Novel mutant glycosyltransferase polypeptides and methods of producing steviol glycosides using the same

Abstract

The present application relates to: a novel variant polypeptide; a polynucleotide encoding the variant polypeptide; a vector comprising the polynucleotide; a microorganism comprising at least one of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide; a method of producing a steviol glycoside using the variant polypeptide or microorganism; a composition for use in the production of at least one of rebaudioside D and rebaudioside M using the same; and use of the microorganism in the production of at least one of rebaudioside D and rebaudioside M.

Description

Technical Field

[0001] This disclosure relates to a novel variant polypeptide; a polynucleotide encoding the variant polypeptide; a carrier containing the polynucleotide; a microorganism containing any one or more of the variant polypeptide, the polynucleotide encoding the variant polypeptide, and the carrier containing the polynucleotide; a method for producing steviol glycosides, the method comprising the step of transferring glucose to lebaudine A by reacting lebaudine A with glucose linked to a nucleotide diphosphate in the presence of the variant polypeptide or the microorganism; a composition for use in the production of any one or more of lebaudine D and lebaudine M; and the use of the microorganism in the production of any one or more of lebaudine D and lebaudine M. Background Technology

[0002] Because the World Health Organization (WHO) has recommended reducing daily sugar intake due to concerns about diseases caused by sugar consumption (obesity), various policies aimed at reducing sugar intake are being actively discussed under the leadership of governments, primarily in developed countries. Consequently, the market demand for various sugar substitutes and high-fructose sweeteners is increasing, and these alternative sweeteners are being continuously developed and commercialized.

[0003] From this perspective, consumer demand for natural sweeteners has been steadily increasing due to concerns about the safety of synthetic sweeteners. However, due to the limitations of natural sweeteners' taste characteristics, such as their distinctive unpleasant flavors and odors, they cannot completely replace existing low-calorie and zero-calorie products based on synthetic sweeteners.

[0004] In this regard, stevia sweeteners extracted from stevia leaves have received considerable attention in recent years (US Patent Publication No. 2023-0270146). Among the sweet components of stevia or its extracts, rebaudiosides D and M have low bitterness and excellent sweetness, making them valuable as alternative sweeteners. However, they are present in very small amounts in stevia leaves, thus presenting a drawback: the methods for extracting and purifying them from the leaves are costly.

[0005] Therefore, there is a growing need for research into new enzymes required for the large-scale production of rebaudioside D and M, as well as the preparation of rebaudioside D and M using said new enzymes. Summary of the Invention

[0006] [Technical Issues] The problem to be solved in this disclosure is to provide a variant polypeptide having glycosyltransferase activity; a polynucleotide encoding the variant polypeptide; a carrier containing the polynucleotide; a microorganism containing any one or more of the polypeptide, the polynucleotide, and the carrier; a method for producing any one or more of rebaudioside D and rebaudioside M, the method comprising the step of culturing the microorganism in a culture medium; a method for producing steviol glycosides, the method comprising the step of transferring glucose to rebaudioside A by reacting rebaudioside A with glucose linked to a nucleotide diphosphate in the presence of the variant polypeptide and / or the microorganism; a composition for use in the production of any one or more of rebaudioside D and rebaudioside M; and the use of the microorganism in the production of any one or more of rebaudioside D and rebaudioside M.

[0007] [Technical Solution] One object of this disclosure is to provide a variant polypeptide having glycosyltransferase activity, said variant polypeptide comprising any one or more of the following substitutions: the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by tryptophan; the amino acid at position 208 is replaced by alanine or methionine; the amino acid at position 380 is replaced by histidine, threonine, alanine or glycine; and the amino acid at position 381 is replaced by phenylalanine, serine, alanine or asparagine.

[0008] Another object of this disclosure is to provide a polynucleotide encoding a variant polypeptide of this disclosure.

[0009] Another object of this disclosure is to provide a vector comprising the said polynucleotide.

[0010] Another object of this disclosure is to provide a microorganism comprising any one or more of the variant polypeptides of this disclosure, the polynucleotide encoding the variant polypeptides, and the vector comprising the polynucleotides.

[0011] Another object of this disclosure is to provide a method for producing steviol glycosides, the method comprising the step of transferring glucose to rebaudioside A by reacting rebaudioside A with glucose linked to a nucleotide diphosphate in the presence of the variant polypeptide and / or microorganisms, wherein the steviol glycoside is any one or more of rebaudioside D and rebaudioside M.

