Novel glycosyltransferase and use thereof

Abstract

The present invention relates to a novel glycosyltransferase and a use thereof.

Description

Novel glycosyltransferase and its uses

[0001] [Cross-reference with related applications]

[0002] The present application claims the benefit of priority based on Korean Patent Application No. 10-2024-0156239 dated November 06, 2024 and Korean Patent Application No. 10-2025-0165165 dated November 05, 2025, and all contents disclosed in the documents of said Korean patent applications are incorporated herein as part of the specification.

[0003] [Technology Field]

[0004] The present application relates to a novel glycosyltransferase and its uses.

[0005] Following the World Health Organization (WHO)'s recommendation to reduce daily sugar intake due to concerns regarding diseases (such as obesity) associated with sugar consumption, government-led policies to reduce the intake of various sugars are being actively discussed, primarily in developed countries. Consequently, there is a growing market demand for various alternative sweeteners to replace sugar and high-fructose corn syrup, leading to the continuous development and commercialization of these materials. The types of alternative sweeteners are continuously evolving, ranging from synthetic high-intensity sweeteners (such as saccharin, aspartame, and sucralose) to synthetic sugar alcohols (such as maltitol and xylitol) and high-intensity sweeteners (such as rebaudioside A and licorice). However, despite the growing customer demand for natural sweeteners due to persistent safety concerns regarding synthetic sweeteners, they have not yet been able to fully replace existing low-calorie and zero-calorie products centered on synthetic sweeteners due to limitations in taste attributes, such as the characteristic off-flavors and odors of natural sweeteners.

[0006] A natural high-intensity sweetener that has recently been receiving significant attention is Stevia, which is extracted from the leaves of the plant Stevia rebaudiana Bertoni. Stevia is a natural material with a sweetness 200 to 300 times that of sugar and contains various steviol glycosides such as stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside E.

[0007] Stevia has potential as an alternative sweetener as it is reported to have no side effects on the human body, has no energy, and has positive effects on blood glucose and insulin levels, but it has the disadvantage of exhibiting a particularly strong bitter taste, which limits its use in sugar reduction. Specifically, among the sweetening components of stevia extract, stevioside, rebaudioside A, and rebaudioside C are relatively high in stevia leaves, making extraction and purification easy and inexpensive, but there is a problem with poor sweetness quality due to a strong bitter taste.

[0008] In contrast, ribaudioside D and ribaudioside M have low bitterness and excellent sweetness, making them highly valuable as alternative sweeteners. However, they exist in extremely small quantities in stevia leaves, and the method of manufacturing them by extracting and purifying from the leaves is expensive. Therefore, there is a need for research on new enzymes required for the mass production of ribaudioside M and manufacturing methods using them.

[0009] Against this technical background, the applicant intends to provide a novel mutant glycosyltransferase capable of effectively producing ribaudioside M by exhibiting significantly superior activity in producing ribaudioside M from ribaudioside D compared to the wild-type glycosyltransferase.

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] Korean Published Patent No. 10-2012-0116460

[0013] The present application aims to provide a variant polypeptide with significantly enhanced rebaudioside M (RebM) conversion activity compared to the wild-type enzyme.

[0014] In addition, the present application aims to provide a polynucleotide encoding a variant polypeptide having glycosyltransfer activity.

[0015] In addition, the present application aims to provide an expression vector comprising a polynucleotide encoding a variant polypeptide having glycosyltransfer activity.

[0016] In addition, the present application aims to provide a transformant comprising a polynucleotide encoding a variant polypeptide having glycosyltransfer activity.

[0017] In addition, the present application aims to provide a method for producing ribaudioside M using one or more of a variant polypeptide having glycosyltransfer activity and a transformant expressing said variant polypeptide.

[0018] In addition, the present application aims to provide a method for producing ribaudioside A using one or more of a variant polypeptide having glycosyltransfer activity and a transformant expressing said variant polypeptide.

[0019] In addition, the present application aims to provide a method for producing ribaudioside D using one or more of a variant polypeptide having glycosyltransfer activity and a transformant expressing said variant polypeptide.

[0020] In addition, the present application aims to provide a composition for producing one or more steviol glycosides selected from the group consisting of ribaudioside M, ribaudioside A, and ribaudioside D, comprising one or more of a variant polypeptide having glycosyltransfer activity and a transformant expressing said variant polypeptide.

[0021] To achieve the above objective, one aspect of the present application provides a polypeptide having glycosyltransfer activity, wherein the variant polypeptide is a polypeptide in which an amino acid corresponding to one or more positions selected from SEQ ID NO. 20, 88, and 126 is substituted with another amino acid.

[0022] Additionally, another aspect of the present application provides a polypeptide having glycosyltransfer activity, wherein the variant polypeptide comprises an amino acid corresponding to any one or more positions selected from 20, 88, and 126 of SEQ ID NO. 1; and an amino acid corresponding to any one or more positions selected from 86, 200, and 379 of SEQ ID NO. 1; which is substituted with another amino acid.

[0023] Additionally, another aspect of the present application aims to provide a polynucleotide encoding the variant polypeptide.

[0024] In addition, the present application provides an expression vector comprising a polynucleotide encoding the variant polypeptide.

[0025] In addition, the present application provides a transformant comprising a polynucleotide encoding the variant polypeptide.

[0026] In addition, the present application provides a method for producing ribaudioside ribaudioside M using one or more of the above-mentioned variant polypeptide and a transformant expressing the above-mentioned variant polypeptide.

[0027] In addition, the present application provides a method for producing ribaudioside A using one or more of the above-mentioned variant polypeptide and a transformant expressing the above-mentioned variant polypeptide.

[0028] In addition, the present application provides a method for producing ribaudioside D using one or more of the above-mentioned variant polypeptide and a transformant expressing the above-mentioned variant polypeptide.

[0029] In addition, the present application provides a composition for producing one or more steviol glycosides selected from the group consisting of ribaudioside M, ribaudioside A, and ribaudioside D, comprising one or more of the variant polypeptide and a transformant expressing the variant polypeptide.

[0030]

[0031] The present application is described in detail below.

[0032]

[0033] One aspect of the present application provides a variant polypeptide having glycosyltransfer activity, wherein the variant polypeptide is a variant polypeptide in which an amino acid corresponding to one or more positions selected from SEQ ID NO. 20, 88, and 126 is substituted with another amino acid.

[0034] The above variant polypeptide may be one in which an amino acid corresponding to one or more positions selected from 86, 200, and 379 of SEQ ID NO. 1 is further substituted with another amino acid.

[0035] The amino acid sequence and / or variant polypeptide of SEQ ID NO. 1 of the present application may be a glycosyltransferase, specifically a UDP-glycosyltransferase (Uridine diphosphate glycosyltransferase; UDP-glycosyltransferase; UGT), and more specifically a UDP-glycosyltransferase A (UGT-A), but is not limited thereto.

[0036] The term "glycosyltransferase" above refers to an enzyme having the activity of transferring a monosaccharide moiety from a glycosyl donor to a glycosyl acceptor molecule, and the UDP-glycosyltransferase above refers to an enzyme that uses a nucleotide diphosphate (e.g., UDP-sugar) to which glucose is bound as a glycosyl donor. In the following application, the term glycosyltransferase may be used interchangeably with "UDP-glycosyltransferase" and "UGT".

[0037] In this application, "UDP-glycotransferase A" may be an enzyme capable of converting stevioside to rebaudioside A and / or rebaudioside D to rebaudioside M (RebM) using a glucose-bound nucleotide diphosphate as a glycosyl donor. In the following application, UDP-glycotransferase A may be used interchangeably with glycotransferase A and UGT-A.

[0038] In the present application, "UDP-glycotransferase B" may be an enzyme capable of converting rebaudioside A (RebA) to rebaudioside D (RebD) using a glucose-bound nucleotide diphosphate as a glycosyl donor. In the following application, UDP-glycotransferase B may be used interchangeably with glycotransferase B and UGT-B.

[0039] The above glycosyltransferases A and B may be glycosyltransferases derived from rice (Oryza sativa), stevia (Stevia rebaudiana Bertoni), bamboo (Bambusa oldhamii), barley (Brachypodium distachyon), barley (Hordeum vulgare), sorghum (Sorghum bicolor), corn (Zea mays), or Arabidopsis thaliana.

[0040] As illustrated in the reaction scheme below, the above-mentioned glycosyltransferase A has the activity of synthesizing Rebaudioside A (RebA) by binding glucose to stevioside using a glucose-bound nucleotide diphosphate, and the activity of synthesizing Rebaudioside M (RebM) by binding glucose to Rebaudioside D (RebD):

[0041] [Reaction Equation]

[0042]

[0043] (UDPG: Uridine-5'-diphosphate-glucose, UDP: Uridine-5'-diphosphate, Fruc: Fructose, Suc: Sucrose, SS: Sucrose Synthase)

[0044] In addition, the glycosyltransferase A has the activity of synthesizing rebaudioside I (RebI) by binding glucose to rebaudioside A, as shown in FIG. 2.

