Novel Glycosyltransferase and Its Use

Abstract

The present application relates to a novel glycosyltransferase B (UGT-B), a polynucleotide encoding the glycosyltransferase B (UGT-B), an expression vector containing the polynucleotide, a microorganism containing the glycosyltransferase B (UGT-B) or a polynucleotide encoding the glycosyltransferase B (UGT-B), and a method for producing rebaudioside D and rebaudioside M using the microorganism.

Description

Technical Field

[0001] This application relates to a novel glycosyltransferase B (UGT-B), a polynucleotide encoding the glycosyltransferase B (UGT-B), an expression vector containing the polynucleotide, a microorganism containing the glycosyltransferase B (UGT-B) or the polynucleotide encoding the glycosyltransferase B (UGT-B), and a method for producing rebaudioside D and rebaudioside M using the microorganism.

Background Art

[0002] The World Health Organization (WHO) recommends reducing the daily sugar intake due to the risk of diseases (obesity) caused by sugar intake, and policies for reducing various sugar intakes are actively discussed mainly in developed countries under government leadership. In the market, the needs for various alternative sweetener materials to replace sugar and high fructose are increasing, and the development and commercialization of alternative sweetener materials are continuing.

[0003] Alternative sweeteners have been continuously changing, including synthetic sweeteners (such as Saccharin, Aspartame, Sucralose), synthetic sugar alcohols (Maltitol, Xylitol), and high-intensity sweeteners (rebaudioside A, Liquorice). However, although consumers' needs for natural sweeteners have been increasing due to concerns about the safety of synthetic sweeteners, the current situation is that conventional low-calorie and zero-calorie products centered on synthetic sweeteners cannot be fully replaced due to the limitations of the taste quality with peculiar odors and off-flavors specific to natural sweeteners.

[0004] Regarding this, in recent years, a natural high-intensity sweetener that has attracted much attention is the stevia sweetener extracted from the leaves of stevia. The stevia sweetener has no calories, has beneficial effects on blood glucose and insulin levels, and has been reported to have no side effects on the human body, and has potential as an alternative sweetener. However, it has the disadvantage of being very bitter, so it has limitations in sugar reduction applications.

[0005] Stevia is a perennial plant of the Asteraceae family native to Paraguay, South America, and its scientific name is Stevia rebaudiana Bertoni. Since the leaves of Stevia contain components with a sweetness more than 200 to 300 times that of sugar, the sweet components are extracted and used as natural sweeteners. The sweet components of Stevia extract include various steviol glycosides such as stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside E.

[0006] Among the sweet components of Stevia extract, stevioside (STV), rebaudioside A (Reb A), and rebaudioside C (Reb C) are relatively abundant in the leaves of Stevia. They have been extracted, purified, industrialized, and commercialized with high purity. However, they have the drawback of being extremely bitter, so there are limitations in applications for sugar reduction.

[0007] On the other hand, rebaudioside D (Reb D) and rebaudioside M (Reb M) have less bitterness and are superior in sweet quality compared to STV, rebaudioside A, and rebaudioside C, and have high value as alternative sweeteners. However, rebaudioside D and rebaudioside M exist only in extremely trace amounts in the leaves of Stevia, and there is a drawback that the method of extracting and purifying them from the leaves requires high costs.

[0008] Therefore, the need for research on novel enzymes necessary for the mass production of rebaudioside D and rebaudioside M and manufacturing methods using them is increasing.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0010] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 2] Rice et al., 2000, Trends Genet. 16: 276 - 277 [Non-Patent Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443 - 453 [Non-Patent Document 4] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-Patent Document 5] Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-Patent Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-Patent Document 7] [Carillo ETA / .](1988) SIAM J Applied Math 48: 1073 [Non-Patent Document 8] Smith and Waterman, Adv. Appl. Math (1981) 2: 482 [Non-Patent Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353 - 358 (1979) [Non-Patent Document 10] Gribskov et al(1986) Nucl. Acids Res. 14: 6745 [Non-Patent Document 11] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 12] F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50 - 9.51, 11.7 - 11.8 [Non-Patent Document 13] “Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981) [Summary of the Invention] [Problems to be Solved by the Invention]

[0011] As a result of intensive research to develop an enzyme having the activity of converting rebaudioside A to rebaudioside D, the applicant has found that a polypeptide sequence whose function has not been disclosed conventionally has glycosyltransferase activity, and has confirmed that the polypeptide has glycosyltransferase activity of converting rebaudioside A to rebaudioside D, thus completing this application. [Means for Solving the Problems]

[0012] This application aims to provide glycosyltransferase B (UGT - B) consisting of the amino acid sequence represented by SEQ ID NO: 1. In addition, this application aims to provide a polynucleotide encoding glycosyltransferase B (UGT - B) of this application.

[0013] Furthermore, this application aims to provide an expression vector containing the polynucleotide. Furthermore, the present application aims to provide a microorganism comprising the glycosyltransferase B (UGT-B) of the present application or a polynucleotide encoding the glycosyltransferase B (UGT-B).

