RU2839979C2 - Method of producing rebaudioside d and rebaudioside m - Google Patents

Abstract

FIELD: biotechnology; food industry.

SUBSTANCE: present group of inventions relates to biotechnology and can be used in food industry. Invention discloses methods for producing glycoside sweeteners, particularly rebaudioside D (Reb D) and rebaudioside M (Reb M) by active bioconversion of more available rebaudioside A (Reb A) in the presence of enzymes of the group of uridine diphosphate(UDP)-glycosyltransferases, characterized by novel amino acid sequences SEQ ID NO: 3 and 4, respectively.

EFFECT: invention may be applicable for production of alternative sweeteners possessing more attractive taste characteristics in comparison with conventional sweeteners, both synthetic and those extracted from natural raw material.

18 cl, 11 dwg, 5 tbl, 5 ex

Description

Field of technology

The present application relates to methods for producing rebaudioside D and rebaudioside M by a reaction using uridine diphosphate (UDP) glycosyltransferases; and compositions for producing rebaudioside D and rebaudioside M containing uridine diphosphate (UDP) glycosyltransferases.

Prior art

Since the World Health Organization (WHO) recommends reducing the amount of daily sugar consumption due to concerns about the disease (obesity) caused by sugar consumption, governments in developed countries are actively discussing various policies aimed at reducing sugar consumption. Thus, since there is a growing demand in the market to develop various alternative sweeteners instead of sugar and high fructose products, alternative sweeteners are constantly being developed and commercialized.

Alternative sweeteners are subject to constant changes in the form of synthetic high-intensity sweeteners (e.g. saccharin, aspartame, sucralose, etc.), synthetic sugar alcohols (e.g. maltitol and xylitol) and high-intensity sweeteners (e.g. rebaudioside A and licorice). However, due to concerns about the safety of synthetic sweeteners, consumer demand for natural sweeteners has steadily increased; however, due to the limitations associated with the specific taste properties of natural sweeteners (i.e. unpleasant odor and taste), natural sweeteners cannot completely replace existing low-calorie and zero-calorie products based on synthetic sweeteners.

A natural high-intensity sweetener that has received considerable attention in recent years is stevia, extracted from the stevia plant. Stevia has potential as an alternative sweetener because it has been reported to generate no calories, have a positive effect on blood glucose and insulin levels, and have no side effects on the human body; however, stevia has limitations in its use for sugar reduction because it has a bitter taste.

Stevia is a perennial plant of the Asteraceae family, native to Paraguay, South America, and its scientific name is Stevia rebaudiana Bertoni. Stevia leaves contain sweetening components that are 200-300 times sweeter than sugar, and these sweetening components are extracted and used as a natural sweetener. The sweetening components of stevia extracts contain various steviol glycosides, such as stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside E, etc.

Stevia leaves contain relatively high levels of stevioside (STV), rebaudioside A (Reb A) and rebaudioside C (Reb C) among the sweetening components of stevia extract, and thus high purity extracted and purified industrial products have been launched, but they have limitations in their use for sugar reduction due to the bitter taste.

Meanwhile, rebaudioside D (Reb D) and rebaudioside M (Reb M) have a less bitter taste than STV, rebaudioside A and rebaudioside C, and have excellent sweetening properties, and are thus very valuable as alternative sweeteners. However, rebaudioside D and rebaudioside M are present in stevia leaves only in very small amounts, which is a disadvantage in that the extraction and purification method of rebaudioside D and rebaudioside M from the leaves and their production are costly.

Prior Art Literature

Patent literature

(Patent Literature 0001) Korean Patent No. 1404728.

Description of the invention

Technical problem

As a result of intensive efforts to develop an enzyme that has an activity of converting rebaudioside A into rebaudioside D, the applicant of the present invention discovered that a polypeptide sequence, the glycosyltransferase activity of which was previously unknown, has a glycosyltransferase activity, and completed this application by confirming that this polypeptide has a glycosyltransferase activity that enables the conversion of rebaudioside A into rebaudioside D.

Technical solution

One of the objectives of the present application is to provide a method for producing rebaudioside D, comprising: reacting a nucleotide diphosphate to which glucose is bound with rebaudioside A in the presence of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) to produce rebaudioside D, wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Another object of the present application is to provide a method for producing rebaudioside M, comprising: reacting a nucleotide diphosphate to which glucose is bound with rebaudioside A in the presence of uridine diphosphate (UDP) glycosyltransferase B (UGT-B) to produce rebaudioside D; and reacting rebaudioside D with a nucleotide diphosphate to which glucose is bound in the presence of uridine diphosphate (UDP) glycosyltransferase A (UGT-A) to produce rebaudioside M, wherein uridine diphosphate (UDP) glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Another object of the present application is to provide a method for producing rebaudioside D from rebaudioside A, comprising: reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase and uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) in situ to produce rebaudioside D.

Another object of the present application is to provide a method for producing rebaudioside M from rebaudioside A, comprising: reacting sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, saccharose synthase, uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) in situ to produce rebaudioside M.

Another object of the present application is to provide a composition for producing rebaudioside D comprising uridine diphosphate (UDP)-glycosyltransferase B (UGT-B).

Furthermore, another object of the present application is to provide a composition for producing rebaudioside M comprising uridine diphosphate (UDP)-glycosyltransferase A (UGT-A).

Furthermore, another object of the present application is to provide the use of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) for converting rebaudioside A to rebaudioside D, wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Positive effects

The method for producing rebaudioside D and rebaudioside M using uridine diphosphate (UDP) glycosyltransferase B (UGT-B) according to the present application can provide rebaudioside D and rebaudioside M with high purity and high yield, with almost no by-products, and thus can be effectively used for mass production of rebaudioside D and rebaudioside M, since it is economical by using inexpensive raw materials, and the procedure is simple and takes less time.

Fig. 1 shows the result of HPLC analysis showing that rebaudioside A is converted to rebaudioside D by uridine diphosphate (UDP)-glycosyltransferase B (UGT-B_6).

Fig. 2 shows the result of HPLC analysis showing that rebaudioside A is converted to rebaudioside D, rebaudioside D isomer and rebaudioside M isomer by uridine diphosphate (UDP)-glycosyltransferase B (UGT-B_7).

Fig. 3 shows the result of HPLC analysis showing that rebaudioside A is converted to rebaudioside D by uridine diphosphate (UDP)-glycosyltransferase B (UGT-B_8).

Fig. 4 shows the result of HPLC analysis showing that rebaudioside D, rebaudioside M, rebaudioside I and rebaudioside A are formed from stevioside via uridine diphosphate (UDP)-glycosyltransferase B (UGT-B_6), uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and sucrose synthase.

Fig. 5 shows the result of HPLC analysis showing that rebaudioside M isomer, rebaudioside D, rebaudioside M, rebaudioside I and rebaudioside A are produced from stevioside by uridine diphosphate (UDP)-glycosyltransferase B (UGT-B_7), uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and sucrose synthase.