[0012] Another object of this disclosure is to provide a composition for producing any one or more of rebaudioside D and rebaudioside M, said composition comprising any one or more of the following: a variant polypeptide of the present disclosure; a polynucleotide encoding said variant polypeptide; a microorganism comprising any one or more of said variant polypeptide, the polynucleotide encoding said variant polypeptide, and a carrier comprising said polynucleotide; and a culture of said microorganism.

[0013] Another object of this disclosure is to provide the use of the said microorganisms in the production of rebaudioside D and rebaudioside M.

[0014] Another object of this disclosure is to provide the use of the composition in the production of rebaudioside D and rebaudioside M.

[0015] [Beneficial Effects] When using the variant peptides of this disclosure or microorganisms containing them, rebaudioside D and / or M can be produced in high yields compared to unmodified peptides or microorganisms. Attached Figure Description

[0016] Figure 1 The results show the comparative RebD synthesis capabilities of 10 monovariates of the modified enzymes of this disclosure; Figure 2 The results show the comparative RebD synthesis capabilities of 16 bivariates of the modified enzyme of this disclosure; and Figure 3 The results show the results of comparing RebD synthesis capabilities using 10 complex variants (double mutations or more) of the modified enzymes of this disclosure. Detailed Implementation

[0017] This disclosure will be described in detail below. Furthermore, each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter covered by this disclosure.

[0018] One aspect of this disclosure provides a variant polypeptide having glycosyltransferase activity, wherein any one or more amino acids at positions 92, 208, 380, and 381 of the amino acid sequence corresponding to SEQ ID NO: 1 are replaced by additional amino acids.

[0019] In one embodiment, the amino acid sequence and / or variant polypeptide of SEQ ID NO: 1 of this disclosure may be a glycosyltransferase, specifically, UDP-glycosyltransferase (uridine diphosphate glycosyltransferase; UGT), and more specifically, UDP-glycosyltransferase B (UGT-B), but not limited thereto.

[0020] As used herein, the term "glycosyltransferase" refers to an enzyme having the activity of transferring a monosaccharide moiety from a glycosyl donor to a glycosyl acceptor molecule, and UDP-glycosyltransferase refers to an enzyme that utilizes glucose linked to a nucleotide diphosphate (e.g., UDP-glucose) as a glycosyl donor. In this disclosure, glycosyltransferase may be used interchangeably with "UDP-glycosyltransferase" and "UGT".

[0021] As used herein, the term "UDP-glycosyltransferase B" can refer to an enzyme capable of converting rebaudioside A (RebA) to rebaudioside D (RebD) using glucose linked to a nucleotide diphosphate as a glycosyl donor. In the following, and in this disclosure, UDP-glycosyltransferase B may be interchangeably referred to as glycosyltransferase B and UGT-B.

[0022] As used herein, the term "UDP-glycosyltransferase A" can refer to an enzyme capable of converting steviol glycosides to rebaudioside A and / or rebaudioside D to rebaudioside M (RebM) using glucose linked to a nucleotide diphosphate as a glycosyl donor. In the following, and in this disclosure, UDP-glycosyltransferase A may be referred to interchangeably as glycosyltransferase A and UGT-A.

[0023] Regarding the variant polypeptides of this disclosure, any one or more amino acids at positions 92, 208, 380, and 381 of the amino acid sequence corresponding to SEQ ID NO: 1, which is the parental sequence, may be replaced by other amino acids.

[0024] As used herein, a parental sequence refers to a reference sequence into which modifications for preparing the variant polypeptide are introduced. In other words, a parental sequence can be a target into which the substitution mutations of this disclosure are introduced. The parental sequence can be naturally occurring or wild-type, or a variant of a natural or wild-type sequence in which one or more substitutions, insertions, or deletions have occurred, or it can be a synthetically produced sequence.

[0025] A polypeptide containing the parental sequence of this disclosure may contain the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70% or higher homology or identity with it. Furthermore, even if a portion of the amino acid sequence of SEQ ID NO: 1 is deleted or substituted, or another sequence is added to the amino acid sequence of SEQ ID NO: 1, it may be included in a polypeptide containing the parental sequence as long as it is a protein exhibiting a function corresponding to glycosyltransferase activity. Specifically, a polypeptide containing the parental sequence may have or contain the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with it, or may be composed of, or substantially composed of, that amino acid sequence. For example, the parental sequence of this disclosure may be a polypeptide having or containing the amino acid sequence of SEQ ID NO: 1, or composed of, or substantially composed of, that amino acid sequence, but is not limited thereto.

