyzed by SDS-PAGE (fig. 6). As shown in FIG. 6, the recombinant HV1 protein (molecular weight: 61.4kD) was purified by affinity chromatography. The polypeptides encoded by the other candidate genes (table 1) did not show any detectable activity in the assays described herein, even though they share about 62-74% sequence identity with the recombinant HV1 polypeptide.

As described herein, the recombinant polypeptide of HV1 transfers the sugar moiety to rebaudioside a to make rebaudioside D under all reaction conditions in the presence or absence of AtSUS 1. Rebaudioside A was completely converted to rebaudioside D by recombinant HV1 polypeptide in UGT-SUS coupled reaction system (FIGS. 3B-E). However, only part of rebaudioside a was converted to rebaudioside D after 24 hours by a separate recombinant HV1 polypeptide that was not coupled to AtSUS1 (fig. 3F-G). Thus, the recombinant HV1 polypeptide showed 1, 2-19-O-glucosylation activity to prepare rebaudioside D from rebaudioside a and AtSUS1 increased the conversion efficiency of UGT-SUS coupling system.

In addition, recombinant HV1 polypeptide conjugated to AtSUS1(SEQ ID NO:9) converted stevioside to rebaudioside E in vitro (FIG. 4). An unexpected compound ("Reb Z") was prepared with HPLC retention time 6.68 minutes (see, fig. 4) different from rebaudioside D and E. This compound is a novel steviol glycoside and is called "rebaudioside Z" ("Reb Z"). To determine the conversion of Reb E to Reb Z, Reb E substrate (0.5mg/ml) was incubated with recombinant HV1 polypeptide (20 μ g) and AtSUS1(20 μ g) in a UGT-SUS coupled reaction system (200 μ L) under conditions similar to those used in the above examples. As shown in fig. 8, Reb Z was prepared by a combination of recombinant HV1 polypeptide and AtSUS 1. These results indicate that HV1 can transfer the glucose moiety to Reb E to form Reb Z.

Example 3: biosynthesis of steviol glycosides using recombinant HV1 polypeptides

As shown in FIGS. 1A-1C, rebaudioside D can also be formed by glycosylation of C-3' of C-13-O-glucose of rebaudioside E. Thus, depending on the order in which the glycosylation reactions occur, rebaudioside D may be prepared by different biosynthetic pathways (e.g., rebaudioside E by rebaudioside a). For example, glycosylation of C-3 'of C-13-O-glucose of stevioside to produce rebaudioside A intermediates may first occur, followed by glycosylation of C-2' of 19-O-glucose of rebaudioside A to produce rebaudioside D. To this end, UGT76G1(SEQ ID NO; 11) from stevia has been identified as an enzyme that transfers sugar residues to C-3' of C-13-O-glucose of stevia to form rebaudioside A.

The codon optimized UGT76G1cDNA was inserted into a bacterial expression vector, recombinant UGT76G1 protein was expressed and purified by affinity chromatography. The purified recombinant UGT76G1 polypeptide (molecular weight: 65.4kD, FIG. 9) was analyzed by SDS-PAGE. The rebaudioside E substrate was incubated with recombinant UGT76G1 with or without AtSUS1 under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 12, rebaudioside D was prepared by recombinant UGT76G 1. The addition of recombinant AtSUS during the reaction improves the conversion efficiency of the UGT-SUS coupling system. Thus, the recombinant UGT76G1 polypeptide showed 1, 3-13-O-glucose glycosylation activity to produce Reb D from Reb E.

Accordingly, the catalytic activity of recombinant HV1 polypeptides for steviol glycoside biosynthesis (e.g., the preparation of rebaudioside D) in combination with UGT76G1 was further determined. Stevia substrates were incubated with recombinant HV1 polypeptide (10 μ G), UGT76G1(10 μ G), and AtSUS1(10 μ G) in a UGT-SUS coupled reaction system (200 μ l) under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 5, rebaudioside D was prepared by a combination of recombinant HV1 polypeptide, UGT76G1, and atasus 1. Thus, recombinant HV1 polypeptides showing at least 1, 2-19-O-glucose glycosylation activity can be used in combination with other UGT enzymes (such as UGT76G1) for complex, multi-step biosynthesis of steviol glycosides.