Fig. 6 shows the result of HPLC analysis showing that rebaudioside D, rebaudioside M, rebaudioside I and rebaudioside A are produced from stevioside by uridine diphosphate (UDP)-glycosyltransferase B (UGT-B_8), uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and sucrose synthase.

Fig. 7 shows the result of HPLC analysis showing that rebaudioside D is converted to rebaudioside M by uridine diphosphate (UDP)-glycosyltransferase A (UGT-A).

Fig. 8 shows the result of LC-MS/MS (liquid chromatography and mass spectrometry) analysis of rebaudioside D isomer.

Fig. 9 shows the result of LC-MS/MS analysis of rebaudioside D.

Fig. 10 shows the result of LC-MS/MS analysis of rebaudioside M isomer.

Fig. 11 shows the result of LC-MS/MS analysis of rebaudioside M.

Detailed Description of Preferred Embodiments

The present application will be described in detail as follows. In this regard, each description and embodiment disclosed herein can be applied to other descriptions and embodiments, respectively. That is, all combinations of various elements disclosed herein are included in the scope of the present application. In addition, the scope of the present application is not limited to the specific description given below. In addition, a number of articles and patent documents are indicated in the present description. The contents of these articles and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present application pertains and the contents of the present application will be described in more detail.

One aspect of the present application provides a method for producing rebaudioside D, comprising: reacting a nucleotide diphosphate to which glucose is bound with rebaudioside A in the presence of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) to produce rebaudioside D, wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins having the amino acid sequences of SEQ ID NOs: 1-3.

As used herein, the term "uridine diphosphate (UDP) glycosyltransferase (UGT)" is an enzyme that catalyzes the transfer of a monosaccharide group from a glycosyl donor to a glycosyl acceptor molecule, and in particular refers to an enzyme that uses UDP-caxap as a glycosyl donor. In the present application, UDP-glycosyltransferase may be used interchangeably with UGT.

UDP-glycosyltransferase can be obtained from recombinant E. coli , Bacillus , yeast, Corynebacterium or Agrobacterium transformed with a vector containing the glycosyltransferase gene, and UDP-glycosyltransferase can be further purified after obtaining from E. coli or the like, or a commercially produced product can be purchased and used, but UDP-glycosyltransferase is not limited to this. In addition, UDP-glycosyltransferase is known in the art, and the protein and the gene sequence of UDP-glycosyltransferase can be obtained from a known database such as GenBank of NCBI, etc., but not limited to this.

In the present application, it was discovered for the first time that a novel uridine diphosphate (UDP)-glycosyltransferase B (UGT-B), which is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3, has an enzymatic activity for converting rebaudioside A to rebaudioside D, thereby providing a new use for UGT-B.

In particular, the uridine diphosphate (UDP) glycosyltransferase B (UGT-B) of the present application may have and/or include the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 3, or consist essentially of or consist of these amino acid sequences.

In addition, the uridine diphosphate (UDP) glycosyltransferase B (UGT-B) may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, or the amino acid sequence of SEQ ID NO: 3. It is further understood that any uridine diphosphate (UDP) glycosyltransferase B (UGT-B) having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added may also be encompassed within the scope of the present application, provided that the amino acid sequence has such homology or identity and exhibits an effect similar to that of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B).

In the context of the present document, although it is described as "a polypeptide or protein comprising an amino acid sequence described by a particular sequence number", "a polypeptide or protein consisting of an amino acid sequence described by a particular sequence number" or "a polypeptide or protein having an amino acid sequence described by a particular sequence number", it is obvious that any protein having an amino acid sequence in which a part of the sequence is deleted, modified, replaced, conservatively replaced or added, can be used in the present application, provided that even if it has the same or corresponding activity as a polypeptide consisting of an amino acid sequence with the corresponding sequence number. For example, this may be the case when a sequence is added to the N-terminus and/or C-terminus of the amino acid sequence that does not change the function of the protein, a natural mutation, a potential mutation (silent mutation) or a conservative substitution thereof.

For example, this may be the case where sequences that do not alter the function of uridine diphosphate (UDP) glycosyltransferase B (UGT-B) according to the present application, a naturally occurring mutation, a potential mutation (silent mutation) or a conservative substitution thereof, are added to or deleted from the N-terminus, C-terminus and/or internal part of the amino acid sequence.

As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and/or chemical properties. Such an amino acid substitution may typically be based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residue. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In addition, amino acids can be divided into amino acids with electrically charged side chains and amino acids with uncharged side chains, and examples of amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further divided into non-polar amino acids or polar amino acids. Examples of non-polar amino acids are glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, and examples of polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In general, conservative substitutions have little or no effect on the activity of the resulting polypeptide. In general, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

In addition, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) can also include the removal or addition of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to a signal (or leader) sequence at the N-terminus involved in co-translational or post-translational transfer of proteins. In addition, the polypeptide can also be conjugated to another sequence or linker for identification, purification or synthesis of the polypeptide.

As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms "homology" and "identity" may often be used interchangeably.

Homology or sequence identity of conserved polynucleotide or polypeptide sequences can be determined using standard alignment algorithms and can be used with the default gap penalty set by the program used. As such, homologous or identical sequences are generally expected to hybridize to all or part of the sequences under moderate to high stringency conditions. Obviously, hybridization to polynucleotides containing a common codon or degenerate codons in the hybridizing polynucleotides is also included.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can, for example, be determined using a known computer algorithm such as the program "FASTA" (Pearson et al., (1988) Proc. Natl. Acad. Sci. USA 85:2444) using default parameters. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is performed using the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet 16:276-277) (preferably version 5.0.0 or higher) (GCG software package). (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information (NCBI).

Homology, similarity, or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using, for example, the GAP computer program such as Needleman et al. (1970) J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines homology, similarity, or identity as the value obtained by dividing the number of identically aligned characters (i.e., nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include 1) a unary comparison matrix (containing the value 1 for identities and 0 for non-identities) and a weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as disclosed in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353–358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version NCBI NUC4.4)); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each character in each gap (or a gap-opening penalty of 10 and a gap-extension penalty of 0.5); and (3) no penalties for gaps at the ends.

In one example of the present application, uridine diphosphate (UDP) glycosyltransferase B (UGT-B) may have the activity of converting rebaudioside A to rebaudioside D.

As used herein, the term "corresponding" refers to an amino acid residue at a position indicated in a peptide, or to an amino acid residue that is similar, identical, or homologous to a residue indicated in a peptide. Identifying an amino acid at a corresponding position may mean identifying a specific amino acid in a sequence that relates to a specific sequence. As used herein, the term "corresponding region" generally refers to an analogous or corresponding position in a related protein or reference protein.

For example, an amino acid sequence is aligned with SEQ ID NO: 1, and based on this alignment, each amino acid residue of the amino acid sequence can be numbered with reference to the numeric position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm, such as that described herein, can identify an amino acid position or a position at which modifications, such as substitutions, insertions, or deletions, occur, compared to a query sequence (also referred to as a "reference sequence").