[0026] For example, SEQ ID NO: 1 of this disclosure may be SEQ ID NO: 1 of US Patent No. 11,414,690, which is incorporated herein by reference.

[0027] Furthermore, the sequence of the polynucleotide encoding the polypeptide containing the parental sequence can be obtained, for example, based on codon information known in the art, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70% or higher homology or identity with it. For example, the polypeptide containing the parental sequence may be encoded by a polynucleotide having or containing a nucleotide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity with the sequence of SEQ ID NO: 2 and / or SEQ ID NO: 127, or being composed of or substantially composed of such nucleotide sequence, but is not limited thereto.

[0028] In this disclosure, a polynucleotide (or gene) containing the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 127 may be used interchangeably with a polynucleotide (or gene) having the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 127 or composed of the nucleotide sequence of SEQ ID NO: 2 and / or SEQ ID NO: 127.

[0029] Meanwhile, in this disclosure, the statement "an amino acid corresponding to a specific position in a specific amino acid sequence is replaced by another amino acid" can be interpreted as meaning that the amino acid sequence is used as a reference sequence.

[0030] As used herein, the term "reference sequence" refers to a sequence used as a reference to specify the Nth position in a particular amino acid sequence. For example, when a particular amino acid sequence and a reference sequence are aligned using sequence alignments known in the art, and based on this, when each amino acid residue in the particular amino acid sequence is numbered with reference to the numerical position of the amino acid residues in the reference sequence, the position of the amino acid corresponding to the Nth position in the reference sequence can be determined in that particular amino acid sequence.

[0031] As used herein, the term "position N" in an amino acid sequence may include, in addition to the Nth position, the amino acid position corresponding to the Nth position. Specifically, the Nth position may include the amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a particular amino acid sequence (e.g., a reference sequence).

[0032] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar to, identical to, or homologous to an amino acid residue listed in the polypeptide. Identifying an amino acid at a corresponding position can be done by identifying a specific amino acid within a defined sequence (referring to a specific sequence). As used herein, "corresponding region" typically refers to a similar or corresponding position in the amino acid sequence of a related or reference protein.

[0033] In this disclosure, the amino acid sequence of SEQ ID NO: 1 can be used as a reference sequence to determine the position of amino acids in any amino acid sequence.

[0034] In other words, the amino acid sequence of SEQ ID NO: 1 disclosed herein can be used to identify the corresponding amino acid residues in any polypeptide. Unless otherwise stated in this disclosure, the residues of a particular amino acid sequence are based on the amino acid sequence number of SEQ ID NO: 1.

[0035] For example, any amino acid sequence can be aligned with the amino acid sequence of SEQ ID NO: 1, and based on the alignment, each amino acid residue in the arbitrary amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm described herein can identify the position of an amino acid or the position where modifications such as substitutions, insertions, deletions, etc., have occurred compared to the query sequence (also known as the "reference sequence").

[0036] In such alignments, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) or the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16: 276-277) can be used, but are not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc., known in the art can be used as appropriate.

[0037] In addition, the corresponding amino acid residues in another polypeptide can be identified by multiple sequence alignment. Examples of known multiple sequence alignment methods in this field include MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: ). The procedures include those using ClustalW's EMBOSS EMMA (version 1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), and can use their respective default parameters, but are not limited thereto.

[0038] Furthermore, when peptides differentiated from the mature peptide of SEQ ID NO: 1 cannot be correlated using traditional sequence-based comparisons, alternative pairwise sequence comparison algorithms can be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity in sequence-based searches can be achieved by using search procedures that utilize probabilistic representations of peptide families (spectrums) to search a database. For example, the PSI-BLAST procedure generates spectra through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even better sensitivity can be achieved if the peptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, J.Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from various sources, such as PSIBLAST, secondary structure prediction, structural alignment spectra, and solvation potential, as input to neural networks predicting the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of these models can be evaluated using various tools developed for this purpose.

[0039] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extensions (CE) (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). Implementations of these algorithms can also be used to query structure databases containing target structures to discover potential structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0040] The above methods are illustrative and not limited thereto.

[0041] For example, the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 of this disclosure can be any amino acid at positions 90 to 100 of the amino acid sequence of the homolog of SEQ ID NO: 1. In this case, a variant polypeptide in which the tyrosine amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by tryptophan can be a variant polypeptide in which the tyrosine amino acid at any one of positions 90 to 100 of the amino acid sequence corresponding to the homolog of SEQ ID NO: 1 is replaced by tryptophan. As another example, the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 of this disclosure can be any amino acid at positions 360 to 381 of the amino acid sequence of the homolog of SEQ ID NO: 1, and the amino acid at position 381 of the amino acid sequence corresponding to SEQ ID NO: 1 of this disclosure can be any amino acid at positions 361 to 382 of the amino acid sequence of the homolog of SEQ ID NO: 1, but is not limited thereto.