[0045] As shown in the reaction scheme above, the above glycosyltransferase B has the activity of synthesizing ribaudioside D by binding glucose to ribaudioside A using a nucleotide diphosphate to which glucose is bound.

[0046] As described above, the variant polypeptide of the present application is one in which an amino acid corresponding to any one or more positions selected from positions 20, 88, and 126 of SEQ ID NO. 1 is substituted with another amino acid. Specifically, the variant polypeptide of the present application may comprise any one or more amino acid substitutions selected from the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) the amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) the amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with isoleucine or tryptophan; and iii) the amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine or tryptophan.

[0047] Additionally, the variant polypeptide of the present application may be one in which an amino acid corresponding to any one or more positions selected from positions 86, 200, and 379 of SEQ ID NO. 1 is further substituted with another amino acid. Specifically, the variant polypeptide of the present application may comprise any one or more amino acid substitutions selected from the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) the amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) the amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with isoleucine or tryptophan; iii) the amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine or tryptophan; iv) the amino acid corresponding to position 86 of SEQ ID NO. 1 is substituted with serine or asparagine; v) the amino acid corresponding to position 200 of SEQ ID NO. 1 is substituted with alanine; and vi) the amino acid corresponding to position 379 of SEQ ID NO. 1 is substituted with methionine.

[0048] The amino acid substitutions described above can modify the shape of the substrate binding site of the parent enzyme glycotransferase A, thereby increasing the enzyme's substrate specificity and significantly increasing the activity of synthesizing ribaudioside M compared to wild-type glycotransferase A.

[0049] In addition, the amino acid substitutions described above can increase the rebaudioside synthesis activity of the glycosyltransferase A even by themselves, and in particular, in the case of glycosyltransferase A having a specific combination of amino acid substitutions, the rebaudioside synthesis activity can be significantly increased compared to wild-type glycosyltransferase A.

[0050] In the case of some amino acid substitutions, they may reduce the rebaudioside synthesis activity of glycosyltransferase A when present alone, but when present with specific combinations of amino acid substitutions, they may significantly increase the rebaudioside synthesis activity compared to wild-type glycosyltransferase A.

[0051] In the present application, the substitutions of i) to vi) may be denoted as i) V20L; ii) M88I or M88W; iii) L126F or L126W; iv) A86S or A86N; v) L200A; and vi) L379M, respectively.

[0052] In one embodiment, the variant polypeptide of the present application may include a V20L amino acid substitution in the amino acid sequence of SEQ ID NO. 1.

[0053] In one embodiment, the variant polypeptide of the present application may include an amino acid substitution of M88I in the amino acid sequence of SEQ ID NO. 1.

[0054] In one embodiment, the variant polypeptide of the present application may include an amino acid substitution of M88W in the amino acid sequence of SEQ ID NO. 1.

[0055] In one embodiment, the variant polypeptide of the present application may include an amino acid substitution of L126F in the amino acid sequence of SEQ ID NO. 1.

[0056] In one embodiment, the variant polypeptide of the present application may include amino acid substitutions of V20L, A86S, M88W, and L126F in the amino acid sequence of SEQ ID NO. 1.

[0057] In one embodiment, the variant polypeptide of the present application may include amino acid substitutions of V20L, A86N, M88I, and L126W in the amino acid sequence of SEQ ID NO. 1.

[0058] In one embodiment, the variant polypeptide of the present application may include amino acid substitutions of V20L, A86S, M88W, L126F, L200A, and L379M in the amino acid sequence of SEQ ID NO. 1.

[0059] The variant polypeptide of the present application may comprise any one amino acid sequence selected from SEQ ID NOs 2 to 9.

[0060] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 2. The amino acid sequence of SEQ ID NO. 2 refers to the amino acid sequence of a variant polypeptide having an amino acid substitution of V20L in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "V20L".

[0061] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 3. The amino acid sequence of SEQ ID NO. 3 refers to the amino acid sequence of a variant polypeptide having an amino acid substitution of M88I in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "M88I".

[0062] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 4. The amino acid sequence of SEQ ID NO. 4 refers to the amino acid sequence of a variant polypeptide having an amino acid substitution of M88W in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "M88W".

[0063] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 5. The amino acid sequence of SEQ ID NO. 5 refers to the amino acid sequence of a variant polypeptide having an amino acid substitution of L126F in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "L126F".

[0064] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 6. The amino acid sequence of SEQ ID NO. 6 refers to the amino acid sequence of a variant polypeptide having an amino acid substitution of L126W in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "L126W".

[0065] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 7. The amino acid sequence of SEQ ID NO. 7 refers to the amino acid sequence of a variant polypeptide having amino acid substitutions of V20L, A86S, M88W, and L126F in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "CM42" or "V20L-A86S-M88W-L126F".

[0066] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 8. The amino acid sequence of SEQ ID NO. 8 refers to the amino acid sequence of a variant polypeptide having amino acid substitutions of V20L, A86N, M88I, and L126W in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "CM48" or "V20L-A86N-M88I-L126W".

[0067] In one embodiment, the variant polypeptide of the present application may comprise the amino acid sequence of SEQ ID NO. 9. The amino acid sequence of SEQ ID NO. 9 refers to the amino acid sequence of a variant polypeptide having amino acid substitutions of V20L, A86S, M88W, L126F, L200A, and L379M in the amino acid sequence of SEQ ID NO. 1, and in the present application, the variant polypeptide may be denoted as "M10" or "V20L-A86S-M88W-L126F-L200A-L379M".

[0068] A variant polypeptide comprising any one of the amino acid sequences selected from SEQ ID NOs 2 to 9 above has superior activity for synthesizing ribaudioside M from ribaudioside D compared to wild-type glycosyltransferase.

[0069] A variant polypeptide comprising any one amino acid sequence selected from SEQ ID NOs 8 and 9 above has an activity for synthesizing ribaudioside M from ribaudioside D that is about 8 times higher than that of wild-type glycosyltransferase A, while having an activity for synthesizing ribaudioside A from stevioside that is about 0.2 times lower than that of wild-type glycosyltransferase, so it can be very effectively used in a selective reaction for converting ribaudioside D to ribaudioside M.

[0070] The variant polypeptide containing the amino acid sequence of SEQ ID NO. 7 has an activity of synthesizing ribaudioside M from ribaudioside D that is about 5 times higher than that of wild-type glycosyltransferase A, and an activity of synthesizing ribaudioside A from stevioside that is also about 1.2 times higher, so it can be effectively used in a complex reaction to produce ribaudioside M from stevioside together with other enzymes such as glycosyltransferase B.

[0071] In addition, the variant polypeptide containing the amino acid sequence of SEQ ID NO. 7 has a significantly lower activity of synthesizing ribaudioside I from ribaudioside A, which is 0.2 times or less compared to wild-type glycosyltransferase A, so it can be effectively used in a complex reaction to produce ribaudioside M from stevioside or ribaudioside A together with other enzymes such as glycosyltransferase B.

[0072] The variant polypeptide of the present application may be produced from recombinant Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed in the form of a vector insertion containing a gene encoding the variant polypeptide or an intrachromosomal insertion of said gene, and said variant polypeptide may be further purified after production from said Escherichia coli, etc.

[0073] Although the variant polypeptide of the present application is defined as a polypeptide in which an amino acid corresponding to one or more positions selected from positions 20, 88, and 126 of SEQ ID NO. 1 is substituted with another amino acid; or a polypeptide in which an amino acid corresponding to one or more positions selected from positions 20, 88, and 126 of SEQ ID NO. 1 is substituted with another amino acid corresponding to one or more positions selected from positions 86, 200, and 379 of SEQ ID NO. 1, this does not exclude meaningless sequence additions before or after the amino acid sequence of SEQ ID NO. 1, naturally occurring mutations, or silent mutations thereof, and it is obvious to those skilled in the art that if it has the same or corresponding activity as the protein composed of the amino acid sequence of SEQ ID NO. 1, it corresponds to the variant polypeptide having glycosyltransfer activity provided in the present application.

[0074] That is, even if the present application is described as ‘a protein or polypeptide having an amino acid sequence described by a specific sequence number’ or ‘a protein or polypeptide containing an amino acid sequence described by a specific sequence number,’ it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added may also be used in the present application if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the said sequence number. For example, in the case of having a sequence addition or deletion that does not alter the function of glycosyltransferase A of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus and / or C-terminus of the amino acid sequence, the N-terminus modification may be a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) attached to the N-terminus of the peptide, and the C-terminus modification may be a hydroxyl group (-OH), an amino group (-NH2), an azide (-NHNH2), etc. attached to the C-terminus of the peptide, but is not limited thereto.

[0075] The term "conservative substitution" above means substituting one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities 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 aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Additionally, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains; amino acids with electrically charged side chains include astragalus, glutamic acid, lysine, arginine, and histidine; amino acids with uncharged side chains can be further classified into nonpolar amino acids or 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. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0076] Additionally, the variant polypeptide of the present application may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated with a signal (or leader) sequence at the N-terminus of a protein involved in the co-translational or post-translational transfer of the protein. Additionally, the polypeptide may be conjugated with another sequence or linker to enable identification, purification, or synthesis of the polypeptide.