An example of alignment can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is performed by the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), etc., but is not limited thereto, and sequence alignment programs such as pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

In addition, the polynucleotide encoding uridine diphosphate (UDP) glycosyltransferase B (UGT-B) of the present invention is not only a nucleotide sequence encoding the amino acids represented by each SEQ ID NO indicated above, but also includes, without limitation, a nucleotide sequence having a homology of 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more with the above sequence and with any sequence of a gene encoding a protein exhibiting the same or corresponding efficiency as each of the above proteins. In addition, it is obvious that any nucleotide sequence in which a part of the sequence is deleted, modified, replaced or added may also fall within the scope of the present application, provided that the nucleotide sequence has such homology.

In this document, the term "polynucleotide", which is a polymer of nucleotides consisting of nucleotide monomers linked into a long chain by a covalent bond, is a DNA or RNA chain having at least a certain length. More specifically, it can refer to a fragment of a polynucleotide encoding uridine diphosphate (UDP)-glycosyltransferase B (UGT-B).

A polynucleotide encoding uridine diphosphate (UDP) glycosyltransferase B (UGT-B) of the present invention may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. As an example of the present invention, the polynucleotide may have or include the sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. Furthermore, the polynucleotide may consist of or essentially consist of the sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

The polynucleotide of the present invention may be subject to various modifications in the coding region to the extent that they do not change the amino acid sequence of the uridine diphosphate (UDP) glycosyltransferase B (UGT-B) of the present invention, due to codon degeneracy or taking into account the codon preferences of the organism in which the uridine diphosphate (UDP) glycosyltransferase B (UGT-B) of the present invention is to be expressed. In particular, a polynucleotide of the present invention may have or include a nucleotide sequence having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 100% or less with the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or may consist of or consist essentially of a nucleotide sequence having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 100% or less with the sequence of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, but is not limited to this.

In addition, the polynucleotide of the present invention may include a probe that can be derived from a known gene sequence, such as any sequence that can hybridize with a sequence complementary to all or part of a polynucleotide sequence of the present invention under stringent conditions without limitation. "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, stringent conditions may include conditions under which genes having high homology and an identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more hybridize with each other and genes having a homology or identity lower than the above homology or identity do not hybridize with each other, or under Southern hybridization washing conditions, that is, washing once, especially two or three times, with a salt concentration and temperature corresponding to 60°C, 1×SSC (citrate buffered saline), 0.1% SDS (sodium dodecyl sulfate), specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, a polynucleotide according to the present application can include isolated nucleotide fragments that are complementary to the entire sequence, as well as nucleic acid sequences that are substantially similar thereto.

In particular, polynucleotides having homology or identity with the polynucleotide of the present application can be detected by using hybridization conditions comprising a hybridization step at a _Tm value of 55°C under the conditions described above. In addition, the _Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately selected by those skilled in the art depending on the purpose.

The appropriate stringency for hybridization of polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (e.g., Sambrook et al).

In one embodiment of the present application, the nucleotide diphosphate to which glucose is bound may be obtained by reacting sucrose and nucleotide diphosphate in the presence of sucrose synthase, but is not limited thereto.

In the present document, the term "sucrose synthase" plays a role in producing sucrose by reversibly transferring glucose, which is bound to nucleotide diphosphate, to fructose in plant metabolism. In the present invention, sucrose synthase exhibits an activity of separating nucleotide diphosphate, to which glucose is bound, and fructose by reacting sucrose and nucleotide diphosphate in a pH range of 5-10.

Sucrose synthase may be obtained from rice, corn, wheat, bamboo, Arabidopsis thaliana , grass, barley, sorghum or potato. Preferably, sucrose synthase is obtained from rice, corn, wheat or barley, and more preferably from rice, in particular Oryza sativa . Sucrose synthase may be obtained from recombinant Escherichia coli , Bacillus , yeast, Corynebacterium or Agrobacterium transformed with a vector containing the sucrose synthase gene, and may be further purified after its production from Escherichia coli and the like. Sucrose synthase may be known in the art or may be commercially purchased, but is not limited thereto.

In particular, the sucrose synthase of the present application may have and/or include the amino acid sequence of SEQ ID NO: 5 or may consist of or consist essentially of this amino acid sequence.

Sucrose is not particularly limited as long as it can serve as a substrate for sucrose synthase to provide glucose for nucleotide diphosphate. Examples of sucrose may include raw sugar or sugar, without limitation.

In the present application, a purine nucleotide or a pyrimidine nucleotide can be used as the nucleotide diphosphate. Preferably, uridine diphosphate is used as the nucleotide diphosphate, but the nucleotide diphosphate is not limited thereto.

The nucleotide diphosphate to which glucose is bound can be reacted with rebaudioside A to produce rebaudioside D using uridine diphosphate (UDP) glycosyltransferase B (UGT-B) of the present application.

In one embodiment of the present application, rebaudioside A can be obtained by reacting a nucleotide diphosphate to which glucose is bound with stevioside in the presence of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A), but is not limited thereto.

Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) can produce rebaudioside A by reacting the nucleotide diphosphate to which glucose is bound with stevioside.

Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) can be obtained from Oryza sativa , Stevia rebaudiana Bertoni , Bambusa oldhamii , Brachypodium distachyon , Hordeum vulgare , Sorghum bicolor , Zea mays or Arabidopsis thaliana . Preferably, uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) can be obtained from Oryza sativa , Stevia rebaudiana Bertoni or Bambusa oldhamii . More preferably, it can be obtained from Stevia rebaudiana Bertoni . Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) can be obtained from recombinant Escherichia coli , Bacillus , yeast, Corynebacterium or Agrobacterium transformed with a vector containing the glycosyltransferase gene, or can be further purified after obtaining from Escherichia coli and the like. Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) may be known in the art or may be commercially purchased, but is not limited thereto.

In particular, the uridine diphosphate (UDP) glycosyltransferase A (UGT-A) of the present application may have and/or include the amino acid sequence of SEQ ID NO: 4 or may consist of or consist essentially of this amino acid sequence.

Stevioside is an extract of Stevia rebaudiana obtained by hot water or aqueous ethanol solution, or a substance purified therefrom, or a by-product after obtaining rebaudioside A from the extract. Steviosides may be those having a stevioside content of 10% by mass or more, preferably 50% by mass or more, particularly preferably 70% by mass or more, and even more preferably 80% by mass or more, based on the total mass of steviol glycoside, but are not limited thereto.

In one embodiment of the present invention, rebaudioside D can be obtained as shown below in chemical reaction 1, but is not limited thereto.

Chemical reaction 1

In particular, the production method can be sequentially carried out in situ .

The term " in situ " as used herein means that the reaction is carried out sequentially in a single reaction system.

The production method of the present invention is a sequential reaction system in which one glucose is specifically bound to the C-3' position of stevioside-13-O-glucose to synthesize rebaudioside A in high yield, and rebaudioside D is synthesized from rebaudioside A according to the chemical reaction 1 above.

In one embodiment of the present invention, without limitation thereto, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) may be a protein consisting of the amino acid sequence of SEQ ID NO: 2, and a rebaudioside D isomer may be further obtained.