[0042] In this disclosure, regarding amino acid sequences, it is apparent that a polypeptide or protein "comprising," "composed of," or "having" an amino acid sequence represented by a specific sequence number may also include polypeptides or proteins in which some of these amino acids are deleted, modified, substituted, or added, provided that it has the same or corresponding activity as the polypeptide or protein with the corresponding sequence number. For example, the polypeptide or protein may also include polypeptides or proteins in which amino acid additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions that do not alter protein function are located internally, upstream, or downstream (N-terminus or C-terminus) of the polypeptide or protein, provided that it has the same or corresponding activity.

[0043] In addition, for example, peptides or proteins conjugated with an N-terminal signal (or leader) sequence that is co-translated or post-translational involved in protein (peptide) translocation, or peptides or proteins conjugated with other sequences or linkers used for the identification, purification or synthesis of peptides or proteins, may also be included in the scope of peptides or proteins with an amino acid sequence represented by a specific sequence number.

[0044] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions often occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues. Generally, conservative substitutions have minimal impact on the activity of peptides or proteins.

[0045] As used herein, the term "variant" or "variant polypeptide" refers to a polypeptide that has a sequence different from that of its parent polypeptide due to conserved substitutions and / or modifications (such as substitution, addition, or deletion) in one or more amino acids of the parent sequence. The function or properties of the variant polypeptide may be increased, unchanged, or decreased compared to the function or properties of the native polypeptide. For example, some variant polypeptides may include variant polypeptides in which one or more amino acids corresponding to positions 92, 208, 380, and 381 of the amino acid sequence of SEQ ID NO: 1 have been substituted with additional amino acids, or one or more regions such as the N-terminal leader sequence or transmembrane domain have been removed. Furthermore, for example, other variant polypeptides may include variants in which a portion of the N- and / or C-terminus has been additionally removed from the mature protein. The term "variant polypeptide" may be used interchangeably with terms such as modification, modified protein, mutant, mutated protein, divergent, variant, etc., and is not limited thereto, as long as it is a term with the meaning of variation.

[0046] Furthermore, the variant peptide may contain the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the peptide. For example, the peptide may be conjugated to an N-terminal signal (or leader) sequence of a peptide that is co-translated or post-translational involved in protein translocation. Additionally, the peptide may be conjugated to other sequences or linkers for peptide identification, purification, or synthesis.

[0047] The variant polypeptides disclosed herein may be, but are not limited to, isolated forms.

[0048] As used herein, the term "isolated" refers to a substance present in the environment that is not naturally occurring or exists in a non-natural form. This includes substantially isolating a substance (sequence or nucleic acid) from a naturally related substance found in nature, such as at least one other component having a sequence or nucleic acid.

[0049] In this disclosure, the variant polypeptide may be, but is not limited to, a non-naturally occurring polypeptide, i.e., a polypeptide that does not exist in nature.

[0050] The variant polypeptides disclosed herein may include bioactive fragments of the variant polypeptides.

[0051] In this disclosure, the term "bioactive fragment or segment" can refer to a "functional fragment" in relation to an amino acid or base (nucleic acid) sequence. A "functional fragment" can also refer to an active fragment, and specifically to a polypeptide containing fewer amino acids than a full-length protein but possessing at least one biological activity corresponding to the full-length protein. For example, a functional fragment of an enzyme can contain the enzyme's catalytic site.

[0052] As used herein, the term "another amino acid" is not limited, as long as it is an amino acid different from the amino acid before substitution.

[0053] The variant polypeptides disclosed herein may have one or more amino acids at positions 92, 208, 380, and 381 of the amino acid sequence corresponding to SEQ ID NO: 1 replaced by other amino acids.

[0054] The variant polypeptides of this disclosure may contain, within an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or higher homology or identity with the amino acid sequence represented by SEQ ID NO: 1, any one or more amino acids corresponding to the amino acid sequence at positions 92, 208, 380, and 381 from the N-terminus of SEQ ID NO: 1 being replaced by additional amino acids. It is also apparent that any variant with partially deleted, substituted, conserved substituted, or added amino acid sequences may fall within the scope of this disclosure, provided that the amino acid sequence contains amino acid substitutions, possesses the aforementioned homology or identity, and exhibits the efficacy corresponding to the variants of this disclosure.