[0077] In this application, the term “corresponding to” refers to an amino acid residue at a listed position in a protein or polypeptide, or an amino acid residue that is similar, identical, or homologous to a listed residue in a protein or polypeptide. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. As used in this application, “corresponding region” generally refers to a similar or corresponding position in a related protein or reference protein.

[0078] In the present application, Sequence No. 1 may be used as a reference sequence to determine the position of an amino acid within any amino acid sequence.

[0079] In this application, the term "reference sequence" refers to a sequence used to determine the position of an amino acid within any amino acid sequence. By aligning any amino acid sequence with the reference sequence, the position of an amino acid corresponding to a specific position of the reference sequence within any amino acid sequence can be determined.

[0080]

[0081] Another aspect of the present application provides a polynucleotide encoding a variant polypeptide having glycosyltransfer activity of the present application.

[0082] The description of the variant polypeptide having glycosyltransfer activity described above can be understood as identical to that described above, so it will not be described again.

[0083] The term "polynucleotide" above refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, and is a DNA or RNA strand of a certain length or longer, more specifically, a polynucleotide fragment encoding the glycosyltransferase A of the present application.

[0084] The polynucleotide encoding the variant polypeptide having glycosyltransfer activity of the present application may include a nucleotide sequence encoding any one amino acid sequence selected from SEQ ID NOs 2 to 9.

[0085] The polynucleotide of the present application may have various modifications made to the coding region within a range that does not alter the amino acid sequence of the variant polypeptide of the present application, taking into account the degeneracy of the codons or the codons preferred by the organism intended to express the variant polypeptide of the present application. Specifically, the polynucleotide of the present application may have or include a nucleotide sequence having 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%, respectively, with respect to a nucleotide sequence encoding any one of the amino acid sequences selected from SEQ ID NOs 2 to 9, or may be composed of or essentially composed of such nucleotide sequences, but is not limited thereto.

[0086] In addition, the polynucleotide of the present application may further include, without limitation, a probe that can be prepared from a known gene sequence, for example, a sequence that can be hybridized under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. For example, it may mean a condition in which polynucleotides with high homology or identity are hybridized with each other, having 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, or 99% or more, and polynucleotides with lower homology or identity are not hybridized with each other, or a washing condition of conventional southern hybridization.

[0087]

[0088] Another aspect of the present application provides an expression vector comprising a polynucleotide encoding a variant polypeptide having glycosyltransfer activity of the present application.

[0089] The description of the variant polypeptide having glycosyltransferable activity and the polynucleotide encoding it is understood to be identical to that described above, so it is not described again.

[0090] The term "expression vector" above refers to a DNA product comprising a sequence of nucleotides of a polynucleotide encoding said target polypeptide, which is operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of said target polypeptide within a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. Any promoter may be used as long as the target polynucleotide of the present application can be appropriately expressed in a host. For example, E. coli or phage-derived promoters such as the trp promoter, lac promoter, PL promoter, or PR promoter; E. coli-infected phage-derived promoters such as the T7 promoter; CaMV35S, MAS, or histone promoters may be used. Additionally, artificially modified promoters such as the tac promoter may also be used.

[0091] By transforming or transfecting a host cell with the above expression vector, the desired proteins can be obtained. After being transformed into a suitable microorganism, the expression vector can replicate or function independently of the host genome and can be incorporated into the genome itself.

[0092] The term "operably connected" above means that a promoter sequence and a polynucleotide sequence are functionally connected to initiate and mediate the transcription of a polynucleotide encoding glycosyltransferase A, which is the subject of this application.

[0093] The expression vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A, etc. can be used as phage vectors or cosmid vectors, and as plasmid vectors, E. coli-derived plasmids (pET28a, pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), and Ti-plasmids that can be used for Agrobacterium-mediated transformation can be used. In addition, as a viral vector, vectors derived from animal viruses such as retroviruses, adenoviruses, or vaccinia viruses, insect viruses such as baculoviruses, double-stranded plant viruses (e.g., CaMV), single-stranded viruses, or Gemini viruses may be used. Furthermore, as an expression vector, a fusion plasmid (e.g., pJG4-5) to which a nucleic acid expression activating protein (e.g., B42) is linked may also be used.

[0094] The expression vector of the present application may additionally include, in addition to the promoter and the polynucleotide of the present application, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), etc.

[0095] To insert the polynucleotide of the present application into a vector, a method may be used in which purified DNA is cleaved with a suitable restriction enzyme and inserted into a restriction site or cloning site of a suitable vector DNA. Additionally, a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome insertion has occurred. The selection marker is intended to select cells transformed with the vector, that is, to confirm whether the target nucleic acid molecule has been inserted, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides may be used. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0096]

[0097] Another aspect of the present application provides a transformant comprising a polynucleotide encoding a variant polypeptide having glycosyltransferable activity of the present application.

[0098] The description of the variant polypeptide having glycosyltransferable activity and the polynucleotide encoding it is understood to be identical to that described above, so it is not described again.

[0099] The transformant of the present application may have the activity of producing ribaudioside A, ribaudioside D and / or ribaudioside M.

[0100] The transformant of the present application may include a variant polypeptide of the present application, a polynucleotide encoding said variant polypeptide, or an expression vector comprising the polynucleotide of the present application.

[0101] The above "transformer" may be in the form of a transformed cell or microorganism comprising the variant polypeptide of the present application, a polynucleotide encoding the variant polypeptide, or an expression vector comprising the polynucleotide of the present application.

[0102] In this application, when the transformant is in the form of a microorganism, it includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. It may be a microorganism in which specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In this application, "microorganism" and "strain" may be used interchangeably without limitation as having the same meaning.

[0103] Specifically, the microorganism may be a microorganism of the genus Escherichia or a microorganism of the genus Corynebacterium, more specifically Escherichia coli or Corynebacterium stationis, but is not limited thereto. Corynebacterium stationis has excellent UDP production capacity and can be used more effectively for the production of various ribaudiosides.

[0104] The transformant of the present application may be, but is not limited to: a strain comprising one or more of the variant polypeptide of the present application, the polynucleotide of the present application, and an expression vector comprising the polynucleotide of the present application; a strain modified to express the variant polypeptide of the present application or the polynucleotide of the present application; a strain expressing the variant polypeptide of the present application or the polynucleotide of the present application (e.g., a recombinant strain); or a strain having glycosyltransferase A activity of the present application (e.g., a recombinant strain).

[0105] In this application, the term "transformation" refers to a phenomenon in which an external polynucleotide is introduced into a cell or microorganism to artificially induce a genetic change, such that the polypeptide encoded by the polynucleotide can be expressed within the introduced cell or microorganism by introducing a vector containing a polynucleotide encoding a target polypeptide into the cell or microorganism. The transformed polynucleotide may include both types of locations, regardless of whether they are inserted into or outside the chromosomes of the microorganism, as long as they can be expressed within the microorganism. Additionally, the polynucleotide may include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

[0106] In this application, any transformation method may be used, and it can be easily performed according to conventional methods in the art. Generally, transformation methods include the CaCl2 precipitation method, the Hanahan method which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 method, electroporation, calcium phosphate precipitation method, protoplasmic fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation method, PEG-mediated transformation method, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods. The method for transforming a vector containing a polynucleotide encoding sucrose phosphorylase according to the present invention is not limited to the above examples, and transformation or transfection methods conventionally used in the art may be used without limitation.

[0107] By the above transformation, an expression vector comprising a polynucleotide encoding the variant polypeptide of the present application, or a portion of said expression vector, may be introduced into a microorganism, wherein the portion of said expression vector refers to a portion of the expression vector comprising a polynucleotide portion encoding glycosyltransferase A so as to confer activity of said glycosyltransferase A into the microorganism. For example, the T-DNA of a Ti plasmid delivered into a host cell in an Agrobacterium-mediated transformation method may be used, but is not limited thereto.

[0108] In one embodiment, the transformant of the present application may further comprise a polynucleotide encoding glycosyltransferase B.

[0109] In one embodiment, the transformant of the present application may further comprise a polynucleotide encoding glycosyltransferase B; and a polynucleotide encoding one or more selected from the group consisting of sucrose synthase and UMPK.

[0110] The above glycosyltransferase B may include, without limitation, an enzyme having the activity of synthesizing ribaudioside D by binding glucose to ribaudioside A using a nucleotide diphosphate to which glucose is bound. For example, the above glycosyltransferase B may have and / or include the amino acid sequence described in SEQ ID NO. 10 or 11, or may be essentially composed of, but is not limited thereto.

[0111] The above-mentioned sucrose synthase refers to an enzyme responsible for producing sucrose in plant metabolism by transferring glucose, to which a nucleotide diphosphate is bound, to fructose. Since this reaction can occur reversibly, the above-mentioned sucrose synthase in this application exhibits activity of reacting sucrose with nucleotide diphosphate in a pH range of 5 to 10 to separate it into a nucleotide diphosphate bound to glucose and fructose.