For example, rebaudioside D may have the following structure, but is not limited to it:

In another aspect of the present invention, there is provided a method for producing rebaudioside M, comprising: reacting a nucleotide diphosphate to which glucose is bound with rebaudioside A in the presence of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) to produce rebaudioside D; and

the reaction of rebaudioside D with nucleotide diphosphate to which glucose is bound in the presence of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) to produce rebaudioside M,

wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Nucleotide diphosphate, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B), rebaudioside A and rebaudioside D are as described above.

In one embodiment of the present invention, the nucleotide diphosphate to which glucose is bound may be obtained by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase as shown in Chemical Reaction 1 above, but is not limited thereto.

In particular, the sucrose synthase of the present invention may have and/or include the amino acid sequence of SEQ ID NO: 5 or may consist of or consist essentially of this amino acid sequence.

In one embodiment of the present invention, rebaudioside A can be obtained by reacting a nucleotide diphosphate to which glucose is bound with stevioside in the presence of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A), as shown in Chemical Reaction 1 above, but is not limited thereto.

In particular, the uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) of the present invention may have and/or include the amino acid sequence of SEQ ID NO: 4 or may consist of or consist essentially of this amino acid sequence.

The uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) of the present invention can produce rebaudioside M by reacting a nucleotide diphosphate to which glucose is bound with rebaudioside D.

In particular, the uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) of the present invention may have and/or include the amino acid sequence of SEQ ID NO: 4 or may consist of or consist essentially of the amino acid sequence.

In one embodiment of the present invention, rebaudioside M can be obtained as shown below in Chemical Reaction 2, but is not limited thereto.

Chemical reaction 2

In particular, the production method can be carried out sequentially in situ .

The production method of the present invention provides a sequential reaction system in which rebaudioside A is synthesized from stevioside in high yield, rebaudioside D is synthesized from rebaudioside A, and rebaudioside M is synthesized from rebaudioside D according to chemical reaction 2 given above.

Accordingly, the production method of the present invention uses raw materials such as stevioside and rebaudioside A, which are inexpensive and can be easily obtained, and can convert stevioside and rebaudioside A, which are bitter components contained in stevia extract, into rebaudioside D and rebaudioside M, which are components with excellent taste and thus can be effectively used in the production of a stevia sweetener with excellent sweetening properties.

In one embodiment of the present invention, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) may be a protein consisting of the amino acid sequence of SEQ ID NO: 2, and additionally, the rebaudioside M isomer may be obtained, without limitation thereto.

For example, rebaudioside M may have the following structure, but is not limited to it:

In another aspect, the present invention provides a method for producing rebaudioside D from rebaudioside A, comprising: reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase and uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) in situ to produce rebaudioside D,

wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B), rebaudioside D and in situ are as described above.

In one embodiment of the present invention, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) may be a protein consisting of the amino acid sequence of SEQ ID NO: 2, and the rebaudioside D isomer may further be obtained, without limitation thereto.

In another aspect of the present invention, there is provided a method for producing rebaudioside M from rebaudioside A, comprising: reacting sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) in situ to produce rebaudioside M,

wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, uridine diphosphate (UDP)-glycosyltransferase A (UGT-A), uridine diphosphate (UDP)-glycosyltransferase B (UGT-B), in situ and rebaudioside M are as described above.

In particular, uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) may be a protein consisting of the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

In one embodiment of the present invention, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) may be a protein consisting of the amino acid sequence of SEQ ID NO: 2, and the rebaudioside M isomer may further be obtained, but is not limited thereto.

In another aspect of the present invention, there is provided a composition for producing rebaudioside D comprising uridine diphosphate (UDP)-glycosyltransferase B (UGT-B), wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins having the amino acid sequences of SEQ ID NO: 1-3.

Uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) and rebaudioside D are as described above.

In one embodiment of the present invention, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) may be a protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and rebaudioside D may be at least one selected from the group consisting of rebaudioside D and an isomer of rebaudioside D, but they are not limited thereto.

In another aspect of the present invention, there is provided a composition for producing rebaudioside M, comprising uridine diphosphate (UDP) glycosyltransferase A (UGT-A) and uridine diphosphate (UDP) glycosyltransferase B (UGT-B), wherein uridine diphosphate (UDP) glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence of SEQ ID NO: 4, and uridine diphosphate (UDP) glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequence of SEQ ID NO: 1-3.

Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A), uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) and rebaudioside M are as described above.

In one embodiment of the present invention, uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) may be a protein consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and rebaudioside M may be at least one selected from the group consisting of rebaudioside M and an isomer of rebaudioside M, but they are not limited thereto.

The composition of the present invention may further comprise any suitable excipient commonly used in a composition for producing amino acids, and such an excipient may be, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizer or an isotonic agent, but is not limited to them.

In another aspect of the present invention, there is provided the use of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) for converting rebaudioside A to rebaudioside D, wherein uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) is at least one protein selected from the group consisting of proteins consisting of the amino acid sequences of SEQ ID NO: 1-3.

Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A), uridine diphosphate (UDP)-glycosyltransferase B (UGT-B), rebaudioside A, rebaudioside M, etc. are as described above.

Method of carrying out the invention

Below, the present application will be described in detail by means of examples. However, these examples are merely preferred examples given for illustrative purposes, and thus the scope of the present invention is not intended to be limited to these examples. However, technical features that are not described herein can be sufficiently understood and easily implemented by those skilled in the art in the technical field of the present invention or in a similar technical field.

Example 1. Cultivation conditions

A recombinant microorganism was obtained by introducing a polynucleotide encoding uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) of the present invention, and then used in the interaction after expression of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B). The conditions for culturing the recombinant microorganism are as follows.

Example 1-1. Conditions for culturing E. coli

Recombinant E. coli was cultured as follows: a test tube containing 5 ml of LB medium containing 50 μg/ml kanamycin was inoculated with recombinant E. coli , followed by culturing in an incubator at 37°C until the absorbance at 600 nm was 2.0. The seed culture solution was added to a flask containing 500 ml of LB medium containing 50 μg/ml kanamycin, and then cultured. Then, 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added and cultured until the absorbance at 600 nm was 0.4, thereby inducing massive expression of the enzymes. The culture conditions were adjusted such that the stirring speed was 180 rpm and the culture temperature was 37°C during the procedure, and the stirring speed was 120 rpm after the addition of IPTG.

Example 1-2. Conditions for culturing Corynebacteria

Recombinant Corynebacteria were inoculated into the medium (Bacto-Tryptone 10 g/L, Bacto-yeast extract 5 g/L, NaCl 5 g/L, Soytone 5 g/L) containing kanamycin at a concentration of 10 μg/ml, with an initial concentration of OD ₆₀₀ = 0.1, and cultured at 30°C for 24 hours to induce enzyme expression. The culture solution obtained in this way was inoculated into a fermenter containing a medium (glucose 80 g/l, soytone 20 g/l, (NH ₄ ) ₂ SO ₄ 10 g/l, KH ₂ PO ₄ 1.2 g/l, MgSO ₄ 1.4 g/l) containing kanamycin at a concentration of 10 μg/ml with OD ₆₀₀ = 0.6 and cultured at 30°C for 24 hours.