[0014] Furthermore, the present application aims to provide a method for producing rebaudioside D, comprising the step of reacting a nucleotide diphosphate bound to glucose with rebaudioside A in the presence of glycosyltransferase B (UGT-B), wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0015] Furthermore, the present application aims to provide a method for producing rebaudioside M, comprising the step of reacting a nucleotide diphosphate bound to glucose with rebaudioside A in the presence of glycosyltransferase B (UGT-B) to produce rebaudioside D, and the step of reacting the rebaudioside D with a nucleotide diphosphate bound to glucose in the presence of glycosyltransferase A (UGT-A) to produce rebaudioside M, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0016] Furthermore, the present application aims to provide a method for producing rebaudioside D from rebaudioside A, comprising the step of reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase and glycosyltransferase B (UGT-B) in the same reaction system, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0017] Furthermore, the present application aims to provide a method for producing rebaudioside M from rebaudioside A, comprising the step of reacting sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A) and glycosyltransferase B (UGT-B) in the same reaction system, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0018] Furthermore, the present application aims to provide a composition for producing rebaudioside D containing the glycosyltransferase B (UGT-B). Furthermore, the present application aims to provide a composition for producing rebaudioside M containing glycosyltransferase A (UGT-A) and the glycosyltransferase B (UGT-B).

[0019] Furthermore, the present application aims to provide the use of the glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO: 1 as a glycosyltransferase for converting rebaudioside A into rebaudioside D.

Advantages of the Invention

[0020] When the glycosyltransferase B (UGT-B) of the present application is used, rebaudioside D and / or rebaudioside M can be produced, and rebaudioside D and rebaudioside M can be provided with high purity and high yield with almost no by-products. Since inexpensive raw materials are used, it is economical, the procedure is simple and time-consuming is less, and it is useful for the mass production of rebaudioside D and rebaudioside M.

Brief Description of the Drawings

[0021]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0022] The following will specifically describe these. Note that each description and embodiment disclosed in this application is applicable to other descriptions and embodiments respectively. That is, all combinations of various elements disclosed in this application are included in this application. Also, this application is not limited to the following specific descriptions. Furthermore, many papers and patent documents are referenced throughout this specification, and their citations are indicated. The entire disclosure content of the cited papers and patent documents is incorporated herein by reference, thereby more clearly explaining the level of the technical field to which this application belongs and the content of this application.

[0023] One aspect of this application provides a glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO: 1. The "glycosyltransferase (UDP (Uridine diphosphate)-glycosyltransferase, UGT)" in this application is an enzyme having the activity of transferring a monosaccharide moiety from a glycosyl donor to a glycosyl acceptor molecule, specifically meaning an enzyme that uses UDP-sugar as a glycosyl donor. The glycosyltransferase in this application is used interchangeably with "UDP-glycosyltransferase" and "UGT".

[0024] The glycosyltransferase is produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector having a glycosyltransferase gene, or is produced from the above-mentioned Escherichia coli etc. and further purified, or is purchased and used as a commercially available product, but is not limited thereto. Also, the glycosyltransferase is known in the art, and the protein and gene sequences of the glycosyltransferase can be obtained from known databases, for example, from GenBank of NCBI etc., but are not limited thereto.

[0025] In the present application, a novel glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO: 1, which has the enzyme activity to convert rebaudioside A into rebaudioside D, has been found.

[0026] Specifically, the glycosyltransferase B (UGT-B) of the present application may have the amino acid sequence represented by SEQ ID NO: 1, may contain the amino acid sequence, may be essentially consisting of the amino acid sequence, or may consist of the amino acid sequence.

[0027] In addition, the glycosyltransferase B (UGT-B) of the present application may contain 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 represented by SEQ ID NO: 1. Furthermore, as long as it has such homology or identity and shows the efficacy corresponding to the glycosyltransferase B (UGT-B) of the present application, it goes without saying that the glycosyltransferase B (UGT-B) having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also included in the present application.

[0028] Even if the present application describes "a polypeptide or protein containing the amino acid sequence represented by a specific SEQ ID NO.", "a polypeptide or protein consisting of the amino acid sequence represented by a specific SEQ ID NO." or "a polypeptide or protein having the amino acid sequence represented by a specific SEQ ID NO.", as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the SEQ ID NO., it goes without saying that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also used in the present application. For example, it includes the addition of a sequence that does not change the function of the protein at the N-terminus and / or C-terminus of the amino acid sequence, naturally occurring mutations, its silent mutations or conservatively substituted ones.

[0029] For example, those having an addition or deletion of a sequence that does not change the function of the glycosyltransferase B (UGT-B) of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or inside of the amino acid sequence are included.

[0030] The "conservative substitution" in the present application means that an amino acid is substituted with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. 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. Also, amino acids are classified into amino acids having an electrically charged side chain and amino acids having an uncharged side chain. Amino acids having an electrically charged side chain include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids having an uncharged side chain are further classified into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Usually, conservative substitutions have little or no effect on the activity of the resulting protein or polypeptide.

[0031] In addition, glycosyltransferase B (UGT-B) may include deletions or additions of amino acids that have a minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be bound to the N-terminal signal (or leader) sequence of a protein involved in the transfer of the protein co-translationally or post-translationally. Further, the polypeptide may be bound to other sequences or linkers so as to enable the polypeptide to be identified, purified, or synthesized.

[0032] As used herein, "homology" or "identity" means the degree to which two given amino acid sequences or nucleotide sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0033] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and the default gap penalties established by the programs used may be used together. Substantially, homologous or identical sequences generally hybridize with the whole or a part of the sequence under moderately or highly stringent conditions. It goes without saying that hybridization includes hybridization with polynucleotides having codons that take into account general codons or codon degeneracy in polynucleotides.