[0112] The above sucrose synthase may be derived from rice, corn, wheat, bamboo, Arabidopsis, grass, barley, sorghum, or potato, preferably from rice, corn, wheat, or barley, and more preferably from rice, particularly Oryza sativa. The above sucrose synthase may be produced from recombinant E. coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector containing a polynucleotide encoding sucrose synthase, and may be further purified after production from said E. coli, etc., and may be a sucrose synthase known in the art or commercially available, but is not limited thereto.

[0113] Specifically, the sucrose synthase of the present application may have and / or include the amino acid sequence described in SEQ ID NO. 12, or may be essentially composed of, but not limited to, said amino acid sequence.

[0114] The above sucrose can be used without limitation as long as it acts as a substrate for sucrose synthase and can provide glucose to nucleotide diphosphate, for example, raw sugar or sugar may be used, but is not limited thereto.

[0115] In this application, the nucleotide diphosphate may be a purine nucleotide or a pyrimidine nucleotide, and preferably, uridine diphosphate (UDP) may be used, but is not limited thereto.

[0116] In addition, the glucose-bound nucleotide diphosphate in this application may be prepared by reacting sucrose and nucleotide diphosphate in the presence of a sucrose synthesizer, but is not limited thereto.

[0117] The above UMPK refers to an enzyme responsible for producing UDP by transferring the phosphate group of ATP to UMP. Since the above UMPK possesses UDP-producing activity, when introduced into the above-mentioned transformant, it can reduce the dependence on UDP during the production reactions of various ribaudiosides.

[0118] The above UMPK may be produced from recombinant E. coli, Bacillus, yeast, Corynebacterium, or Agrobacterium transformed with a vector containing a polynucleotide encoding UMPK, or may be further purified after production from said E. coli, etc., and may be a UMPK known in the art or commercially available, but is not limited thereto.

[0119] Specifically, the UMPK of the present application may have and / or include the amino acid sequence described in SEQ ID NO. 13, or may be essentially composed of, but is not limited to.

[0120] Meanwhile, in the present application, the stevioside may be a hot water or ethanol aqueous extract of Stevia rebaudiana, a purified product thereof, or a byproduct after the production of rebaudioside A from the extract, containing a stevioside content of 10% or more by weight, preferably 50% or more by weight, particularly preferably 70% or more by weight, and even more particularly preferably 80% or more by weight based on the total weight of the steviol glycoside, but is not limited thereto.

[0121] The transformant of the present application may be, but is not limited to: a strain comprising one or more of a polynucleotide encoding glycosyltransferase B, sucrose synthase, and UMPK, respectively, and an expression vector comprising said polynucleotide; a strain modified to express glycosyltransferase B, sucrose synthase, and UMPK, or each polynucleotide encoding glycosyltransferase B, sucrose synthase, and UMPK; a strain expressing glycosyltransferase B, sucrose synthase, and UMPK, or each polynucleotide encoding glycosyltransferase B, sucrose synthase, and UMPK (e.g., a recombinant strain); or a strain having the activity of glycosyltransferase B, sucrose synthase, and UMPK (e.g., a recombinant strain).

[0122] According to one embodiment, since the transformant of the present application can not only express the variant polypeptide of the present application but also express glycotransferase B, sucrose synthase and UMPK, it can produce ribaudioside M using ribaudioside D as a substrate without the addition of other enzymes, and can be widely used for the production of ribaudioside A, ribaudioside D, or ribaudioside M from stevioside, and for the production of ribaudioside D or ribaudioside M from ribaudioside A (see reaction scheme below).

[0123] That is, the transformant of the present application may have one or more production capabilities selected from the group consisting of ribaudioside A, ribaudioside D, ribaudioside I, and ribaudioside M.

[0124] [Reaction Equation]

[0125]

[0126] (UDPG: Uridine-5'-diphosphate-glucose, UDP: Uridine-5'-diphosphate, Fruc: Fructose, Suc: Sucrose, SS: Sucrose Synthase)

[0127] Accordingly, by reacting sucrose, nucleotide diphosphate, and stevioside in the presence of the transformant of the present application, or by culturing the transformant of the present application in the presence of sucrose, nucleotide diphosphate, stevioside, and / or glucose, one or more selected from the group consisting of ribaudioside A, ribaudioside D, and ribaudioside M can be effectively produced.

[0128] In addition, one or more selected from the group consisting of ribaudioside D and ribaudioside M can be effectively produced by reacting sucrose, nucleotide diphosphate and ribaudioside A in the presence of the transformant of the present application, or by culturing the transformant of the present application in the presence of sucrose, nucleotide diphosphate, ribaudioside A and / or glucose.

[0129] In addition, ribaudioside M can be effectively produced by reacting sucrose, nucleotide diphosphate, and ribaudioside D in the presence of the transformant of the present application, or by culturing the transformant of the present application in the presence of sucrose, nucleotide diphosphate, ribaudioside D, and / or glucose.

[0130]

[0131] Another aspect of the present application provides a method for producing one or more steviol glycosides selected from the group consisting of ribaudioside M, ribaudioside A, and ribaudioside D, comprising the step of culturing a transformant expressing the variant polypeptide of the present application in a medium.

[0132] The description of the variant polypeptide having glycosyltransferable activity and the transformant expressing it is understood to be identical to that described above, so it is not described again.

[0133] In this application, the term "culture" means growing microorganisms under appropriately controlled environmental conditions. The culture process may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the microorganism selected. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0134] In this application, the term "medium" refers to a substance mixed with nutrients as the main component required for culturing microorganisms, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, any medium and other culture conditions used for culturing the microorganisms of this application may be used without special limitations as long as they are media commonly used for culturing microorganisms. For example, the microorganisms of this application may be cultured under aerobic conditions while controlling temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0135] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used in various ways without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0136] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.

[0137] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0138] In addition, during the cultivation of the transformant of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during cultivation, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress the formation of bubbles. Furthermore, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto.

[0139] In one embodiment, after culturing the transformant of the present application, a substrate for the synthesis of ribaudioside M, ribaudioside A, and / or ribaudioside D can be reacted in the presence of said transformant to produce said ribaudioside M, ribaudioside A, and / or ribaudioside D.

[0140] In one embodiment, the transformant of the present application can be cultured in a medium containing a substrate for the synthesis of ribaudioside M, ribaudioside A, and / or ribaudioside D to produce said ribaudioside M, ribaudioside A, and / or ribaudioside D.

[0141] The above medium may include one or more of stevioside, ribaudioside A, ribaudioside D, glucose-conjugated nucleotide diphosphate, sucrose, and nucleotide diphosphate.

[0142] Specifically, the medium for producing ribaudioside A may include stevioside and a nucleotide diphosphate conjugated with glucose; stevioside, sucrose, and a nucleotide diphosphate; or stevioside, a nucleotide diphosphate conjugated with glucose, sucrose, and a nucleotide diphosphate. Sucrose and a nucleotide diphosphate may be included in place of or additionally substitute for the nucleotide diphosphate conjugated with glucose.

[0143] The above medium for producing ribaudioside D may include ribaudioside A and a nucleotide diphosphate conjugated with glucose; ribaudioside A, sucrose, and a nucleotide diphosphate; or ribaudioside A, a nucleotide diphosphate conjugated with glucose, sucrose, and a nucleotide diphosphate. A medium for producing ribaudioside A may be included in place of or additionally included with ribaudioside A, and sucrose and a nucleotide diphosphate may be included in place of or additionally included with the nucleotide diphosphate conjugated with glucose.

[0144] The above medium for producing ribaudioside M may include ribaudioside D and a nucleotide diphosphate bound to glucose; or ribaudioside D, sucrose, and a nucleotide diphosphate. A medium for producing ribaudioside D may be included in place of ribaudioside D, and may include sucrose and a nucleotide diphosphate in place of the nucleotide diphosphate bound to glucose or additionally.

[0145] In the culture of the present application, the culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and culture may be carried out for about 10 to 160 hours, but is not limited thereto.

[0146] In the present application, the term "culture" means a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, wherein the culture solution means containing the specific microorganism, and the culture filtrate means not substantially containing the specific microorganism (wherein "substantially" means excluding the specific microorganism separated by filtration, etc., and does not mean that the microorganism is completely excluded from the filtrate). The formulation of the culture is not limited and may be, for example, a liquid, an emulsion, or a solid.

[0147] In this application, the term "fermentation" refers to a process in which microorganisms decompose organic matter using their own enzymes, excluding putrefaction. Although fermentation and putrefaction proceed through similar processes, if useful substances are produced as a result of the decomposition, it is called fermentation, whereas if foul odors are emitted or harmful substances are produced, it is called putrefaction.

[0148] In this application, the method for obtaining a fermented product from the microorganism is not particularly limited and can be obtained according to methods commonly used in the relevant technical field or similar fields.

[0149] In this application, the term "fermented product" includes not only the fermented substance itself, but also all types of substances containing a fermented product generated from said microorganisms, such as a substance containing a fermented microorganism, a culture produced from a fermented microorganism, a fermented product of a culture, a concentrated fermented product, a dried product of a fermented product, a filtrate of a fermented product, a filtrate of a concentrated fermented product, or a dried product of a filtrate of a fermented product, an extract of a fermented product, or a diluted solution of a fermented product.