Example 2. Measurement of the enzymatic activity of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) for rebaudioside A as a raw material

Example 2-1. Purification of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B)

For uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) (UGT-B; SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) of the present invention, which converts rebaudioside A into rebaudioside D, each recombinant plasmid (vector-pET28a) containing the gene (SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8) encoding uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) was prepared and cloned into E. coli BL21 (DE3) to induce mass expression of each enzyme, and then the enzymes were purified and used.

Specifically, the recombinant strain BL21(DE3) was inoculated into a test tube containing 5 ml of LB medium, followed by culturing the seed in an incubator at 37°C until the absorbance at 600 nm became 2.0. The seed culture solution was added to a flask containing 500 ml of LB medium and then cultured. Then, 0.1 mM IPTG (isopropyl-β-D-1-thiogalactopyranoside) was added until the absorbance at 600 nm became 0.4, thereby inducing massive expression of the enzymes. The culture conditions were adjusted such that the stirring speed was 180 rpm and the culture temperature was 37°C during the procedure, and the stirring speed was 120 rpm and the culture temperature was 16°C after the addition of IPTG. The culture solution of the transformed strain was centrifuged at 6000 g at 4°C for 20 minutes to separate the cell supernatant as an enzyme solution. In order to accurately determine the properties of the enzymes, the enzyme solution was purified using a Ni-NTA Superflow column.

Each enzyme, mass-expressed using a recombinant plasmid containing SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, was named UGT-B_6, UGT-B_7, and UGT-B_8, respectively.

Meanwhile, the recombinant Corynebacteria culture was inoculated into the medium (Bacto-Tryptone 10 g/L, Bacto-yeast extract 5 g/L, NaCl 5 g/L, Soytone 5 g/L) containing kanamycin at a concentration of 10 μg/ml with an initial concentration of OD ₆₀₀ = 0.1 and cultured at 30°C for 24 hours to induce enzyme expression. The culture solution obtained in this way was inoculated into a fermenter containing a medium (glucose 80 g/l, soytone 20 g/l, (NH ₄ ) ₂ SO ₄ 10 g/l, KH ₂ PO ₄ 1.2 g/l, MgSO ₄ 1.4 g/l) containing kanamycin at a concentration of 10 μg/ml, with OD ₆₀₀ = 0.6 and cultured at 30°C for 24 hours.

Example 2-2. Measurement of the enzymatic activity of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) for rebaudioside A as a raw material

The enzymatic activity of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) for rebaudioside A as a starting material was measured using each of the enzymes obtained in Example 2-1.

Specifically, the raw material used in the enzymatic reaction was RebA (Daepyeong), which was dissolved in water to a concentration of 2 mM. The reaction was carried out at 37°C for 16 hours using each of the enzymes expressed in microorganisms and prepared in Example 2-1, and then analyzed by HPLC. The aqueous solution of the raw material may contain UDP-glucose or UDP (uridine diphosphate) in an amount of 2 mM.

The conditions for HPLC analysis are as follows:

- Detector wavelength: 210 nm,

- Flow rate: 1 ml/min,

- Volume of injected sample: 10 µl,

- Column: Capcell pak C18 MG II (Shiseido, 250 mm × 4.6 mm, particle size: 5 μm),

- Solvent: Acetonitrile 30%.

The measurement results are shown in Table 1 below and Fig. 1-3.

As shown in Table 1 above and Figs. 1-3, it was confirmed that rebaudioside A was converted into rebaudioside D by the action of uridine diphosphate (UDP) glycosyltransferase B (UGT-B_6 and UGT-B_8) of the present invention, and that rebaudioside A was converted into rebaudioside M isomer, rebaudioside D isomer and rebaudioside D by the action of uridine diphosphate (UDP) glycosyltransferase B (UGT-B_7).

Example 3. Measurement of the enzymatic activity of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) using rebaudioside A as a raw material

Uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) and sucrose synthase were purified using the method disclosed in the prior patent literature (WO 2014/133248), and their sequences were shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

The enzymatic activity of uridine diphosphate (UDP) glycosyltransferase A (UGT-A) and uridine diphosphate (UDP) glycosyltransferase B (UGT-B) using rebaudioside A as a raw material was measured using uridine diphosphate (UDP) glycosyltransferase A (UGT-A), sucrose synthase, and each of uridine diphosphate (UDP) glycosyltransferase B (UGT-B) obtained in Example 2-1.

Specifically, the raw materials used for the enzymatic reaction were 2 mM stevioside (Haigen) and 50 mM aqueous sugar solution (CJ Cheiljedang). The reaction was carried out at 37°C for 24 hours using uridine diphosphate (UDP) glycosyltransferase A (UGT-A), sucrose synthase, and each of the prepared uridine diphosphate (UDP) glycosyltransferase B (UGT-B) in Example 2-1, which were expressed in microorganisms, and then analyzed by HPLC. The aqueous raw material solution may contain UDP-glucose or UDP (uridine diphosphate) at a concentration of 2 mM.

The conditions for HPLC analysis are as follows:

- Detector wavelength: 210 nm,

- Flow rate: 1 ml/min,

- Sample injection volume: 10 µl,

- Column: Capcell pak C18 MG II (Shiseido, 250 mm × 4.6 mm, particle size: 5 μm),

- Solvent: Acetonitrile 30%.

The measurement results are shown in Table 2 below and Fig. 4-6.

As shown in Table 2 above and Fig. 4-6, it was confirmed that 100% of stevioside was converted into rebaudioside M isomer, rebaudioside D, rebaudioside M and rebaudioside I, and rebaudioside A relative to the molar concentration.

Example 4. Measurement of the enzymatic activity of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) for rebaudioside D as a raw material

The rate of conversion of rebaudioside M from rebaudioside D was measured using uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) according to Example 3.

Specifically, the raw material used for the enzymatic reaction was rebaudioside D (Haigen), which was dissolved in water to a concentration of 1 mM. The reaction was carried out at 37°C for 16 hours using uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) expressed in microorganisms and then analyzed by HPLC. The aqueous solution of the raw material may contain UDP-glucose or UDP (uridine diphosphate) at a concentration of 2 mM.

The conditions for HPLC analysis are as follows:

- Detector wavelength: 210 nm,

- Flow rate: 1 ml/min,

- Sample injection volume: 10 µl,

- Column: Capcell pak C18 MG II (Shiseido, 250 mm × 4.6 mm, particle size: 5 μm),

- Solvent: Acetonitrile 30%.

The measurement results are shown in Fig. 7.

As shown in Fig. 7, it was confirmed that most of rebaudioside D was converted into rebaudioside M by the action of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A) of the present invention.