[0034] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as default parameters like those in Non-Patent Document 1 and the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3) as performed by the needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later versions) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7)). For example, homology, similarity or identity can be determined using BLAST or Clustal W of the National Center for Biotechnology Information.

[0035] The homology, similarity or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8 for example. Briefly, the GAP program defines it as the value obtained by dividing the number of similar sequence symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program include (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Non-Patent Document 10 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0036] As an example of this application, the glycosyltransferase B (UGT-B) of this application may have the activity of converting rebaudioside A to rebaudioside M. As used herein, "corresponding to" means an amino acid residue at the position listed in the polypeptide, or an amino acid residue similar to, identical to, or corresponding to the residue listed in the polypeptide. Identifying the amino acid at the corresponding position determines the specific amino acid of the sequence with reference to a specific sequence. As used herein, "corresponding region" generally means a similar or corresponding position in a related protein or reference protein.

[0037] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, based thereon, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, the sequence alignment algorithm herein can identify the position of an amino acid or the position where modifications such as substitution, insertion, or deletion occur when compared with a query sequence (also referred to as a "reference sequence").

[0038] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 3), the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2), etc. can be used, but it is not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0039] Another aspect of the present application provides a polynucleotide encoding the glycosyltransferase B (UGT-B) of the present application. As used in this application, "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, meaning a DNA or RNA strand longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the said glycosyltransferase B (UGT-B).

[0040] The polynucleotide encoding the glycosyltransferase B (UGT-B) of this application may contain a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of this application, the polynucleotide may have the sequence of SEQ ID NO: 4, or may contain the sequence of SEQ ID NO: 4. Further, the polynucleotide may consist of the sequence of SEQ ID NO: 4, or may be substantially composed of the sequence of SEQ ID NO: 4.

[0041] Due to the degeneracy of codons or considering the preferred codons in the organism in which the glycosyltransferase B (UGT-B) of this application is to be expressed, various modifications can be made to the coding region within the range where the amino acid sequence of the glycosyltransferase B (UGT-B) of this application does not change. Specifically, the polynucleotide of this application has a base sequence with 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 less than 100% with the sequence of SEQ ID NO: 4, or contains the said base sequence, or consists of a base sequence with 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 less than 100% with the sequence of SEQ ID NO: 4, or is substantially composed of the said base sequence, but is not limited thereto.

[0042] In addition, the polynucleotide of the present application may be any probe prepared from a known gene sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions for hybridizing polynucleotides with high homology or identity, such as polynucleotides having 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 homology or identity, and not hybridizing polynucleotides with lower homology or identity, or washing conditions for ordinary Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, and washing once, specifically 2 to 3 times, at the corresponding salt concentration and temperature can be mentioned.

[0043] Hybridization requires that two nucleic acids have complementary sequences, even if base mismatches are possible depending on the stringency of hybridization. "Complementary" is used to represent the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotide of the present application may include not only nucleic acid sequences that are substantially similar, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0044] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. Further, the Tm value may be 60°C, 63°C or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0045] The appropriate stringency for hybridizing the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the variables are known in the art (for example, Non-Patent Document 11).

[0046] Still another aspect of the present application provides a vector containing the polynucleotide of the present application. The vector is an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0047] The "vector" in the present application may include a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host. The expression regulatory region includes a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When the vector is transformed into a suitable microorganism, it can replicate and function regardless of the host genome and can be integrated into the genome itself.

[0048] The vector used in this application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0049] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming whether the polynucleotide has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, that is, for confirming whether the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show different phenotypes, so transformed cells can be selected.

[0050] As used in this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into the interior of a microorganism, so as to cause the microorganism to express the polypeptide encoded by the polynucleotide. The transformed polynucleotide may be any polynucleotide as long as it is expressed in the microorganism, regardless of whether it is inserted into the chromosome of the microorganism or located outside the chromosome. Further, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the microorganism in any form as long as it is introduced into the microorganism and expressed therein. For example, the polynucleotide is introduced into the microorganism in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. Usually, the expression cassette includes a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicable expression vector. Further, the polynucleotide may be introduced into the microorganism in its own form and operably linked to the sequences necessary for expression in the microorganism, but is not limited thereto.

[0051] Further, the "operably linked" means that the promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target glycosyltransferase B (UGT-B) of this application is functionally linked to the polynucleotide sequence.

[0052] Still another aspect of this application provides a microorganism containing the glycosyltransferase B (UGT-B) of this application or a polynucleotide encoding the glycosyltransferase B (UGT-B). The microorganism of this application may contain the glycosyltransferase B (UGT-B) of this application, a polynucleotide encoding the glycosyltransferase B (UGT-B), or a vector containing the polynucleotide of this application.

[0053] As used in this application, the term "microorganism" encompasses all wild-type microorganisms and those that have been genetically modified either naturally or artificially. It refers to microorganisms in which specific mechanisms have been weakened or enhanced due to reasons such as the insertion of foreign genes or the strengthening or inactivation of endogenous gene activity, and which have been genetically modified for the production of a target polypeptide, protein, or product. In this application, the terms "microorganism", "strain", and "microbe" are used interchangeably with the same meaning.

[0054] Specifically, the microorganism is a microorganism belonging to the genus Escherichia or Corynebacterium, and more specifically Escherichia coli or Corynebacterium glutamicum, but is not limited thereto.