[0150] In the method of the present application, any culture conditions and methods known in the art may be used for the culture of microorganisms. Such a culture process can be easily adjusted and used by those skilled in the art depending on the microorganism selected.

[0151] Ribaudioside M, ribaudioside A, and / or ribaudioside D produced by the culture of the present application may be secreted into the medium or remain within the cell.

[0152]

[0153] Another aspect of the present application provides a method for preparing ribaudioside M, comprising the step of reacting ribaudioside D with a glucose-conjugated nucleotide diphosphate in the presence of one or more of the variant polypeptide having glycosyltransferable activity of the present application and a transformant expressing said variant polypeptide.

[0154] The description of the variant polypeptide having glycosyltransferable activity and the transformant expressing it is understood to be identical to that described above, so it is not described again.

[0155] As illustrated in the reaction scheme described above, the ribaudioside M can be synthesized from ribaudioside D in the presence of glycosyltransferase A. Accordingly, ribaudioside M can be prepared by reacting ribaudioside D with a glucose-conjugated nucleotide diphosphate in the presence of one or more of the variant polypeptide having glycosyltransfer activity of the present application and a transformant expressing said variant polypeptide.

[0156] The above variant polypeptide may be included in the transformant of the present application or obtained therefrom (e.g., purified).

[0157] In one embodiment, the method for preparing ribaudioside M of the present application may involve reacting ribaudioside D and a nucleotide diphosphate combined with glucose in the presence of the transformant after culturing the transformant.

[0158] In one embodiment, the method for producing ribaudioside M of the present application may involve culturing the transformant in a medium comprising ribaudioside D and a nucleotide diphosphate combined with glucose.

[0159] The method for preparing rebaudioside M of the present application may additionally include, for example, the step of preparing a transformant of the present application, the step of preparing a medium for culturing said transformant, or a combination thereof (in any order), prior to the culturing step.

[0160] The descriptions of the above polynucleotide, expression vector, culture, and medium can be understood as identical to those previously described, so they will not be repeated.

[0161] In one embodiment, the method for preparing ribaudioside M of the present application may further include the step of preparing glucose-linked nucleotide diphosphate by reacting sucrose with nucleotide diphosphate in the presence of a sucrose synthesizing enzyme, but is not limited thereto.

[0162] The descriptions of the above sucrose synthase, sucrose, and nucleotide diphosphate can be understood as identical to those described above, so they are not repeated.

[0163] The method for producing ribaudioside M of the present application may further include the step of producing ribaudioside D by reacting ribaudioside A and a nucleotide diphosphate combined with glucose in the presence of one or more of glycosyltransferase B and a transformant expressing said glycosyltransferase B.

[0164] In this case, ribaudioside D can be synthesized from ribaudioside A, and ribaudioside M can be synthesized from ribaudioside D.

[0165] The method for preparing ribaudioside M of the present application may further include the step of preparing ribaudioside A by reacting stevioside and a glucose-conjugated nucleotide diphosphate in the presence of at least one of the glycosyltransferase B of the present application and a transformant expressing said variant polypeptide; and the step of preparing ribaudioside D by reacting ribaudioside A and a glucose-conjugated nucleotide diphosphate in the presence of at least one of the glycosyltransferase B and a transformant expressing said glycosyltransferase B.

[0166] In this case, ribaudioside A can be synthesized from stevioside, ribaudioside D can be synthesized from ribaudioside A, and ribaudioside M can be synthesized from ribaudioside D.

[0167] In one embodiment, the preparation of the ribaudioside A, D and / or M may be carried out continuously or discontinuously in the same reaction system and may be carried out in one pot, but is not limited thereto.

[0168] The method for producing ribaudioside M of the present application may further include the step of recovering ribaudioside M from the reaction solution in which the enzymatic reaction occurred, the cultured transformant, the culture of the transformant, the fermented product of the transformant, or the culture medium. The recovery step may be performed after the ribaudioside production reaction is completed.

[0169] The above recovery may involve collecting the desired ribaudioside using a suitable method known in the art according to the culture method of the transformant of the present application, such as a batch, continuous, or fed-batch culture method. For example, various chromatographic methods such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired ribaudioside may be recovered from the culture medium or the transformant using a suitable method known in the art.

[0170] In addition, the manufacturing method of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the manufacturing method 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 simultaneously or integrated into a single step, but are not limited thereto.

[0171]

[0172] Another aspect of the present application provides a method for producing ribaudioside M, comprising the step of reacting ribaudioside A; a glucose-bound nucleotide diphosphate; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of one or more of the variant polypeptide having glycosyltransferase activity of the present application and a transformant expressing said variant polypeptide.

[0173] The descriptions of the variant polypeptide having glycosyltransferable activity, the transformant expressing it, and glycosyltransferase B are understood to be identical to those described above, so they are not repeated.

[0174] As illustrated in the aforementioned reaction scheme, ribaudioside D can be synthesized from ribaudioside A in the presence of glycotransferase B, and ribaudioside M can be synthesized from ribaudioside D in the presence of glycotransferase A.

[0175] Accordingly, ribaudioside M can be prepared by reacting ribaudioside A; a glucose-bound nucleotide diphosphate; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of any one or more of the variant polypeptide having glycosyltransferase activity of the present application and said variant polypeptide.

[0176] The above variant polypeptide may be included in the transformant of the present application or obtained therefrom (e.g., purified).

[0177] In one embodiment, the method for preparing ribaudioside M of the present application may involve reacting ribaudioside A; a nucleotide diphosphate bound to glucose; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of said transformant after culturing said transformant.

[0178] In one embodiment, the method for preparing ribaudioside M of the present application may involve culturing the transformant in a medium comprising ribaudioside A; a nucleotide diphosphate bound to glucose; and at least one of glycosyltransferase B and a transformant expressing glycosyltransferase B; and the diphosphate.

[0179] The method for preparing rebaudioside M of the present application may additionally include, for example, the step of preparing a transformant of the present application, the step of preparing a medium for culturing said transformant, or a combination thereof (in any order), prior to the culturing step.

[0180] The descriptions of the above polynucleotide, expression vector, culture, and medium can be understood as identical to those previously described, so they will not be repeated.

[0181] In one embodiment, the method for preparing ribaudioside M of the present application may further include the step of preparing glucose-linked nucleotide diphosphate by reacting sucrose with nucleotide diphosphate in the presence of a sucrose synthesizing enzyme, but is not limited thereto.

[0182] The descriptions of the above sucrose synthase, sucrose, and nucleotide diphosphate can be understood as identical to those described above, so they are not repeated.

[0183] The method for preparing ribaudioside M of the present application may further include the step of preparing ribaudioside A by reacting a nucleotide diphosphate combined with stevioside and glucose in the presence of one or more of the variant polypeptide having glycosyltransfer activity of the present application and a transformant expressing said variant polypeptide.

[0184] In this case, ribaudioside A can be synthesized from stevioside, ribaudioside D can be synthesized from ribaudioside A, and ribaudioside M can be synthesized from ribaudioside D.

[0185] In one embodiment, the preparation of the ribaudioside A, D and / or M may be carried out continuously or discontinuously in the same reaction system and may be carried out in one pot, but is not limited thereto.

[0186] In the present application, the manufacturing method may further include a step of recovering rebaudioside M.

[0187] In addition, the manufacturing method in the present application may additionally include a purification step.

[0188] The description of the recovery and / or purification above can be understood as identical to that described above, so it will not be described again.

[0189]

[0190] Another aspect of the present application provides a method for producing ribaudioside M, comprising the step of reacting stevioside; a glucose-bound nucleotide diphosphate; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of any one or more of the variant polypeptide having glycosyltransferase activity of the present application and a transformant expressing said variant polypeptide.

[0191] The descriptions of the variant polypeptide having glycosyltransferable activity, the transformant expressing it, and glycosyltransferase B are understood to be identical to those described above, so they are not repeated.

[0192] As shown in the reaction scheme described above, ribaudioside A can be synthesized from stevioside in the presence of glycotransferase A, ribaudioside D can be synthesized from ribaudioside A in the presence of glycotransferase B, and ribaudioside M can be synthesized from ribaudioside D in the presence of glycotransferase A.

[0193] Accordingly, ribaudioside M can be prepared by reacting stevioside; a glucose-bound nucleotide diphosphate; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of any one or more of the variant polypeptide having glycosyltransferase activity of the present application and a transformant expressing said variant polypeptide.

[0194] The above variant polypeptide may be included in the transformant of the present application or obtained therefrom (e.g., purified).

[0195] In one embodiment, the method for preparing ribaudioside M of the present application may involve reacting one or more of stevioside; a nucleotide diphosphate bound to glucose; a glycosyltransferase B; and a transformant expressing the glycosyltransferase B in the presence of the transformant after culturing the transformant.

[0196] In one embodiment, the method for preparing ribaudioside M of the present application may involve culturing the transformant in a medium comprising one or more of stevioside; a nucleotide diphosphate bound to glucose; glycosyltransferase B; and a transformant expressing glycosyltransferase B.