Example 5. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of Rebaudioside D isomer and Rebaudioside M isomer

In order to confirm whether the compound converted in Examples 2 and 3 was rebaudioside D isomer or rebaudioside M isomer, the rebaudioside D isomer and rebaudioside M isomer converted in Examples 2 and 3 were compared with rebaudioside D and rebaudioside M, respectively, using liquid chromatography-mass spectrometry (LC-MS/MS) analysis.

Specifically, Acquity UPLC (Waters) and Xe-vo G2-XS Q-Tof mass spectrometer were used. The LC-MS/MS analysis conditions are shown in Table 3 below, the elution conditions are shown in Table 4 below, and the MS conditions are shown in Table 5 below.

As a result, as shown in Figs. 8 and 9, the comparison of the RebD standard and Unknown Peak 1 showed that both the RebD standard and Unknown Peak 1 have the same molecular weight, and their MSMS fragments are consistent with each other but differ only in the peak retention time, thereby confirming that Unknown Peak 1 is an isomer of RebD.

In addition, as shown in Fig. 10 and 11, comparison of the RebM standard and Unknown Peak 2 showed that both the RebM standard and Unknown Peak 2 have the same molecular weight, and their MSMS fragments are consistent with each other but differ only in peak retention time, thereby confirming that Unknown Peak 2 is an isomer of RebM.

From the above, a person skilled in the art to which this application pertains will understand that the present application can be embodied in other specific forms without changing the technical concepts or essential characteristics of the present invention. In this regard, the typical embodiments disclosed herein are intended for illustrative purposes only and should not be considered as limiting the scope of the present invention. Accordingly, the scope of the present invention is determined by the appended claims rather than by the above description. All changes that fall within the meaning and range of equivalence of the claims are intended to be included within the scope of the present invention.

--->

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<INSDSeq_sequence>MENKTETTVRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTN

FNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVS

CLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKD

IKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLD

HDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFL

GERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLE

NGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL</INSDSeq_s

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>808</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..808</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q9">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Sucrose synthase</INSDQualifier_value>

<NonEnglishQualifier_value>sucrose synthase</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..808</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>unidentified</INSDQualifier_value>

<NonEnglishQualifier_value>not set</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MAAKLARLHSLRERLGATFSSHPNELIALFSRYVNQGKGMLQRHQLLAE

FDALIEADKEKYAPFEDILRAAQEAIVLPPWVALAIRPRPGVWDYIRVNVSELAVEELSVSEYLAFKEQL

VDGHTNSNFVLELDFEPFNASFPRPSMSKSIGNGVQFLNRHLSSKLFQDKESLYPLLNFLKAHNHKGTTM

MLNDRIQSLRGLQSSLRKAEEYLMGIPQDTPYSEFNHRFQELGLEKGWGDCAKRVLDTIHLLLLLDLEAPD

PANLEKFLGTIPMMFNVVILSPHGYFAQSNVLGYPDTGGQVVYILDQVRALENEMLLRIKQQGLDITPKI

LIVTRLLPDAVGTTCGQRVEKVIGTEHTDILRVPFRSENGILRKWISRFDVWPFLETYTEDVANEIMREM

QAKPDLIIGNYSDGNLVATLLAHKLGVTQCTIAHALEKTKYPNSDIYLDKFDSQYHFSCQFTADLIAMNH

TDFIITSTFQEIAGSKDTVGQYESHIAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSVYFPYTEADKRLT

AFHPEIEELLYSEVENDEHKFVLKDKNKPIIFSMARLDRVKNMTGLVEMYGKNAHLRDLANLVIVCGDHG

NQSKDREEQAEFKKMYGLIDQYKLKGHIRWISAQMNRVRNGELYRYICDTKGVFVQPAFYEAFGLTVIEA

MTCGLPTIATCHGGPAEIIVDGVSGLHIDPYHSDKAADILVNFFEKCKQDSTYWDNISQGGLQRIYEKYT

WKLYSERLLMTLTGVYGFWKYVSNLERRETRRYIEMFYALKYRSLASAVPLAVDGESTSK</INSDSeq_s

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>1326</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1326</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q11">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>UGT-B</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1326</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q21">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>unidentified</INSDQualifier_value>

<NonEnglishQualifier_value>not set</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggcgaccttgcgcgtactgatgtttccctggttggcgtatggtcaca

tttctccttttctcaacatagccaagcagctcgcggatagaggattcctgatctacctttgtagcacgct

gatcaacctcgaaagcatcatcaagaaaatccccgaaaaatactctgagtcaattcgttttgtggagctt

cacctgcccgaattgcctgaacttcctcctcattaccacacaaccaatggtctgccgccccatctgaacc

acacacttcataaagccctgaaaatgtccaaaccaaacttttccaaaatcctgcagaatctgaaacccga

tttggtgatttatgacatattgcagccgtgggccgagcatgtcgtaaatgagcagaatattccggcagtc

aagatattaacgtcgggtgcagctctgttttcgtatttcttcaactttctaaaaaatccaggggttgaat

ttccgtttccggcaatttatctcccgaaagtggagcaagtcaagatgcgagaaatgttcgagaaggaacc

taatgaagaggatcgtctcgccgagggcaacatgcagattatgttaatgtgcacctctaggaccattgag

gccaagtatctagattattgtactgaactgagcaattggaaagttgttccagttggtccaccgttccaag

acccgatcaccaacgacgtggacgacatggagctcattgattggctaggcacaaaggatgagaattcaac

tgttttcgtctgctttggaagtgagtatttcttgtcacgggaagatatggaagaagtagcgtttggtttg

gagctgagtaatgtgaatttcatctgggtagcccgctttccgaaaggtgaggagcagaaccttgaggatg

tattgccgaaaggttttcttgaaagaattggagagcggggccgtgtgttagacaaatttgcaccacagcc

acgcattttaaatcatccgtccaccggcggattcataagtcattgtggctggaattcagtgatggagagt

ttagattttggcgttcctatcatcgccatgcctatgcataacgatcaaccaatcaacgcgaagttgatag

tggaattgggggtggcaatggaaattgttcgcgatgatgatgggaatattcaccggggtgaaattacgga

aactcttaaagatgtcataacgggggaaacaggggagatttgcgtggcaaagtgcgtgacatcagcaaa

aatttgaagtctatccgcgaggaagagatgaacgctgcagctgaagagctgattcagctgtgccggaata

gcaataagtacaaatag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>1326</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1326</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q22">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>UGT-B</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1326</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q23">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>unidentified</INSDQualifier_value>