[0055] In this disclosure, when it is stated that "the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid" and / or "the amino acid at position 208 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid", it can be replaced by an amino acid other than tyrosine. When it is stated that "the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid", it can be replaced by an amino acid other than phenylalanine. And when it is stated that "the amino acid at position 381 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid", it can be replaced by an amino acid other than glycine. Furthermore, it can be replaced by any one of alanine, glutamic acid, phenylalanine, glycine, arginine, aspartic acid, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, tryptophan, valine, methionine, threonine, or leucine, but is not limited thereto.

[0056] In one embodiment, the variant polypeptide of this disclosure may comprise any one or more of the following substitutions: the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is substituted with tryptophan; the amino acid at position 208 is substituted with alanine or methionine; the amino acid at position 380 is substituted with histidine, threonine, alanine or glycine; and the amino acid at position 381 is substituted with phenylalanine, serine, alanine or asparagine.

[0057] For example, a variant polypeptide comprising the substitution of tryptophan for the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 of this disclosure may comprise an amino acid sequence having at least 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher; and less than 100% homology or identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid at position 92 of the amino acid sequence shown in SEQ ID NO: 1 is fixed with tryptophan. Furthermore, it is apparent that variant polypeptides having amino acid sequences containing deletions, modifications, substitutions, conservative substitutions, or additions of some sequences may also fall within the scope of this disclosure, provided that the amino acid sequence has the same efficacy as the variant polypeptide of this disclosure and also possesses such homology or identity.

[0058] The variant polypeptides disclosed herein may contain one to four amino acids at positions 92, 208, 380, and 381 of the amino acid sequence corresponding to SEQ ID NO: 1, specifically any one or more, two or more, three or more, or four amino acids may be replaced by additional amino acids.

[0059] The variant polypeptides disclosed herein may include any variant polypeptide without limitation, provided that it contains any one or more amino acid substitutions at positions 92, 208, 380, and 381 of the amino acid sequence corresponding to SEQ ID NO: 1 as described above.

[0060] In one embodiment, the variant polypeptide may also include, in addition to comprising any one or more of the following: the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by tryptophan; the amino acid at position 208 being replaced by alanine or methionine; and the amino acid at position 381 being replaced by phenylalanine, serine, alanine, or asparagine, the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by glycine.

[0061] In one embodiment, the variant polypeptide may comprise the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by tryptophan; the amino acid at position 208 being replaced by alanine or methionine; the amino acid at position 380 being replaced by histidine, threonine, or alanine; the amino acid at position 381 being replaced by phenylalanine, serine, alanine, or asparagine; or the amino acid at position 380 being replaced by histidine, threonine, or glycine, and the amino acid at position 381 being replaced by phenylalanine, serine, alanine, or asparagine.

[0062] In one embodiment, the variant polypeptide, in addition to comprising the substitution of amino acid position 381 corresponding to SEQ ID NO: 1 with phenylalanine, serine, alanine, or asparagine, may also comprise the substitution of amino acid position 380 corresponding to SEQ ID NO: 1 with histidine, glycine, threonine, or alanine. Furthermore, the variant polypeptide may also comprise the substitution of amino acid position 92 corresponding to SEQ ID NO: 1 with tryptophan, the substitution of amino acid position 208 with alanine, or combinations thereof, but is not limited thereto. In this regard, in one embodiment, the variant polypeptide may comprise the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by alanine, and the amino acid at position 381 being replaced by alanine; the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by threonine, and the amino acid at position 381 being replaced by phenylalanine; or the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by threonine, and the amino acid at position 381 being replaced by serine, but is not limited thereto.

[0063] In one embodiment, the variant polypeptide may comprise the following: the amino acid corresponding to position 208 is replaced by alanine; the amino acid corresponding to position 380 is replaced by threonine or alanine; and the amino acid corresponding to position 381 is replaced by phenylalanine, serine, or alanine; the amino acid corresponding to position 92 is replaced by tryptophan; the amino acid corresponding to position 380 is replaced by threonine or alanine; and the amino acid corresponding to position 381 is replaced by phenylalanine, serine, or alanine; the amino acid corresponding to position 92 is replaced by tryptophan; and the amino acid corresponding to position 208 is replaced by alanine; or the amino acid corresponding to position 92 is replaced by tryptophan; the amino acid corresponding to position 208 is replaced by alanine; the amino acid corresponding to position 380 is replaced by threonine or alanine; and the amino acid corresponding to position 381 is replaced by phenylalanine, serine, or alanine.