[0055] The microorganism of this application is a strain that contains at least one of the glycosyltransferase B (UGT-B) of this application, the polynucleotide of this application, and a vector containing the polynucleotide of this application, or is a strain modified to express the glycosyltransferase B (UGT-B) of this application or the polynucleotide of this application, or is a strain (e.g., a recombinant strain) that expresses the glycosyltransferase B (UGT-B) of this application or the polynucleotide of this application, or is a strain (e.g., a recombinant strain) having the activity of the glycosyltransferase B (UGT-B) of this application, but is not limited thereto.

[0056] The strain of this application may be a microorganism that produces rebaudioside D and / or rebaudioside M. As used in this invention, the term "microorganism that produces rebaudioside D and / or rebaudioside M" means a prokaryotic or eukaryotic microbial strain that produces rebaudioside D and / or rebaudioside M in vivo. For the purpose of this invention, the microorganism may be either a prokaryotic cell or a eukaryotic cell as long as it contains the glycosyltransferase B (UGT-B) and produces rebaudioside D and / or rebaudioside M.

[0057] The microorganism that produces rebaudioside D and / or rebaudioside M and contains the glycosyltransferase B (UGT-B) may be any microorganism that contains the chromosomal sequence encoding the glycosyltransferase B (UGT-B), and / or the microorganism that contains the vector containing the polynucleotide encoding the glycosyltransferase B (UGT-B), but is not limited thereto.

[0058] For example, the strain of the present application is a cell or microorganism transformed with the polynucleotide of the present application or a vector containing the polynucleotide encoding the glycosyltransferase B (UGT-B) of the present application, and expresses the glycosyltransferase B (UGT-B) of the present application. For the purpose of the present application, the strain of the present application may be any microorganism that contains the glycosyltransferase B (UGT-B) of the present application and produces rebaudioside D and / or rebaudioside M. For example, the strain of the present application may be a natural wild-type microorganism, or a recombinant strain in which the polynucleotide encoding the glycosyltransferase B (UGT-B) of the present application is introduced into a microorganism that produces rebaudioside D and / or rebaudioside M, resulting in the expression of the glycosyltransferase B (UGT-B) and an improved ability to produce rebaudioside D and / or rebaudioside M.

[0059] In the microorganisms of the present application, partial or total modification of polynucleotides can be induced by (a) homologous recombination using a chromosomal integration vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet rays or radiation and / or chemical substances, but is not limited thereto. The methods for partial or total modification of the said gene include methods by DNA recombination techniques. For example, partial or total deletion of a gene is carried out by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to cause homologous recombination. The introduced nucleotide sequence or vector may include a dominant selectable marker, but is not limited thereto.

[0060] Regarding glucosyltransferase B (UGT-B), polynucleotides, rebaudioside D, rebaudioside M, etc. in the microorganisms of the present application, it is as described above. Still another aspect of the present application provides a method for producing rebaudioside D, which includes a step of reacting a nucleotide diphosphate bound to glucose with rebaudioside A in the presence of glucosyltransferase B (UGT-B), and the glucosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1.

[0061] Specifically, the method for producing rebaudioside D of the present application includes, but is not limited to, a step of culturing a microorganism containing glucosyltransferase B (UGT-B), the polynucleotide of the present application, or the vector of the present application in a medium.

[0062] In this application, "cultivation" means growing the microorganisms of this application under appropriately adjusted environmental conditions. The cultivation process of this application is carried out using a suitable medium and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used according to the selected strain by those skilled in the art. Specifically, the cultivation may be batch, continuous, and / or fed-batch cultivation, but is not limited thereto.

[0063] In this application, "medium" means a substance mixed mainly with the nutrients necessary for culturing the microorganisms of this application, and supplies nutrients such as water, which is indispensable for survival and growth, and growth factors. Specifically, the medium and other cultivation conditions used for culturing the microorganisms of this application can be any that are used for culturing ordinary microorganisms. The microorganisms of this application can be cultured by adjusting temperature, pH, etc. under aerobic conditions in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0064] For example, the culture medium for Corynebacterium strains is disclosed in Non-Patent Document 13. Examples of the carbon source in this application include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and any other appropriate amount of carbon source can be used. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0065] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used as organic nitrogen sources. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0066] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or their corresponding sodium-containing salts. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. can be used, and in addition, amino acids, vitamins and / or suitable precursors, etc. can be used. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0067] Also, during the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in a suitable manner. Furthermore, during the cultivation, a defoaming agent such as a fatty acid polyglycol ester can be used to suppress the generation of bubbles. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic states, it may not be necessary to inject gas, and nitrogen, hydrogen or carbon dioxide gas may be injected, but it is not limited thereto.

[0068] The cultivation of the present application maintains the cultivation temperature at 20 to 45 °C, specifically 25 to 40 °C, and cultivates for about 10 to 160 hours, but is not limited thereto. Rebaudioside D produced by the cultivation of the present application is secreted into the medium or remains intracellularly.

[0069] As an example of an embodiment of the present application, the nucleotide diphosphate to which glucose is bound is prepared by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase, but is not limited thereto.

[0070] The "sucrose synthase" in the present application plays a role of reversibly transferring glucose to which nucleotide diphosphate is bound to fructose in plant metabolism to produce sucrose. In the present invention, it shows an activity of reacting sucrose with nucleotide diphosphate in the pH range of 5 to 10 to separate into nucleotide diphosphate to which glucose is bound and fructose.