<NonEnglishQualifier_value>not set</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctactctgaaagtactgatgtttccgtttttggcttatggccaca

tttctccctatctgaatgtagccaagaagctcgccgatcggggcttcctgatttacttctgctcaacacc

gataaatctgaagagcattatcaaaaaaatccctgaaaaatacgcgaatagcattcatcttatcgagctt

cacttaccggagttaccggagttaccccctcattaccatacgacgaatggtctcccgccaaacctcaatc

acatccttcagaaagcactgaaaatgtcaaagccaaacttctcgaagatcttacagaacctgaaacctga

tctggtgatttatgacgtattgcagcggtgggcgaaacatgtcgccaatgaacagaatatcccagcagtt

aagctcctgacgagtggtgcagccgtgtttagctattttttcaatgtcttgaaaaagccaggggtcgaat

ttccattcccgggaatttatctgcgcaaaatcgaacaagtaagactgagtgaaatgatgaaagaactgga

ggacgacgatgacgatgatgatcttctggtggatggcaatatgcaaataatgttgatgagtacctctcgc

accatcgaagccaaatatatcgatttttgcactgcgttgacgaactggaaagttgttccggttggtcctc

cggttcaggatctgataaccaacgacgcggacgatatggaacttatcgattggctaggcacaaaagatga

gaactcaactgtgtttgtctcctttggtagtgagtatttcctgtccaaggaagatatggaagaagtggct

tttgcgttggaattatcgaacgtgaactttatctgggttgccagatttccgaaaggggaagagcgaaatc

tggaagacgcactgccgaagggatttcttgagcggattggagaaaggggacgtgttttggataaatttgc

cccccagccacgtattctgaaccatccgtctaccggcggttttatatcccattgtggttggaattcagca

atggaatctatagacttcggggttcctatcatcgcgatgcctatgcacctagatcagcccatgaacgctc

gtctcatcgtagaattaggcgttgcggtcgagattgttcgtgatgatgatggtaagatccacagaggaga

aatcgcggaaaccttgaaaggtgtgataacagggaaaacaggtggaaagctccgtgctaaagtgcgcgac

attagtaaaaacttaaaaaccatacgcgacgaagagatggatgcagctgctgaggagctaatccagcttt

gtcgcaatagcaattag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>1386</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1386</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q24">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>UGT-B</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1386</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q25">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>unidentified</INSDQualifier_value>

<NonEnglishQualifier_value>not set</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggatgcgtcggaaagcgagtcatctccgctccacgttgttatatttc

cttggttagcaatgggtcatctgctcccgtgtctggaactggcagaacgattggcagcacgtggtcatcg

ggtttcgttcgttagcactcccagaaatttagcacgtttaccaccagttcgtcctgcattggcatcactc

gttcatctggttgcactgccgttgcctcgtgttgcaggtctgcccgatggagcagaaagtacatctgatg

ttccgccggataaatttcaccttcatcgtgttgctttcgatggtttggctgcaccatttaccgcatttct

ggatgcagcttgcgcaggaggtaggcgtcctgattgggttgtggcagattttgtacatcattgggttgct

gcagttgcacaagcacgtaaggtaccgtgtgcaatgttactgccttgcgcagcaggtgttgcagcactaa

gcggtccaccaccggaaagtcgtgcagaggaacatcaggctgttagtcgaagcatggatgcggcaccacg

ttttgaagcagaactgactaccgaggaatttgctgcggaagatgcgagtggtctgagtattatgggacgt

tttttcatgacgctgaaaagatctaaattagttgctctacgttcatcaccggaattagaaccggatgcat

ttccgctgctgacccgtctttatggtaaaccggcagttccgcttggactgttaccgccccctcctaacgg

agatagaagcgcaagcgaaaatagcgaggatgttgcaattatccagtggttggatgcacgtccagccaaa

tctgttgtttacgttgcactgggtagcgaagcacccctccgtgcagaactgttacgtgaattagcacatg

gacttgaactgtctggtacgcgatttttatgggttctgcgtaatccagttaacggtgatgcagatacgat

tttacctgatggttttatggaacgtacagcagaacgtggtcttgttgcagttcgttgggtacctcaagta

agcatcctggcacacggtgcagttggtgcattcttaacccactgtgggtggggttcagttgttgaaggtc

ttcaatttggtcatccatgattatgcttccgcttgcaggcgatcaggggcccaacgctcgcttaatgga

agaacgtaaagttggtgttttggtaccccgtgacgaaaaggatggttcgtttacacgtgatggtgttgca

ggtgcagttagagcagttgttgttgaagaaggtggtcgtgtatttgcagataatgcaaaacgtctgcagg

gtatagttgcgagtgttgaatgcaatgagagatgcattgatctgtttgtacagcatctacgtagctgtcg

taattag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>1377</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1377</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q26">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>UGT-A</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1377</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q27">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>unidentified</INSDQualifier_value>

<NonEnglishQualifier_value>not set</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggaaaataaaacggagaaccaccgttcgccggcgccggagaataatat

tattcccggtaccatttcaaggccacattaacccaattcttcagctagccaatgtgttgtactctaaagg

attcagtatcaccatctttcacaccaacttcaacaaacccaaaacatctaattaccctcacttcactttc

agattcatcctcgacaacgacccacaagacgaacgcatttccaatctaccgactcatggtccgctcgctg

gtatgcggattccgattatcaacgaacacggagctgacgaattacgacgcgaactggaactgttgatgtt

agcttctgaagaagatgaagaggtatcgtgtttaatcacggatgctctttggtacttcgcgcaatctgtt

gctgacagtcttaacctccgacggcttgttttgatgacaagcagcttgtttaattttcatgcacatgttt

cacttcctcagtttgatgagcttggttacctcgatcctgatgacaaaacccgtttggaagaacaagcgag

tgggtttcctatgctaaaagtgaaagacatcaagtctgcgtattcgaactggcaaatactcaaagagata

ttagggaagatgataaaacaaacaaaagcatcttcaggagtcatctggaactcatttaaggaactcgaag

agtctgagctcgaaactgttatccgtgagatcccggctccaagtttcttgataccactccccaagcattt

gacagcctcttccagcagcttactagaccacgatcgaaccgtttttcaatggttagaccacaaccgcca

agttcggtactgtatgttagttttggtagtactagtgaagtggatgagaaagatttcttggaaatagctc

gtgggttggttgatagcaagcagtcgtttttatgggtggttcgacctgggtttgtcaagggttcgacgtg

ggtcgaaccgttgccagatgggttcttgggtgaaagaggacgtattgtgaaatgggttccacagcaagaa

gtgctagctcatggagcaataggcgcattctggactcatagcggatggaactctacgttggaaagcgttt

gtgaaggtgttcctatgattttctcggattttgggctcgatcaaccgttgaatgctagatacatgagtga

tgttttgaaggtaggggtgtatttggaaaatgggtgggaaagaggagagatagcaaatgcaataagaaga

gttatggtggatgaagaaggagaatacattagacagaatgcaagagttttgaaacaaaaggcagatgttt

ctttgatgaagggtggttcgtcttacgaatcattagagtctctagtttcttacatttcatcgttgtaa</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>2427</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..2427</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q28">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>Sucrose synthase</INSDQualifier_value>

<NonEnglishQualifier_value>sucrose synthase</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..2427</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q29">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>unidentified</INSDQualifier_value>