[0197] The method for preparing rebaudioside M of the present application may additionally include, for example, the step of preparing a transformant of the present application, the step of preparing a medium for culturing said transformant, or a combination thereof (in any order), prior to the culturing step.

[0198] The descriptions of the above polynucleotide, expression vector, culture, and medium can be understood as identical to those previously described, so they will not be repeated.

[0199] In one embodiment, the method for preparing ribaudioside M of the present application may further include the step of preparing glucose-linked nucleotide diphosphate by reacting sucrose with nucleotide diphosphate in the presence of a sucrose synthesizing enzyme, but is not limited thereto.

[0200] The descriptions of the above sucrose synthase, sucrose, and nucleotide diphosphate can be understood as identical to those described above, so they are not repeated.

[0201] In one embodiment, the preparation of the ribaudioside A, D and / or M may be carried out continuously or discontinuously in the same reaction system and may be carried out in one pot, but is not limited thereto.

[0202] In the present application, the manufacturing method may further include a step of recovering rebaudioside M.

[0203] In addition, the manufacturing method in the present application may additionally include a purification step.

[0204] The description of the recovery and / or purification above can be understood as identical to that described above, so it will not be described again.

[0205]

[0206] Another aspect of the present application provides a method for producing ribaudioside A, comprising the step of reacting a nucleotide diphosphate combined with stevioside and glucose in the presence of one or more of the variant polypeptide having glycosyltransferable activity of the present application and a transformant expressing said variant polypeptide.

[0207] The description of the variant polypeptide having glycosyltransferable activity and the transformant expressing it is understood to be identical to that described above, so it is not described again.

[0208] As shown in the aforementioned reaction scheme, ribaudioside A can be synthesized from stevioside in the presence of glycosyltransferase A.

[0209] Accordingly, ribaudioside A can be prepared by reacting a nucleotide diphosphate combined with stevioside and glucose in the presence of one or more of the variant polypeptide having glycosyl transfer activity of the present application and a transformant expressing said variant polypeptide.

[0210] The above variant polypeptide may be included in the transformant of the present application or obtained therefrom (e.g., purified).

[0211] In one embodiment, the method for preparing ribaudioside A of the present application may involve reacting a nucleotide diphosphate combined with stevioside and glucose in the presence of the transformant after culturing the transformant.

[0212] In one embodiment, the method for producing ribaudioside A of the present application may involve culturing the transformant in a medium containing nucleotide diphosphate combined with stevioside and glucose.

[0213] The method for preparing rebaudioside A of the present application may additionally include, for example, the step of preparing a transformant of the present application, the step of preparing a medium for culturing said transformant, or a combination thereof (in any order), prior to the culturing step.

[0214] The descriptions of the above polynucleotide, expression vector, culture, and medium can be understood as identical to those previously described, so they will not be repeated.

[0215] In one embodiment, the method for preparing ribaudioside A of the present application may further include the step of preparing glucose-linked nucleotide diphosphate by reacting sucrose with nucleotide diphosphate in the presence of a sucrose synthesizing enzyme, but is not limited thereto.

[0216] The descriptions of the above sucrose synthase, sucrose, and nucleotide diphosphate can be understood as identical to those described above, so they are not repeated.

[0217] In one embodiment, the preparation of the ribaudioside A may be carried out continuously or discontinuously in the same reaction system and may be carried out in one pot, but is not limited thereto.

[0218] In the present application, the manufacturing method may further include the step of recovering rebaudioside A.

[0219] In addition, the manufacturing method in the present application may additionally include a purification step.

[0220] The description of the recovery and / or purification above can be understood as identical to that described above, so it will not be described again.

[0221]

[0222] Another aspect of the present application provides a method for producing ribaudioside D, comprising the step of reacting stevioside; a glucose-bound nucleotide diphosphate; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of one or more of the variant polypeptide having glycosyltransferase activity of the present application and a transformant expressing said variant polypeptide.

[0223] The descriptions of the variant polypeptide having glycosyltransferable activity, the transformant expressing it, and glycosyltransferase B are understood to be identical to those described above, so they are not repeated.

[0224] As shown in the reaction scheme described above, ribaudioside A can be synthesized from stevioside in the presence of glycosyltransferase A, and ribaudioside D can be synthesized from ribaudioside A in the presence of glycosyltransferase B.

[0225] Accordingly, ribaudioside D can be prepared by reacting stevioside; a glucose-bound nucleotide diphosphate; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of at least one of the variant polypeptide having glycosyltransferase activity of the present application and a transformant expressing said variant polypeptide.

[0226] The above variant polypeptide may be included in the transformant of the present application or obtained therefrom (e.g., purified).

[0227] In one embodiment, the method for producing ribaudioside D of the present application may involve reacting stevioside; a nucleotide diphosphate bound to glucose; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B in the presence of said transformant after culturing said transformant.

[0228] In one embodiment, the method for producing ribaudioside D of the present application may involve culturing the transformant in a medium comprising: stevioside; a nucleotide diphosphate bound to glucose; and one or more of glycosyltransferase B and a transformant expressing glycosyltransferase B.

[0229] The method for preparing rebaudioside D of the present application may additionally include, for example, the step of preparing a transformant of the present application, the step of preparing a medium for culturing said transformant, or a combination thereof (in any order), prior to the culturing step.

[0230] The descriptions of the above polynucleotide, expression vector, culture, and medium can be understood as identical to those previously described, so they will not be repeated.

[0231] In one embodiment, the method for preparing ribaudioside D of the present application may further include the step of preparing glucose-linked nucleotide diphosphate by reacting sucrose with nucleotide diphosphate in the presence of a sucrose synthesizing enzyme, but is not limited thereto.

[0232] The descriptions of the above sucrose synthase, sucrose, and nucleotide diphosphate can be understood as identical to those described above, so they are not repeated.

[0233] The method for preparing ribaudioside D of the present application may comprise: a step of preparing ribaudioside A by reacting stevioside and a glucose-conjugated nucleotide diphosphate in the presence of at least one of the glycosyltransferase B of the present application and a transformant expressing said variant polypeptide; and a step of preparing ribaudioside D by reacting ribaudioside A and a glucose-conjugated nucleotide diphosphate in the presence of at least one of the glycosyltransferase B and a transformant expressing said glycosyltransferase B.

[0234] In this case, ribaudioside A can be synthesized from stevioside, ribaudioside D can be synthesized from ribaudioside A, and ribaudioside M can be synthesized from ribaudioside D.

[0235] In one embodiment, the preparation of ribaudioside A and / or D may be carried out continuously or discontinuously in the same reaction system and may be carried out in one pot, but is not limited thereto.

[0236] In the present application, the manufacturing method may further include a step of recovering rebaudioside D.

[0237] In addition, the manufacturing method in the present application may additionally include a purification step.

[0238] The description of the recovery and / or purification above can be understood as identical to that described above, so it will not be described again.

[0239]

[0240] Another aspect of the present application provides a composition for producing one or more steviol glycosides selected from the group consisting of ribaudioside M, ribaudioside A, and ribaudioside D, comprising one or more of the variant polypeptide having glycosyltransferable activity of the present application and a transformant expressing said variant polypeptide.

[0241] The composition of the present application may further include any suitable excipients commonly used in compositions for producing amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, but are not limited thereto.

[0242] The variant polypeptide having glycosyltransferable activity of the present application has significantly superior activity in producing ribaudioside M from ribaudioside D compared to wild-type glycosyltransferable enzyme A, so it can effectively convert raw materials that are easy to obtain but have a strong bitter taste, such as stevioside and ribaudioside A contained in stevia extract, into ribaudioside M, which is a component with excellent taste, and thus can be usefully utilized for the mass production of stevia sweeteners with excellent sweetness.

[0243] However, the effects of the present application are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0244] Figure 1 shows the results of evaluating the RebM conversion rate of wild-type glycosyltransferase A and glycosyltransferase A having a single mutation.

[0245] Figure 2 shows the structures of ribaudioside A and ribaudioside I.

[0246] Figure 3 shows the results of evaluating the RebI conversion rate of glycosyltransferase A and complex mutant enzyme CM42.

[0247] Figure 4 shows the results of evaluating the production of ribaudioside M in Corynebacterium stationis strains into which wild-type or mutant glycosyltransferase A, glycosyltransferase B, sucrose synthase, and UMPK genes were introduced.

[0248] The present application will be explained in detail below through examples.

[0249] However, the following examples and experimental examples are merely for illustrating the invention of the present application, and the content of the invention of the present application is not limited by the following examples and experimental examples.

[0250]

[0251] Example 1. Design and activity measurement of a novel mutant glycosyltransferase A (UGT-A)

[0252] 1-1. Design of a Novel Mutant Glycotransferase A (UGT-A)

[0253] The UGT-A enzyme has the activity to convert stevioside into rebaudioside A (RebA) and rebaudioside D (RebD) into rebaudioside M (RebM), which is high-quality stevia, but it has a problem in that the efficiency of the reaction for producing RebM is relatively low due to low substrate specificity for RebD.