[0071] The sucrose synthase is a sucrose synthase derived from rice, corn, wheat, bamboo, Arabidopsis thaliana, Miscanthus sinensis, barley, sorghum or potato, preferably a sucrose synthase derived from rice, corn, wheat or barley, and more preferably a sucrose synthase derived from rice, particularly Oryza sativa. The sucrose synthase is produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium or Agrobacterium transformed with a vector having a sucrose synthase gene, or is produced from the above Escherichia coli etc. and further purified, or is a sucrose synthase known in the art, or is commercially available, but is not limited thereto.

[0072] Specifically, the sucrose synthase of the present application may have an amino acid sequence represented by SEQ ID NO: 3, may contain the amino acid sequence, may be essentially consisting of the amino acid sequence, or may consist of the amino acid sequence.

[0073] The sucrose may be any sucrose that can act as a substrate for sucrose synthase to supply glucose to a nucleotide diphosphate. For example, raw sugar or granulated sugar may be used, but it is not limited thereto.

[0074] As the nucleotide diphosphate in the present application, a purine nucleotide or a pyrimidine nucleotide is used, and uridine diphosphate is preferred, but it is not limited thereto.

[0075] The nucleotide diphosphate to which the glucose is bound may react with rebaudioside A by the glycosyltransferase B (UGT-B) of the present application to produce rebaudioside D.

[0076] As an example of the present application, the rebaudioside A is prepared by reacting a nucleotide diphosphate to which glucose is bound with stevioside in the presence of glycosyltransferase A (UGT-A), but it is not limited thereto.

[0077] The glycosyltransferase A (UGT-A) may react a nucleotide diphosphate to which glucose is bound with stevioside to produce rebaudioside A. The glycosyltransferase A (UGT-A) may be a glycosyltransferase derived from Oryza sativa, Stevia rebaudiana Bertoni, Bambusa oldhamii, Brachypodium distachyon, Hordeum vulgare, Sorghum bicolor, Zea mays, or Arabidopsis thaliana. The glycosyltransferase is preferably a glycosyltransferase derived from Oryza sativa, Stevia rebaudiana Bertoni, or Bambusa oldhamii. More preferably, the glycosyltransferase is a glycosyltransferase derived from Stevia rebaudiana Bertoni. The glycosyltransferase A (UGT-A) is produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector having a glycosyltransferase gene, or is produced from the like and further purified, or is a glycosyltransferase known in the art or commercially available, but is not limited thereto.

[0078] Specifically, the glycosyltransferase A (UGT-A) of the present application may have an amino acid sequence represented by SEQ ID NO: 2, may contain the amino acid sequence, may be essentially consisting of the amino acid sequence, or may consist of the amino acid sequence.

[0079] The stevioside is a by-product of the production of rebaudioside A from a hot water or ethanol aqueous solution extract of Stevia rebaudiana or a purified product thereof or an extract, and a stevioside content of 10% by weight or more, preferably 50% by weight or more, more preferably 70% by weight or more, and even more preferably 80% by weight or more of the total weight of steviol glycosides is used, but is not limited thereto.

[0080] As an example of the present application, the rebaudioside D is prepared as shown in Reaction Scheme 1, but is not limited thereto.

[0081] [ka]

[0082] Specifically, the production method may be carried out continuously in the same reaction system. In this application, "same reaction system" means that reactions occur sequentially in one reaction system or reaction system.

[0083] The production method of the present application provides a continuous reaction system for synthesizing rebaudioside A in high yield by specifically binding one glucose to the C-3' position of 13-O-glucose of stevioside according to Reaction Scheme 1, and synthesizing rebaudioside D from rebaudioside A.

[0084] The method for producing rebaudioside D of the present application may further include a step of providing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, prior to the culturing step.

[0085] The method for producing rebaudioside D of the present application may further include a step of recovering rebaudioside D from the culture medium (culture medium) or the microorganism of the present application as described above. The recovering step may be further included after the culturing step.

[0086] The recovery may be performed by using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, the target rebaudioside D can be collected from the medium or the microorganism using a suitable method known in the art, such as centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof.

[0087] The method for producing rebaudioside D of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the method for producing rebaudioside D of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0088] The glycosyltransferase B (UGT-B), polynucleotide, vector, microorganism, etc. used in the method of the present application are as described above. Yet another aspect of the present application provides a method for producing rebaudioside M, comprising the steps of reacting a glucose-linked nucleotide diphosphate with rebaudioside A in the presence of glycosyltransferase B (UGT-B) to produce rebaudioside D, and reacting the rebaudioside D with a glucose-linked nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A) to produce rebaudioside M, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0089] In one embodiment of the present application, the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, but is not limited thereto. In one example of the present application, the glucose-linked nucleotide diphosphate is prepared by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase, but is not limited thereto.

[0090] Specifically, the sucrose synthase of the present application may have the amino acid sequence shown in SEQ ID NO: 3, may contain the amino acid sequence, may essentially consist of the amino acid sequence, or may consist of the amino acid sequence.

[0091] In one example of the present application, rebaudioside A is prepared by reacting a glucose-linked nucleotide diphosphate with stevioside in the presence of glycosyltransferase A (UGT-A), but is not limited thereto.

[0092] Specifically, the glycosyltransferase A (UGT-A) may have the amino acid sequence shown in SEQ ID NO: 2, may contain the amino acid sequence, may essentially consist of the amino acid sequence, or may consist of the amino acid sequence.