[0064] In one embodiment of the above embodiments, the variant polypeptide may comprise the following: the amino acid at position 380 of SEQ ID NO: 1 is replaced by histidine, and the amino acid at position 381 is replaced by phenylalanine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by histidine, and the amino acid at position 381 is replaced by asparagine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by histidine, and the amino acid at position 381 is replaced by serine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by histidine, and the amino acid at position 381 is replaced by alanine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by glycine, and the amino acid at position 381 is replaced by phenylalanine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by glycine, and the amino acid at position 381 is replaced by asparagine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by glycine, and the amino acid at position 381 is replaced by asparagine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by glycine, and the amino acid at position 381 is replaced by asparagine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by asparagine; the amino acid at position 381 of SEQ ID NO: ... The amino acid at position 380 of SEQ ID NO: 1 is replaced by glycine, and the amino acid at position 381 is replaced by serine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by glycine, and the amino acid at position 381 is replaced by alanine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by threonine, and the amino acid at position 381 is replaced by phenylalanine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by threonine, and the amino acid at position 381 is replaced by asparagine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by threonine, and the amino acid at position 381 is replaced by serine; the amino acid at position 380 of SEQ ID NO: 1 is replaced by threonine, and the amino acid at position 381 is replaced by alanine ... The amino acid at position 380 of the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, and the amino acid at position 381 is replaced by phenylalanine; the amino acid at position 380 of the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, and the amino acid at position 381 is replaced by asparagine; the amino acid at position 380 of the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, and the amino acid at position 381 is replaced by serine; the amino acid at position 380 of the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, and the amino acid at position 381 is replaced by alanine.The amino acid at position 208 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by alanine, the amino acid at position 380 is replaced by alanine, and the amino acid at position 381 is replaced by alanine; the amino acid at position 208 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by alanine, the amino acid at position 380 is replaced by threonine, and the amino acid at position 381 is replaced by phenylalanine; the amino acid at position 208 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by alanine, the amino acid at position 380 is replaced by threonine, and the amino acid at position 381 is replaced by serine; the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by tryptophan, the amino acid at position 380 is replaced by alanine, and the amino acid at position 381 is replaced by alanine ... The amino acid sequence of SEQ ID NO: 1 has the following amino acid sequence: amino acid position 92 is replaced by tryptophan, amino acid position 380 is replaced by threonine, and amino acid position 381 is replaced by phenylalanine; the amino acid sequence of SEQ ID NO: 1 has the following amino acid sequence: amino acid position 92 is replaced by tryptophan, amino acid position 380 is replaced by threonine, and amino acid position 381 is replaced by serine; the amino acid sequence of SEQ ID NO: 1 has the following amino acid sequence: amino acid position 92 is replaced by tryptophan, amino acid position 208 is replaced by alanine, amino acid position 380 is replaced by alanine, and amino acid position 381 is replaced by alanine; the amino acid sequence of SEQ ID NO: The amino acid sequence of 1 is modified such that amino acid position 92 is replaced by tryptophan, amino acid position 208 is replaced by alanine, amino acid position 380 is replaced by threonine, and amino acid position 381 is replaced by phenylalanine; or amino acid position 92 is replaced by tryptophan, amino acid position 208 is replaced by alanine, amino acid position 380 is replaced by threonine, and amino acid position 381 is replaced by serine, but not limited to these.

[0065] In one embodiment of the above-described embodiments, the variant polypeptide may be composed of the following: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO: The amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 or SEQ ID NO: 73.

[0066] Specifically, the variant polypeptides of this disclosure may have or contain an amino acid sequence having at least 70%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with the sequence number, or may be composed of, or may be substantially composed of, the amino acid sequence.