[0254] Accordingly, to increase the activity of the conversion reaction from ribaudioside D to ribaudioside M, a major amino acid site among the enzyme active sites was selected, and eight amino acid residue positions (V20, F22, A86, M88, L126, I199, L200, L379) that can affect the conversion rate by changing the shape around the substrate binding site of the UGT-A enzyme were selected by considering the consensus sequence through sequence comparison analysis with homologous enzymes, and a combinational library was designed by selecting amino acids to be substituted.

[0255]

[0256] 1-2. Measurement of single mutant UGT-A enzyme activity

[0257] Experiments were performed to confirm the ribaudioside M conversion activity of the modified UGT-A enzyme having a single mutation at the above amino acid residue position.

[0258] V20L, M88I, M88W, L126F, and L126W were selected as single mutants, and single mutant UGT-A enzymes were constructed using the site-directed mutagenesis method. Specifically, PCR was performed using AccuPower® Pfu PCR premix (Bionia), 10 ng of template plasmid (pET-28a vector), and 0.5 μM each of forward and reverse primers. PCR was performed using a Mastercycler® X50s (Eppendorf) in the following sequence: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, 20 cycles of extension at 72°C for 1 minute / kb, and final extension at 72°C for 5 minutes.

[0259] Subsequently, the template plasmid was digested using 1 μl of DpnI (NEB, R0176L), and 5 μl of the PCR reaction solution was injected into the E. coli DH5α strain via heat shock to induce transformation. The transformed E. coli strains were cultured in LB medium containing 50 mg / L of kanamycin. After colony selection, mutations were confirmed through plasmid DNA sequencing (Bionia).

[0260] The sequenced mutant enzyme expression plasmid was injected into the E. coli BL21 strain via heat shock, and colonies were obtained by plating on solid LB medium containing 50 μg / ml of kanamycin. Subsequently, pre-culture was performed for 16 hours at 37°C with stirring at 900 RPM. The pre-culture solution was inoculated into 0.7 mL of LB kanamycin autoinduction medium (GRiSP) at 2 (v / v)%, and main culture was performed for 24 hours at 25°C with stirring at 700 RPM.

[0261] Subsequently, the main culture medium was centrifuged (4000 RPM, 5 min) and the supernatant was discarded. The cell pellet was then resuspended in 0.35 mL of lysozyme solution (1 g / L in 0.1 M sodium phosphate buffer pH 8.0) to prepare the enzyme solution. 0.1 mL of the enzyme solution was added to 0.4 mL of reaction premix (Rebaudioside D 0.0625%, UDP-glucose 18.75 mM in 0.1 M sodium phosphate buffer pH 8.0) and reacted at 40°C at 800 RPM for 4 to 8 hours, after which the solution was diluted 50-fold. Subsequently, the RebM conversion activity was compared by measuring the concentration of UDP generated in the diluted reaction solution using the UDP-glo™ (Promega) assay kit. Relative activity was measured with the activity of the UGT-A wild-type enzyme set to 1.

[0262] Measurement results confirmed that the conversion activity of the enzyme with each single variant was increased compared to the wild-type enzyme (Fig. 1).

[0263]

[0264] Example 2. Screening and activity measurement of mutant UGT-A enzyme with enhanced ribaudioside M conversion activity

[0265] Experiments were conducted to screen mutant enzymes with enhanced ribaudioside M conversion activity among mutant enzymes containing two or more diverse amino acid substitutions, rather than a single mutation. Specifically, mutant enzymes with enhanced ribaudioside M conversion activity were screened among mutant enzymes containing one or more substitutions of V20L, A86S, A86N, M88I, M88W, L126F, L126W, L200A, and L379M. First, the modified UGT-A enzyme library designed in Example 1-1 above was synthesized in the form of DNA fragments (TWIST Bioscience). The synthesized DNA fragments were cloned into the pET28a vector using the Golden Gate assembly method. Specifically, 0.5 WU of T4 DNA ligase (NEB, M0202T), HF BsaI restriction enzyme (NEB, R3733L), and T4 DNA ligase buffer were added to a vector and fragment in a 1:2 molar ratio, and the reaction was carried out sequentially at 37°C for 1 hour, 50°C for 20 minutes, and 60°C for 1 minute. Subsequently, the cloned UGT-A library was introduced into the E. coli DH5α strain via the heatshock method to induce transformation. The colony pool of the transformed strain was mixed and cultured in LB medium containing 50 μg / ml kanamycin; library diversity was confirmed by extracting and sequencing the plasmid (Bionia).

[0266] Subsequently, the UGT-A library plasmid prepared as described above was injected into the E. coli BL21 strain via a heat-shock method using the same method as in Examples 1-2 to obtain approximately 4,000 colonies. The obtained colonies were cultured using the same method as in Examples 1-2, and the rebaudioside M conversion activity was compared by measuring the concentration of UDP generated in the diluted reaction solution using the UDP-glo™ (Promega) assay kit. Relative activity was measured with the activity of the wild-type UGT-A enzyme set to 1, and mutants with improved activity were selected.

[0267] Each strain selected as described above was inoculated into 50 mL of LB kanamycin autoinduction medium and cultured at 37°C for 24 hours to collect cell pellets. The collected pellets were resuspended in 10 mL of lysozyme solution, and 20 (v / v)% of the suspension was added to a solution (0.1 M sodium phosphate, pH 8.0) containing 1 mg / mL of ribaudioside D (RebD) and 10 mM UDP-glucose. The mixture was then reacted at 40°C for 2 to 4 hours. The reaction solution was heat-treated at 100°C for 10 minutes, diluted, and filtered to measure the conversion rate of ribaudioside M (RebM) in the reaction solution using HPLC. The HPLC analysis conditions were as follows:

[0268] - Detector wavelength: 210nm

[0269] - Flow rate: 1ml / min

[0270] - Sample injection vol.: 20㎕

[0271] - Column: Capcell pak C18 MG II (Shiseido, 250 x 4.6 mm, particle size: 5μm)

[0272] - Solvent: Acetonitrile 30%.

[0273] The conversion rate from RebD to RebM measured as described above was compared with the UGT-A wild-type enzyme, and a total of three enzymes with the highest activity were finally selected. In addition, the conversion rate from stevioside to RebA was measured using 10 mg / ml of stevioside instead of RebD under the same conditions as above for the suspensions of each selected enzyme.

[0274] The level of change in activity for each mutation is shown in Table 1 below when the activity of the wild-type UGT-A enzyme is set to 1. In Table 1 below, "RebD → RebM" refers to the level of change in activity with respect to the conversion rate from RebD to RebM, and "stevioside → RebA" refers to the level of change in activity with respect to the conversion rate from stevioside to RebA.

[0275] Mutant amino acid substitution RebD → RebM stevioside → RebACM42(SEQ No. 7)V20L-A86S-M88W-L126F4.61.2CM48(SEQ No. 8)V20L-A86N-M88I-L126W7.80.2M10(SEQ No. 9)V20L-A86S-M88W-L126F-L200A-L379M9.010.19

[0276] As shown in Table 1 above, it was confirmed that the CM48 and M10 mutant enzymes showed increased substrate specificity for RebD, resulting in a significantly higher conversion rate from RebD to RebM—more than 8 times higher than the wild type. The conversion rate from stevioside to RebA was reduced to 0.2 times or less, making them advantageous for the selective conversion reaction from RebD to RebM. Meanwhile, the CM42 mutant enzyme was found to have a conversion rate from RebD to RebM that increased by about 5 times compared to the wild type, while the conversion rate from stevioside to RebA was also 1.2 times higher. Therefore, when reacted with the UGT-B enzyme, it can be effectively utilized in the process of producing ribaudioside M from stevioside.

[0277] Through the above experiment, it was confirmed that the UGT-A enzyme of the present application, having multiple mutations simultaneously, has a significantly superior conversion rate from RebD to RebM compared to the wild-type enzyme, and can be effectively used for the production of ribaudioside M.

[0278]

[0279] Example 3. Measurement of Ribaudioside I production activity of mutant UGT-A enzyme

[0280] The UGT-A enzyme can use ribaudioside A as a substrate, in addition to stevioside and ribaudioside D, in which case it can produce ribaudioside I (RebI) by attaching a glucose molecule to the glucose connected to R1 of ribaudioside A (RebA) via a β1-3 bond (Fig. 2).

[0281] It is known that the conversion rate of wild-type UGT-A enzymes from RebA to RebI is very low, so an experiment was performed to confirm whether the complex mutant UGT-A enzymes prepared in Example 2 above could effectively produce RebI using RebA as a substrate.

[0282] First, E. coli BL21 strains into which the UGT76G1 (UGT-A) or CM42 enzyme was inserted in the form of a pET-28a vector were each inoculated into 50 mL of LB kanamycin autoinduction medium and cultured at 37°C for 24 hours to collect cell pellets. The collected pellets were added at a concentration of 20 mg / mL to a solution (0.1 M sodium phosphate pH 8.0) containing 10 mg / mL of RebA, 10 mM UDP-glucose, and 1 mg / mL of lysozyme, and reacted sufficiently at 40°C for 24 hours. The reaction mixture was heat-treated at 100°C for 10 minutes, diluted and filtered, and the RebA conversion rate in the reaction mixture was measured by HPLC. The HPLC analysis conditions were the same as those in Example 2 above.