Example 4: NMR analysis of the Structure of Reb Z

Materials prepared by enzymatic conversion with rebaudioside E and purified by HPLC for characterization of rebaudioside z (reb z).

Generating HRMS data by using an LTQ Orbitrap Discovery HRMS instrument, wherein the resolution ratio is set to be 30 k; the data scanned in the cationic electrospray mode ranged from m/z 150 to 1500. The pin voltage was set to 4 kV; other source conditions were 25 sheath flow gas, 0 assist gas, 5 off gas (all gases flowing in arbitrary units), 30V capillary voltage, 300 c capillary temperature, and 75 tube lens voltage. The sample was diluted with 2:2:1 acetonitrile, methanol, water (same as the eluent injected) and injected with 50 microliters.

NMR spectra were obtained on a Bruker Avance DRX 500MHz or Varian INOVA 600MHz instrument with standard pulse sequences. At C₅D₅In N, 1D (¹H and¹³C) and 2D (COSY, TOCSY, HMQC, and HMBC) NMR spectra.

Shown as a mixture of Reb Z1 and Reb Z2Compound Reb Z of compound is shown in figure 10. The molecular formula of the compound Reb Z has been deduced as C based on its positive High Resolution (HR) mass spectrum₅₀H₈₀O₂₈Its positive High Resolution (HR) mass spectrum shows a correspondence at M/z1151.4713 to [ M + Na [ ]]⁺The adduct ion of (1); the composition quilt¹³C NMR spectral data support. Of Reb Z¹The H NMR spectral data show the presence of a mixture of the two compounds (Reb Z1 and Reb Z2) in a ratio between 60:40 and 70: 30. Thus of Reb Z¹H and¹³the C NMR spectral data shows a set of peaks for each proton and carbon present in its structure. With 5% H₂SO₄Acid hydrolysis of Reb Z provided D-glucose, which was identified by direct comparison with authentic samples by TLC. Enzymatic hydrolysis of Reb Z provides the aglycone by comparison with the standard compound¹H NMR and co-TLC identified the aglycone as steviol. Assignment of Compound Reb Z based on TOCSY, HMQC and HMBC data¹H and¹³the large coupling constants observed for the 5 terminal protons of the glucose moiety indicate their β -localization as reported for steviol glycosides.

TABLE 2 for rebaudioside Z ("Reb Z") and rebaudioside E^a-cIs/are as follows¹H and¹³c NMR spectral data (chemical migration and coupling constants).

^aAssignments based on TOCSY, HMQC and HMBC correlations;^bchemical mobility value in (ppm);^ccoupling constants in Hz.

Based on NMR spectral data from Reb Z and the results of hydrolysis experiments and of Reb Z with rebaudioside E¹H and¹³a detailed comparison of the CNMR values shows that the mixture of the two compounds prepared by the enzymatic conversion is presumed to be 13- [ (2-O- β -D-glucopyranosyl-2-O- β -D-glucopyranosyl- β -D-glucopyranosyl) oxygen]Ent-kauri-16-ene-19-carboxylic acid 2-O- β -D-glucopyranosyl- β -D-glucopyranosyl ester (Reb Z1) or 13- [ (2-O- β -D-glucopyranosyl- β -D-glucopyranosyl) oxy]Ent-kauri-16-ene-19-carboxylic acid- [ (2-O- β -D-glucopyranosyl-2-O- β -D-glucopyranosyl- β -D-glucopyranosyl) ester (Reb Z2).

Acid hydrolysis of compound Reb Z. To a solution of Compound Reb Z (5mg) in methanol (10ml) was added 3ml of 5% H₂SO₄And the mixture was refluxed for 24 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 25ml) to yield a water fraction containing the sugar and an EtOAc fraction containing the aglycone moiety. The aqueous phase was concentrated and the TLC system EtOAc/n-butanol/water (2:7:1) and CH was used₂Cl₂Comparison of/MeOH/water (10:6:1) with standard sugars; the sugar was identified as D-glucose.