[0093] The glycosyltransferase A (UGT-A) of the present application may react a glucose-linked nucleotide diphosphate with rebaudioside D to produce rebaudioside M. Specifically, the glycosyltransferase A (UGT-A) of the present application may have the amino acid sequence shown in SEQ ID NO: 2, may contain the amino acid sequence, may essentially consist of the amino acid sequence, or may consist of the amino acid sequence.

[0094] In one embodiment of the present application, the rebaudioside M is prepared as shown in Reaction Scheme 2, but is not limited thereto.

[0095] [ka]

[0096] Specifically, the production method may be carried out continuously in the same reaction system. The production method of the present application provides a continuous reaction system for synthesizing rebaudioside A from stevioside in high yield, synthesizing rebaudioside D from rebaudioside A, and synthesizing rebaudioside M from rebaudioside D, according to Reaction Scheme 2.

[0097] The rebaudioside M production method of the present application may further include, for example, prior to the culturing step, a step of providing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order).

[0098] The method for producing rebaudioside M of the present application may further include a step of recovering rebaudioside M from the culture medium (culture medium) or the microorganism of the present application as described above. The recovering step may be further included after the culturing step.

[0099] The recovery may involve collecting the target rebaudioside M using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used to recover the target rebaudioside M from the medium or the microorganism using a suitable method known in the art.

[0100] In addition, the method for producing rebaudioside M of the present application may further include a purification step. The purification can be carried out by a suitable method known in the art. For example, when the method for producing rebaudioside M of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously regardless of the order, or may be carried out simultaneously or integrated as one step, but are not limited thereto.

[0101] Regarding the glycosyltransferase B (UGT-B), polynucleotide, vector, microorganism, etc. in the method of the present application, it is as described above. Therefore, the production method of the present application can convert bitter components such as stevioside and rebaudioside A contained in stevia extract into rebaudioside D and rebaudioside M, which are delicious components, using easily available and inexpensive raw materials such as stevioside and rebaudioside A. Therefore, it is useful for the production of stevia sweeteners with good sweetness quality.

[0102] Still another aspect of the present application includes a step of reacting sucrose, nucleotide diphosphate, rebaudioside A, sucrose synthase, and glycosyltransferase B (UGT-B) in the same reaction system to produce rebaudioside D, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and provides a method for producing rebaudioside D from rebaudioside A.

[0103] Still another aspect of the present application includes a step of reacting sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A), and glycosyltransferase B (UGT-B) in the same reaction system to produce rebaudioside M, wherein the glycosyltransferase B is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and provides a method for producing rebaudioside M from rebaudioside A.

[0104] The sucrose, nucleotide diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, glycosyltransferase A (UGT-A), glycosyltransferase B (UGT-B), identical reaction system, rebaudioside M, and the like are as described above.

[0105] Specifically, the glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, but is not limited to this. Yet another aspect of the present application provides a composition for producing rebaudioside D, which comprises a glycosyltransferase B (UGT-B) of the present application, a polynucleotide encoding the glycosyltransferase B (UGT-B), a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present application, or a medium in which any of these has been cultured, or a combination of two or more thereof.

[0106] Yet another aspect of the present application provides a composition for producing rebaudioside M, which comprises the glycosyltransferase B (UGT-B) and glycosyltransferase A (UGT-A) of the present application, a polynucleotide encoding the glycosyltransferase B (UGT-B) and a polynucleotide encoding the glycosyltransferase A (UGT-A), a vector comprising the polynucleotides, or a microorganism comprising the polynucleotide of the present application, a medium in which the microorganisms are cultured, or a combination of two or more thereof.

[0107] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0108] The glycosyltransferase B (UGT-B), glycosyltransferase A (UGT-A), polynucleotide, vector, strain, medium, L-valine, and the like in the composition of the present application are as described above.

[0109] Still another aspect of the present application provides the use of the glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO:1 as a glycosyltransferase that converts rebaudioside A into rebaudioside D.

[0110] SEQ ID NO:1, glycosyltransferase B (UGT-B), rebaudioside A, rebaudioside D, etc. are as described above.

Examples

[0111] Hereinafter, the present application will be described in more detail with reference to examples. However, these examples are merely preferred embodiments for illustrating the present application, and the present application is not limited thereto. Technical matters not described in this specification can be fully understood and easily implemented by those skilled in the art in the technical field of the present application or similar technical fields.

Examples

[0112] Design of novel glycosyltransferase B (UGT-B) Example 1-1. Design of novel glycosyltransferase B (UGT-B) In order to develop excellent enzymes that produce rebaudioside D and rebaudioside M, two types of plant-derived enzymes known to recognize stevia and rebaudioside A as acceptors of glycosyltransferase reactions and UDP-glucose as donors were selected, and their expression in microorganisms and conversion activity from rebaudioside A to rebaudioside D were examined. The results are shown in Table 1.

[0113]

Table 1

[0114] As shown in Table 1, the two types of plant-derived enzymes have low enzyme expression levels and conversion rates, and are not efficient for industrial production of rebaudioside D. Therefore, a new enzyme was designed to improve them.