[0283] As a result, it was confirmed that the conversion rate of ribaudioside I by the CM42 mutant enzyme was reduced to less than 0.2 times compared to UGT76G1 after a 2-hour reaction (Fig. 3).

[0284] From the above results, in the case of the CM42 mutant enzyme, the conversion rate of ribaudioside A from stevioside and the conversion rate of ribaudioside M from ribaudioside D increased, but the activity of producing ribaudioside I from ribaudioside A decreased, so it may be used more advantageously than other mutant enzymes when synthesizing ribaudioside M from stevioside or ribaudioside A through a complex reaction with the UGT-B enzyme, etc.

[0285]

[0286] Example 4. Production of Ribaudioside M using Mutant UGT-A Enzyme

[0287] Experiments were conducted to evaluate the actual production level of ribaudioside M by performing a complex reaction to produce RebM from stevioside using the mutant enzymes prepared in the above experiments, UGT-B MM9 enzyme (SEQ No. 11), sucrose synthase (RSS, SEQ No. 12), and UMPK (uridine monophosphate kinase, SEQ No. 13).

[0288] The strain used was Corynebacterium staionis. The wild-type UGT-A enzyme or the mutant UGT-A enzyme gene of the present application was introduced into the strain, and the UGT-B enzyme (SEQ No. 11), RSS (SEQ No. 12), and UMPK (SEQ No. 13) genes were introduced. The mutant UGT-A enzyme used was the CM42 complex mutant UGT-A enzyme (CM42(V20L-A86S-M88W-L126F)), and the same genes were introduced for the RSS and UMPK genes. The composition of the UGT-A enzyme and UGT-B enzyme introduced into each strain is as shown in Table 2 below.

[0289] Strain name UGT-AUGT-BC Wild type UGT-AUGT-B MM9DCM42(V20L-A86S-M88W-L126F)UGT-B MM9

[0290] The C and D Corynebacterium stationaryis strains were pre-cultured by inoculating them into 3 ml of Corynebacterium genus microbial medium (glucose 80 g / L, soytone 20 g / L, (NH4)2SO4 10 g / L, KH2PO 41.2 g / L, MgSO4 1.4 g / L) containing 50 μg / ml kanamycin. Pre-culture was performed using a shaking incubator at 30°C at 200 rpm for 24 hours, and subsequently, 10% (v / v) of the total volume of the pre-culture medium was inoculated into 50 ml of new Corynebacterium medium (containing 50 μg / ml kanamycin) (glucose 80 g / L, soytone 20 g / L, (NH4)2SO4 10 g / L, KH2PO4 1.2 g / L, MgSO4 1.4 g / L) and cultured for 24 hours under the same conditions. After centrifuging the culture medium and removing the supernatant, the reaction was carried out using the bacterial cells. A total of 1 ml of reaction solution containing 20% ​​(w / v) bacterial cells, 10 mM UDP (Uridine-5'-diphosphate-glucose; Carbosynth), 1% (w / v) stevioside (Daepyeong), 30% (w / w) sucrose, and 100 mM sodium phosphate (pH 8.0) was reacted at 40°C for a total of 40 hours. Samples were extracted at reaction times of 16, 24, and 40 hours, diluted with triple-distilled water at a 1:10 ratio, inactivated with enzymes at 100°C for 5 minutes, and pretreated using a filter. The pretreated reaction solution was analyzed using HPLC (Agilent). The HPLC analysis conditions were the same as those in Example 2 above.

[0291] As a result of the analysis, it was measured that the conversion rate of ribaudioside M was higher in strain D, into which the CM42 gene was introduced, compared to strain C, into which the wild-type UGT-A enzyme gene was introduced, and in particular, the conversion rate of ribaudioside M was found to be significantly higher at about 96% after a 40-hour reaction.

[0292] Through the above experiments, the mutant enzymes of the present application exhibit superior rebaudioside M conversion activity compared to wild-type enzymes, and can be effectively utilized for the production of rebaudioside M.

[0293]

[0294] Although representative embodiments of the present application have been described above by way of example, the scope of the present application is not limited to such specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of the present application.

Claims

1. A variant polypeptide having glycosyltransfer activity, wherein the variant polypeptide is a variant polypeptide in which an amino acid corresponding to one or more positions selected from 20, 88, and 126 of SEQ ID NO. 1 is substituted with another amino acid.

2. In Claim 1, The above variant polypeptide is a variant polypeptide in which an amino acid corresponding to one or more positions selected from 86, 200, and 379 of SEQ ID NO. 1 is further substituted with another amino acid.

3. In Claim 1, The above variant polypeptide comprises one or more amino acid substitutions selected from the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) The amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) the amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with isoleucine or tryptophan; and iii) The amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine or tryptophan.

4. In Claim 2, The above variant polypeptide comprises one or more amino acid substitutions selected from the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) The amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) The amino acid corresponding to position 86 of SEQ ID NO. 1 is substituted with serine or asparagine; iii) The amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with isoleucine or tryptophan; iv) The amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine or tryptophan; v) Substitution of the amino acid corresponding to position 200 of SEQ ID NO. 1 with Alanine; and vi) The amino acid corresponding to position 379 of sequence number 1 is substituted with methionine.

5. In Claim 1, The above variant polypeptide comprises any one amino acid substitution selected from the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) The amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) the amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with isoleucine or tryptophan; and iii) The amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine or tryptophan.

6. In Claim 2, The above variant polypeptide comprises the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) The amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) The amino acid corresponding to position 86 of sequence number 1 is substituted with serine; iii) The amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with tryptophan; and iv) The amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine.

7. In Claim 2, The above variant polypeptide comprises the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) The amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) The amino acid corresponding to position 86 of SEQ ID NO. 1 is substituted with asparagine; iii) The amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with isoleucine; and iv) The amino acid corresponding to position 126 of sequence number 1 is substituted with tryptophan.

8. In Claim 2, The above variant polypeptide comprises the following amino acid substitutions in the amino acid sequence of SEQ ID NO. 1: i) The amino acid corresponding to position 20 of SEQ ID NO. 1 is substituted with leucine; ii) The amino acid corresponding to position 86 of sequence number 1 is substituted with serine; iii) The amino acid corresponding to position 88 of SEQ ID NO. 1 is substituted with tryptophan; iv) The amino acid corresponding to position 126 of SEQ ID NO. 1 is substituted with phenylalanine; v) Substitution of the amino acid corresponding to position 200 of SEQ ID NO. 1 with Alanine; and vi) The amino acid corresponding to position 379 of sequence number 1 is substituted with methionine.

9. In Claim 1, The above variant polypeptide comprises any one amino acid sequence selected from SEQ ID NOs 2 to 6.

10. In Claim 2, The above variant polypeptide comprises any one amino acid sequence selected from SEQ ID NOs 7 to 9.

11. A polynucleotide encoding a variant polypeptide of any one of claims 1 to 10.

12. An expression vector comprising a polynucleotide encoding a variant polypeptide of any one of claims 1 to 10.

13. A transformant comprising a polynucleotide encoding a variant polypeptide of any one of claims 1 to 10.

14. In Claim 13, The above transformant is a transformant further comprising a polynucleotide encoding glycosyltransferase B, sucrose synthase, and UMPK.

15. In the presence of one or more of the variant polypeptide of any one of claims 1 to 10 and a transformant expressing said variant polypeptide, A method for preparing ribaudioside M, comprising the step of reacting ribaudioside D with a glucose-conjugated nucleotide diphosphate.

16. In the presence of one or more of the variant polypeptide of any one of claims 1 to 10 and a transformant expressing said variant polypeptide, Ribaudioside A; Glucose-bound nucleotide diphosphate; and A method for producing ribaudioside M, comprising the step of reacting at least one of glycosyltransferase B and a transformant expressing glycosyltransferase B.

17. In the presence of one or more of the variant polypeptide of any one of claims 1 to 10 and a transformant expressing said variant polypeptide, Stevioside; Glucose-bound nucleotide diphosphate; and A method for producing ribaudioside M, comprising the step of reacting at least one of glycosyltransferase B and a transformant expressing glycosyltransferase B.

18. In the presence of one or more of the variant polypeptide of any one of claims 1 to 10 and a transformant expressing said variant polypeptide, A method for preparing ribaudioside A, comprising the step of reacting a nucleotide diphosphate combined with stevioside and glucose.

19. In the presence of one or more of the variant polypeptide of any one of claims 1 to 10 and a transformant expressing said variant polypeptide, Stevioside; Glucose-bound nucleotide diphosphate; and A method for producing ribaudioside D, comprising the step of reacting at least one of glycosyltransferase B and a transformant expressing said glycosyltransferase B.

20. A composition for preparing one or more steviol glycosides selected from the group consisting of ribaudioside M, ribaudioside A, and ribaudioside D, comprising one or more of the variant polypeptide of any one of claims 1 to 10 and a transformant expressing said variant polypeptide.

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