Enzymatic hydrolysis of compound Reb Z. Compound Reb Z (1mg) was dissolved in 10ml of 0.1M sodium acetate buffer (pH4.5) and natural pectinase (50uL, Sigma-Aldrich, P2736) from Aspergillus niger was added. The mixture was stirred at 50 ℃ for 96 hours. The product precipitated during the reaction and was filtered and then crystallized. The resulting product obtained from hydrolysis of 1 was identified as steviol by comparing common TLC and 1H NMR spectral data (fig. 11) with standard compounds.

A mixture of two compounds, called Reb Z, was prepared by biotransformation of rebaudioside E using an enzymatic method and its structural characterization, based on extensive 1D and 2D NMR and high resolution mass spectrometric data and hydrolysis studies, was 13- [ (2-O- β -D-glucopyranosyl) oxy ] ent-kaurel-16-ene-19-carboxylic acid-2-O- β -D-glucopyranosyl-19-carboxylic acid ester (Reb Z1), or 13- [ (2-O- β -D-glucopyranosyl) oxy ] ent-kaurel-16-ene-19-carboxylic acid- [ (2-O- β -D-glucopyranosyl) oxy ] 2-O-beta-D-glucopyranosyl) ester (Reb Z2).

Thus, the 1, 2-19-O-glucose glycosylation activity of the recombinant HV1 polypeptide was determined by its ability to transfer the second sugar moiety to C-2' of the 19-O-glucose of stevioside to make rebaudioside E. The HV1 recombinant polypeptide also has activity to transfer a third glucose moiety to C-2 'of 13-O-glucose or C-2' of 19-O-glucose of rebaudioside E to make Reb Z1 and RebZ 2.

Claims (45)

1. A recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6.

2. The recombinant polypeptide of claim 1, wherein the amino acid sequence has at least 90% identity to SEQ ID NO 6.

3. The recombinant polypeptide of claim 1, wherein the amino acid sequence has at least 95% identity to SEQ ID NO 6.

4. The recombinant polypeptide of claim 1, wherein the amino acid sequence consists of SEQ ID NO 6.

5. An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant polypeptide of claim 1.

6. The isolated nucleic acid of claim 5, wherein the nucleotide sequence has at least 80% identity to SEQ ID NO 7.

7. A vector comprising the isolated nucleic acid of claim 5.

8. A host cell comprising the vector of claim 7.

9. The host cell of claim 8, wherein the host cell is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria, and plant cells.

10. The host cell of claim 9, wherein the host cell is selected from the group consisting of escherichia, salmonella, bacillus, acinetobacter, streptomyces, corynebacterium, methylcampylobacter, methylomonas, rhodococcus, pseudomonas, rhodobacter, synechocystis, saccharomyces, zygosaccharomyces, kluyveromyces, candida, hansenula, debaryomyces, mucor, pichia, torulopsis, aspergillus, nematocide, brevibacterium, microbacterium, arthrobacter, citrobacter, escherichia, klebsiella, pantoea, salmonella, clostridium, and clostridium acetobutylicum.

11. The host cell of claim 9, wherein the host cell is a cell of a plant isolated from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oat, sorghum, rice, broccoli, cauliflower, cabbage, parsnip, melon, carrot, celery, parsley, tomato, potato, strawberry, peanut, grape, a grass seed crop, sugar beet, sugarcane, bean, pea, rye, flax, broad leaf, conifer, gramineous forage, arabidopsis, rice (Oryza sativa), barley, switchgrass (Panicum virtuum), brachypodium, brassica, and crambe.

12. A method of making a steviol glycoside composition, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6.

13. The method of claim 12, further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide.

14. The method of claim 13, wherein the recombinant sucrose synthase comprises an amino acid sequence having at least 80% identity to SEQ ID No. 9.