<NonEnglishQualifier_value>not set</NonEnglishQualifier

_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctgccaagctagctcgcctccacagtctccgcgaacgcctcggtg

ccaccttctcgtctcatcccaatgagttgattgcactcttctctaggtatgttaaccagggaaagggaat

gctccagcgtcaccagctgcttgcggagttcgatgccttgatcgaagctgacaaagagaaatatgctccc

tttgaagacattctccggggctgctcaggaagccattgtgctgccgccctgggttgcactggccatcaggc

caaggcctggtgtctgggactacattcgggtgaatgtaagtgagttggcagtggaagagctgagtgtttc

tgagtacttggcattcaaggaacagcttgttgatggacacaccaacagcaactttgttcttgagcttgat

tttgagcccttcaatgcctccttcccgcgcccgtccatgtccaagtccatcggaaatggggtgcagttcc

ttaaccgtcacctgtcgtccaagttgttccaggacaagggagagcctctctaccccttgctgaacttcctgaa

agcccataaccacaagggcacgacaatgatgctgaatgacagaattcagagccttcgtgggctccaatca

tcccttagaaaggcagaagaatatctgatgggcattcctcaagacacgccctactcggagttcaaccaca

ggttccaagagctcggtttggagaagggttggggtgactgtgcaaagcgtgtgcttgacaccatccactt

gcttcttgaccttcttgaggcccctgatccggccaacttggagaagttccttggaactattccaatgatg

ttcaatgttgttatcctgtctccgcatggatactttgcccaatccaatgtgttgggataccctgatactg

gtggtcaggttgtgtacattttggaccaagtccgcgctttggagaatgagatgcttttgaggatcaagca

gcaaggccttgatatcacacctaagatcctcattgtaaccaggctgttgcctgatgctgttggtactaca

tgcggccagcgtgtggagaaggttattggaactgagcacactgacattctcttcgtgttccattcaggagtg

agaatggtatcctccgcaagtggatctcccgttttgatgtctggccattcctggaaacatacactgagga

tgttgcaaacgaaattatgagggaaatgcaagccaaacctgatctcatcattggcaattacagtgatgga

aaccttgttgccactctgctggctcacaaattaggagttacccagtgtaccattgctcatgccttggaga

aaaccaaataccccaactcagacatatacttggacaagtttgacagccagtaccacttctcatgccaatt

cactgctgatcttatcgccatgaatcacactgatttcatcatcaccagtacattccaagaaattgctgga

agcaaggacactgtggggcagtatgaatcacacattgcattcacccttcctgggctttaccgagttgtgc

atggcatagatgtttttgatcccaagttcaacattgtctctcctggagctgacatgagtgtctacttccc

gtacaccgaggctgacaagaggctcactgctttccaccctgaaattgaggagcttctctacagtgaagtc

gagaacgatgaacacaagtttgtattgaaggacaagaacaagccaatcatcttctccatggctcgtcttg

accgagtgaagaacatgacaggtctggttgagatgtatggtaagaatgcacatctcagggatttggcaaa

ccttgtgattgtttgtggtgaccacggcaatcagtccaaggacagggaggagcaggctgagttcaagaag

atgtacggtctcattgaccagtacaagttgaaggggcatatccgctggatctcagctcagatgaaccgtg

ttcgtaacggggagttgtaccgatacatttgtgacaccaagggagtctttgtccagcctgcattctatga

agcgtttggtctgactgtcatcgaagccatgacatgtggtttgccaacaatcgcaacatgccatggtggc

cctgctgagattattgttgatggggtgtctggtctgcacattgatccttaccacagtgacaaggctgctg

atatcttggtcaacttctttgagaagtgcaagcaggattcaacctactgggacaatatttcacagggagg

tctgcagaggatttacgagaagtacacctggaagctgtactctgagaggctgatgaccttgactggtgta

tacggattctggaagtacgtaagcaaccttgagaggcgcgagactcgccgttacattgagatgttctatg

ctctgaaataccgcagcctggccagcgccgtcccattggctgtcgatggagagagcacatccaagtaa</

INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

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Claims (29)

1. A method for obtaining rebaudioside D from rebaudioside A, comprising: the interaction of nucleotide diphosphate, to which glucose is bound, with rebaudioside A in the presence of uridine diphosphate (UDP)-glycosyltransferase B (UGT-B) to produce rebaudioside D, wherein UGT-B is a protein consisting of the amino acid sequence of SEQ ID NO: 3. 2. The method according to claim 1, wherein the nucleotide diphosphate to which glucose is bound is obtained by reacting sucrose and nucleotide diphosphate in the presence of sucrose synthase. 3. The method according to claim 1, wherein rebaudioside A is obtained by reacting a nucleotide diphosphate to which glucose is bound with stevioside in the presence of uridine diphosphate (UDP)-glycosyltransferase A (UGT-A). 4. The method according to claim 2, wherein sucrose synthase is a protein consisting of the amino acid sequence SEQ ID NO: 5. 5. The method according to claim 3, wherein UGT-A is a protein consisting of the amino acid sequence SEQ ID NO: 4. 6. The method according to any one of paragraphs 1-5, which is carried out sequentially in situ . 7. A method for obtaining rebaudioside M from rebaudioside A, comprising: reacting the nucleotide diphosphate to which glucose is bound with rebaudioside A in the presence of UGT-B to yield rebaudioside D; and the interaction of rebaudioside D with nucleotide diphosphate to which glucose is bound in the presence of UGT-A to yield rebaudioside M, wherein UGT-B is a protein consisting of the amino acid sequence of SEQ ID NO: 3. 8. The method according to claim 7, wherein UGT-A is a protein consisting of the amino acid sequence SEQ ID NO: 4. 9. The method according to claim 7, wherein the nucleotide diphosphate to which glucose is bound is obtained by reacting sucrose and nucleotide diphosphate in the presence of sucrose synthase. 10. The method according to claim 7, wherein rebaudioside A is obtained by reacting nucleotide diphosphate, to which glucose is bound, with stevioside in the presence of UGT-A. 11. The method according to claim 9, wherein sucrose synthase is a protein consisting of the amino acid sequence SEQ ID NO: 5. 12. The method according to claim 10, wherein UGT-A is a protein consisting of the amino acid sequence SEQ ID NO: 4. 13. The method according to any one of claims 7-12, which is carried out sequentially in situ . 14. A method for producing rebaudioside D from rebaudioside A, comprising: interaction of sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase and UGT-B in situ to produce rebaudioside D, wherein UGT-B is a protein consisting of the amino acid sequence of SEQ ID NO: 3. 15. A method for producing rebaudioside M from rebaudioside A, comprising: interaction of sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, UGT-A and UGT-B in situ to produce rebaudioside M, wherein UGT-B is a protein consisting of the amino acid sequence of SEQ ID NO: 3. 16. The method according to claim 15, wherein UGT-A is a protein consisting of the amino acid sequence SEQ ID NO: 4. 17. A composition for producing rebaudioside D from rebaudioside A, comprising UGT-B, which is a protein consisting of the amino acid sequence of SEQ ID NO: 3. 18. A composition for obtaining rebaudioside M from rebaudioside A, comprising: UGT-B, which is a protein consisting of the amino acid sequence of SEQ ID NO: 3, and UGT-A, which is a protein consisting of the amino acid sequence SEQ ID NO: 4.