[0115] Specifically, based on the chemical structure and reaction mechanism of the substrate used in the glycosyltransferase reaction of the enzyme, the tertiary structure of each protein was predicted to belong to the GT-B fold in terms of classification. Since proteins with the GT-B fold structure have the characteristic of being clearly divided into an N-domain that recognizes the sugar receptor and a C-domain that recognizes the sugar donor, a total of 12 improved enzymes were prepared by mixing the N-domain and C-domain of each protein to create a chimera, and their expression and activity were evaluated.

[0116] As a result, compared with the wild-type enzyme, a novel glycosyltransferase B (UGT-B), which has good protein expression in microorganisms and improved activity, was prepared as shown in the following schematic diagram. Its sequence is shown in SEQ ID NO: 1.

[0117]

Table 2

[0118] Example 1-2. Production and Activity Measurement of Novel Glycosyltransferase B (UGT-B) For the novel enzyme that converts rebaudioside A designed in Example 1-1 to rebaudioside D and the W1 and W2 enzymes, a recombinant plasmid (vector - pET24a) having a gene encoding UDP - glycosyltransferase was prepared, cloned into Escherichia coli BL21(DE3) to highly express the enzyme, and then purified for use.

[0119] The recombinant strain BL21(DE3) was inoculated into a test tube containing 5 ml of LB medium, and the inoculum was cultured in an incubator at 37°C until the absorbance at 600 nm reached 2.0. The culture solution of the inoculum was added to a flask containing 500 ml of LB medium for main culture. When the absorbance at 600 nm reached 0.4, 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to induce the massive expression of the enzyme. In the above process, the stirring speed was adjusted to 180 rpm and the culture temperature was maintained at 37°C. After the addition of IPTG, the culture was carried out at a stirring speed of 120 rpm and a culture temperature of 16°C. As described above, the culture solution of the transformed strain was centrifuged at 6000×g for 20 minutes at 4°C, and only the cell supernatant was separated as the enzyme solution. In order to accurately grasp the characteristics of the enzyme, it was purified using a Ni-NTA superflow column.

[0120] On the other hand, the recombinant Corynebacterium was inoculated into a medium (Bacto-Trypton 10 g / L, Bacto-yeast extract 5 g / L, NaCl 5 g / L, Soytone 5 g / L) containing 10 μg / ml of kanamycin at an initial concentration of O.D.600 = 0.1 and cultured at 30°C for 24 hours to induce the expression of the enzyme. The culture solution obtained as described above was inoculated into a fermenter containing a medium (glucose 80 g / L, soytone 20 g / L, (NH4)2SO4 10 g / L, KH2PO4 1.2 g / L, MgSO4 1.4 g / L) containing 10 μg / ml of kanamycin at O.D.600 = 0.6 and cultured at 30°C for 24 hours.

[0121] The raw material used for the enzyme reaction was Reb A (Depion Co., Ltd.), which was dissolved in water to a concentration of 1, 5, 10, and 20 mM for use. The enzyme (glycosyltransferase B (UGT-B), W1, W2) expressed in the microorganism was used to carry out the reaction at 37°C for 24 hours and then analyzed by HPLC. UDP-glucose (Carbosynth Co., Ltd.) was added to the aqueous solution of the above raw material at 2, 10, 20, and 40 mM. A purified enzyme was used as the enzyme, and the reaction was carried out at an enzyme concentration of 0.1 mg / ml.

[0122] The measurement results are shown in Table 2 and Table 3.

[0123]

Table 3

[0124]

Table 4

[0125] As shown in Table 2 and Table 3, it was confirmed that in the novel glycosyltransferase B (UGT-B) of the present application, the conversion yield from rebaudioside A to rebaudioside D and the protein expression level were improved.

Examples

[0126] Measurement of the enzyme activity of glycosyltransferase B (UGT-B) against rebaudioside A raw material Using the glycosyltransferase B (UGT-B) prepared in Examples 1-2, the enzyme activity of glycosyltransferase B (UGT-B) against rebaudioside A raw material was measured.

[0127] Specifically, the raw material used in the enzyme reaction was Reb A (Depion Co., Ltd.), which was dissolved in water to a concentration of 2 mM and used. Using the enzyme (sequence No04) expressed in microorganisms, the reaction was carried out at 37 °C for 16 hours and then analyzed by HPLC. The aqueous solution of the raw material contained 2 mM of UDP-glucose or UDP (Uridine-diphosphate).

[0128] The HPLC analysis conditions are as follows. ·Detector wavelength: 210 nm ·Flow rate: 1 ml / min ·Sample injection vol.: 10 μl ·Column: Capcell pak C18MG II (Shiseido, 250×4.6 mm, particle size: 5 μm) [[ID=**46**]] ·Solvent:Acetonitrile 30% The measurement results are shown in Table 4 and Figure 1.

[0129]

Table 5

[0130] As shown in Table 4 and Figure 1, it was confirmed that the glucosyltransferase B of the present application converted rebaudioside A to rebaudioside D almost (98.6%).

Example

[0131] Measurement of the enzyme activities of glucosyltransferase A (UGT-A) and glucosyltransferase B (UGT-B) against stevioside raw materials Glucosyltransferase A (UGT-A) and sucrose synthase were purified and used by the method of the prior art document (Patent Document 2). Their sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0132] Using the glucosyltransferase A (UGT-A), the sucrose synthase, and the glucosyltransferase B (UGT-B) prepared in Examples 1-2, the enzyme activities of glucosyltransferase A (UGT-A) and glucosyltransferase B (UGT-B) against stevioside raw materials were measured.