15. The method of claim 13, further comprising incubating a recombinant UDP-glycosyltransferase with the sucrose synthase, the substrate, and the recombinant polypeptide.

16. The method of claim 15, wherein the recombinant UDP-glycosyltransferase comprises an amino acid sequence having at least 80% identity to SEQ ID No. 11.

17. The method of claim 12, wherein the substrate is selected from the group consisting of stevioside, rebaudioside a, rebaudioside E, and combinations thereof.

18. A steviol glycoside composition prepared by the method of claim 12.

19. The steviol glycoside composition of claim 18, comprising a steviol glycoside compound selected from rebaudioside D, rebaudioside E, rebaudioside Z and combinations thereof.

20. A sweetener comprising the steviol glycoside composition of claim 18.

21. A method of making rebaudioside Z, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID No. 6.

22. The method of claim 21, wherein the substrate is selected from the group consisting of rubusoside, stevioside, and combinations thereof.

23. The method of claim 21, further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide.

24. The method of claim 23, wherein the recombinant sucrose synthase comprises an amino acid sequence having at least 80% identity to SEQ ID No. 9.

25. The method of claim 23, further comprising incubating a recombinant UDP-glycosyltransferase with the sucrose synthase, the substrate, and the recombinant polypeptide.

26. Rebaudioside Z prepared by the method of claim 21.

27. The rebaudioside Z compound of claim 26, wherein the rebaudioside Z compound comprises a mixture of rebaudioside Z1(Reb Z1) and rebaudioside Z2(Reb Z2).

28. A method of synthesizing rebaudioside Z from rebaudioside E, the method comprising:

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 HV 1;

incubating the reaction mixture for a sufficient time to produce rebaudioside Z, wherein glucose is covalently coupled to rebaudioside E to produce rebaudioside Z, glucose is covalently coupled to C2 '-13-O-glucose of rebaudioside E to produce rebaudioside Z1, and glucose is covalently coupled to C2' -19-O-glucose of rebaudioside E to produce rebaudioside Z2.

29. The method of claim 28, further comprising adding a sucrose synthase to the reaction mixture.

30. The method of claim 29, wherein the sucrose synthase is selected from the group consisting of an arabidopsis sucrose synthase 1, an arabidopsis sucrose synthase 3, and a mungbean sucrose synthase.

31. The method of claim 30, wherein the sucrose synthase is an arabidopsis sucrose synthase 1.

32. A method of synthesizing rebaudioside D from rebaudioside E, the method comprising:

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 UGT76G 1; and

incubating the reaction mixture for a sufficient time to produce rebaudioside D, wherein glucose is covalently coupled to rebaudioside E to produce rebaudioside D.

33. The method of claim 32, further comprising adding a sucrose synthase to the reaction mixture.

34. The method of claim 33, wherein the sucrose synthase is selected from the group consisting of an arabidopsis sucrose synthase 1, an arabidopsis sucrose synthase 3, and a mungbean sucrose synthase.

35. The method of claim 34, wherein the sucrose synthase is an arabidopsis sucrose synthase 1.

36. A rebaudioside Z1 compound comprising the structure:

37. a rebaudioside Z2 compound comprising the structure:

38. a sweetener comprising a compound having the chemical structure:

39. the sweetener of claim 38, further comprising at least one of a filler, a bulking agent, and an anti-caking agent.

40. A consumable comprising a sweetening amount of the sweetener of claim 38.

41. The consumable of claim 40, said consumable being selected from the group consisting of a beverage, a dessert, a baked product, a biscuit and a chewing gum.

42. A sweetener comprising a compound having the chemical structure:

43. the sweetener of claim 42, further comprising at least one of a filler, a bulking agent, and an anti-caking agent.

44. A consumable comprising a sweetening amount of the sweetener of claim 42.

45. The consumable of claim 44, selected from the group consisting of a beverage, a dessert, a baked product, a biscuit and a chewing gum.