[0067] In one embodiment, among the variant peptides of this disclosure, a variant peptide comprising a single substitution of the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 with tryptophan can be SEQ ID NO: 3; a variant peptide comprising a single substitution of the amino acid at position 208 of the amino acid sequence corresponding to SEQ ID NO: 1 with alanine or methionine can be SEQ ID NO: 5 or SEQ ID NO: 7, respectively; a variant peptide comprising a single substitution of the amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 with histidine, threonine, or alanine can be SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, respectively; a variant peptide comprising a single substitution of the amino acid at position 381 of the amino acid sequence corresponding to SEQ ID NO: 1 with phenylalanine, serine, alanine, or asparagine can be SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21, respectively; and a variant peptide comprising a double substitution, wherein the double substitution comprises a single substitution of the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 108 with tryptophan or methionine ... The amino acid sequence of SEQ ID NO: 1 is substituted with histidine at position 380 and with phenylalanine, asparagine, serine, or alanine at position 381, which can be SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29, respectively; a variant polypeptide containing double substitutions, wherein the amino acid sequence of SEQ ID NO: 1 is substituted with glycine at position 380 and with phenylalanine, asparagine, serine, or alanine at position 381, which can be SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, respectively; a variant polypeptide containing double substitutions, wherein the amino acid sequence of SEQ ID NO: 1 is substituted with threonine at position 380 and with phenylalanine, asparagine, serine, or alanine at position 381, which can be SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 29, respectively. NO: 43 or SEQ ID NO: 45; a variant polypeptide comprising a double substitution comprising the substitution of amino acid at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 with alanine, and the substitution of amino acid at position 381 with phenylalanine, asparagine, serine, or alanine, which may be SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, or SEQ ID NO: 53, respectively;A variant polypeptide comprising the substitution of amino acid position 208 (corresponding to SEQ ID NO: 1) with alanine, amino acid position 380 (corresponding to SEQ ID NO: 1) with alanine, and amino acid position 381 (corresponding to SEQ ID NO: 1) with alanine can be SEQ ID NO: 55; a variant polypeptide comprising the substitution of amino acid position 208 (corresponding to SEQ ID NO: 1) with alanine, amino acid position 380 (corresponding to threonine), and amino acid position 381 (corresponding to phenylalanine) with phenylalanine can be SEQ ID NO: 57; a variant polypeptide comprising the substitution of amino acid position 208 (corresponding to SEQ ID NO: 1) with alanine, amino acid position 380 (corresponding to threonine), and amino acid position 381 (corresponding to serine) with serine can be SEQ ID NO: 59; a variant polypeptide comprising the substitution of amino acid position 92 (corresponding to SEQ ID NO: 1) with tryptophan, amino acid position 380 (corresponding to SEQ ID NO: 1) with alanine, and amino acid position 381 (corresponding to SEQ ID NO: 1) with alanine can be SEQ ID NO: 59. 61; A variant polypeptide comprising the amino acid sequence corresponding to SEQ ID NO: 1, wherein the 92nd amino acid is replaced by tryptophan, the 380th amino acid by threonine, and the 381st amino acid by phenylalanine, may be SEQ ID NO: 63; A variant polypeptide comprising the amino acid sequence corresponding to SEQ ID NO: 1, wherein the 92nd amino acid is replaced by tryptophan, the 380th amino acid by threonine, and the 381st amino acid by serine, may be SEQ ID NO: 65; A variant polypeptide comprising the amino acid sequence corresponding to SEQ ID NO: 1, wherein the 92nd amino acid is replaced by tryptophan, and the 208th amino acid by alanine, may be SEQ ID NO: 67; A variant polypeptide comprising the amino acid sequence corresponding to SEQ ID NO: 1, wherein the 92nd amino acid is replaced by tryptophan, and the 208th, 380th, and 381st amino acids by alanine, may be SEQ ID NO: 69 ... The variant polypeptide of SEQ ID NO: 71 can be formed by replacing amino acid 92 with tryptophan, amino acid 208 with alanine, amino acid 380 with threonine, and amino acid 381 with phenylalanine.The variant polypeptide comprising, but not limited to, the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by tryptophan, the amino acid at position 208 being replaced by alanine, the amino acid at position 380 being replaced by threonine, and the amino acid at position 381 being replaced by serine can be SEQ ID NO: 73.

[0068] In one embodiment, the variant polypeptide of this disclosure may comprise any one or more of the following: the amino acid tyrosine at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by tryptophan; the amino acid tyrosine at position 208 is replaced by alanine or methionine; the amino acid phenylalanine at position 380 is replaced by histidine, threonine or alanine; and the amino acid glycine at position 381 is replaced by phenylalanine, serine, alanine or asparagine.

[0069] In one embodiment of the above-described embodiments, the variant polypeptide may further comprise the following: the amino acid phenylalanine at position 380 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by glycine; the amino acid tyrosine at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by tryptophan; the amino acid tyrosine at position 208 is replaced by alanine or methionine; and the amino acid glycine at position 381 is replaced by phenylalanine, serine, alanine or asparagine, but is not limited thereto.

[0070] Another aspect of this disclosure provides a polynucleotide encoding a variant polypeptide of this disclosure.