[0133] Specifically, as the raw materials used in the enzyme reaction, an aqueous solution of 2 mM stevioside (haigen) and 50 mM sugar (CJ CheilJedang) was used. Using the glucosyltransferase A (UGT-A), the sucrose synthase, and the glucosyltransferase B (UGT-B) expressed in microorganisms, the reaction was carried out at 37 degrees for 16 hours and then analyzed by HPLC. The aqueous solution of the raw materials contains 2 mM UDP-glucose or UDP (Uridine-diphosphate).

[0134] The measurement results are shown in Table 5 and Figure 2.

[0135]

Table 6

[0136] As shown in Table 5 and FIG. 2, it was confirmed that stevioside and rebaudioside A were converted to rebaudioside D, rebaudioside M, and rebaudioside I at 100% molar concentration.

Example

[0137] Measurement of the enzyme activity of glycosyltransferase A (UGT-A) with respect to the rebaudioside D raw material Using the glycosyltransferase A (UGT-A) of Example 3, the conversion rate from rebaudioside D to rebaudioside M was measured.

[0138] Specifically, as the raw material used in the enzyme reaction, rebaudioside D (manufactured by Haigen) was dissolved in water to a concentration of 1 mM and used. Using the glycosyltransferase A (UGT-A) expressed in microorganisms, the reaction was carried out at 37° C. for 16 hours and then analyzed by HPLC. The aqueous solution of the raw material contains 2 mM of UDP-glucose or UDP (Uridine-diphosphate).

[0139] The measurement results are shown in FIG. 3. As shown in FIG. 3, it was confirmed that the glycosyltransferase A of the present application almost converts rebaudioside D to rebaudioside M.

[0140] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not restrictive. The present application should be construed as including all changes and modified forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the description.

Claims

1. Uridine diphosphate glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO:

1.

2. A polynucleotide encoding uridine diphosphate glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO:

1.

3. An expression vector containing a polynucleotide encoding uridine diphosphate glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO:

1.

4. A microorganism containing uridine diphosphate glycosyltransferase B (UGT-B) consisting of the amino acid sequence represented by SEQ ID NO: 1, or a polynucleotide encoding said uridine diphosphate glycosyltransferase B (UGT-B).

5. A step of reacting a nucleotide diphosphate bound to glucose with rebaudioside A in the presence of uridine diphosphate glycosyltransferase B (UGT-B) to produce rebaudioside D, wherein said uridine diphosphate glycosyltransferase B (UGT-B) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, a method for producing rebaudioside D.

6. The method according to claim 5, wherein the nucleotide diphosphate bound to glucose is produced by reacting sucrose with a nucleotide diphosphate in the presence of sucrose synthase.

7. The method according to claim 5, wherein said rebaudioside A is produced by reacting a nucleotide diphosphate bound to glucose with stevioside in the presence of uridine diphosphate glycosyltransferase A (UGT-A).

8. The method according to claim 6, wherein said sucrose synthase is a protein consisting of the amino acid sequence represented by SEQ ID NO:

3.

9. The method according to claim 7, wherein said uridine diphosphate glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

2.

10. The method according to any one of claims 5 to 9, wherein said production method is continuously carried out in the same reaction system.

11. A step of reacting a nucleotide diphosphate bound to glucose with rebaudioside A in the presence of uridine diphosphate glycosyltransferase B (UGT-B) to produce rebaudioside D, and A step of reacting the rebaudioside D with a nucleoside diphosphate to which glucose is bound in the presence of uridine diphosphate glycosyltransferase A (UGT-A) to produce rebaudioside M, The method for producing rebaudioside M, wherein the uridine diphosphate glycosyltransferase B (UGT-B) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

1.

12. The method according to claim 11, wherein the uridine diphosphate glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

2.

13. The method according to claim 11, wherein the nucleoside diphosphate to which glucose is bound is produced by reacting sucrose with a nucleoside diphosphate in the presence of sucrose synthase.

14. The method according to claim 11, wherein the rebaudioside A is produced by reacting a nucleoside diphosphate to which glucose is bound with stevioside in the presence of uridine diphosphate glycosyltransferase A (UGT-A).

15. The method according to claim 13, wherein the sucrose synthase is a protein consisting of the amino acid sequence represented by SEQ ID NO:

3.

16. The method according to claim 14, wherein the uridine diphosphate glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

2.

17. The method according to any one of claims 11 to sixteen, wherein the production method is continuously performed in the same reaction system.

18. A step of reacting sucrose, a nucleoside diphosphate, rebaudioside A, sucrose synthase and uridine diphosphate glycosyltransferase B (UGT-B) in the same reaction system to produce rebaudioside D, The method for producing rebaudioside D from rebaudioside A, wherein the uridine diphosphate glycosyltransferase B (UGT-B) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

1.

19. A step of reacting sucrose, a nucleoside diphosphate, rebaudioside A, rebaudioside D, sucrose synthase, uridine diphosphate glycosyltransferase A (UGT-A) and uridine diphosphate glycosyltransferase B (UGT-B) in the same reaction system to produce rebaudioside M, A method for producing rebaudioside M from rebaudioside A, wherein the uridine diphosphate glycosyltransferase B (UGT-B) is a protein consisting of the amino acid sequence represented by SEQ ID NO:

1.

20. The method for producing rebaudioside M from rebaudioside A according to claim 19, wherein the uridine diphosphate glycosyltransferase A (UGT-A) is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2.

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