[0071] As used herein, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or longer that is a polymer of nucleotides, wherein nucleotide monomers are linked together in the long chain by covalent bonds, and more specifically, refers to a polynucleotide fragment encoding the variant polypeptide.

[0072] The polynucleotides disclosed herein may include any polynucleotide sequence without limitation, as long as it is a polynucleotide sequence encoding a variant polypeptide of this disclosure. The polynucleotides may be prepared based on codon information known in the art, but are not limited thereto. For example, the polynucleotide may comprise a nucleotide sequence encoding an amino acid sequence represented by any one or more of the following: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59. SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 73.For example, the polynucleotides of this disclosure may have any one or more of the following sequences, comprise any one or more of the following sequences, consist of any one or more of the following sequences, or consist substantially of any one or more of the following sequences: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 128, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54. SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74.

[0073] Considering codon degeneracy or preferred codons in organisms intended to express variant polypeptides of this disclosure, the polynucleotides of this disclosure may undergo various modifications in the coding region without altering the amino acid sequence of the variant polypeptides of this disclosure. Therefore, it is apparent that, due to codon degeneracy, polynucleotides may also comprise polypeptides to be translated into polypeptides consisting of the amino acid sequence of the variant polypeptides of this disclosure, or polypeptides homologous to or identical with them. For example, the polynucleotides disclosed herein may be the following nucleotide sequences: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 128, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 or SEQ ID NO: 74, or their degenerate sequences. For example, SEQ ID NO: 2 and SEQ ID NO: 44 can target the genus Corynebacterium ( Corynebacterium Codon optimization was performed on microorganisms, and SEQ ID NO: 127 and SEQ ID NO: 128 can target Escherichia coli ( ). Escherichia Codon optimization is performed on microorganisms (such as Escherichia coli).

[0074] In another instance, the polynucleotides disclosed herein may have or contain SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 128, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, any one or more of the sequences of SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74 having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, or may be composed of or substantially composed of such nucleotide sequences, but not limited thereto.

[0075] For example, the polynucleotide of this disclosure may comprise a fixed nucleotide sequence having any one or more of the following fixed sequences: the codon encoding the amino acid corresponding to position 92 of SEQ ID NO: 1 is fixed with one of the codons encoding tryptophan; the codon encoding the amino acid corresponding to position 208 is fixed with one of the codons encoding alanine or methionine; the codon encoding the amino acid corresponding to position 380 is fixed with one of the codons encoding histidine, threonine, or alanine; and the codon encoding the amino acid corresponding to position 381 is fixed with one of the codons encoding phenylalanine, serine, alanine, or asparagine.

[0076] In one embodiment, the polynucleotide sequences encoding SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 73 may be SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 128 (both sequences can encode SEQ ID NO: 43), SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQSEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74, but not limited to these.

[0077] Furthermore, the polynucleotides disclosed herein may comprise probes that can be prepared from known gene sequences; for example, they may comprise any sequence without limitation, as long as it is a sequence capable of hybridizing under stringent conditions with all or part of the complementary sequence of the polynucleotide sequence of this disclosure and encoding a variant polypeptide of this disclosure. The stringent conditions are as described above.

[0078] As used herein, the terms “identity” or “homology” refer to the correlation between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. In this disclosure, “homology” and “identity” are often used interchangeably.

[0079] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms and can be used in conjunction with default gap penalties established by the program used.

[0080] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms, such as the “FASTA” procedure with default parameters as described in, for example, Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used, such as the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) or by using GAP computer programs, such as the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) (including the GCG package (Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL)

[215] :403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073) determine by comparing sequence information. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0081] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be determined by Southern hybridization experiments under appropriate hybridization conditions, which can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto. For example, under stringent conditions, homologous or identical polynucleotide sequences can typically be hybridized along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or full-length sequence.

[0082] As used herein, the term “strict conditions” refers to conditions under which specific hybridization between polynucleotides can occur. Such conditions are described in detail in the literature (see Sambrook et al., above, 9.50-9.51, 11.7-11.8). For example, the stringent conditions may include polynucleotides with high homology or identity, i.e., polynucleotides with 60% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity hybridizing with each other, and polynucleotides with lower homology or identity than the above conditions not hybridizing with each other, or washing conditions for general Southern hybridization, i.e., washing once, specifically twice or three times, at salt concentrations and temperatures corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0083] The hybridization can occur between nucleotides with bases that have complementary sequences, but depending on the strictness of the hybridization, the hybridized polynucleotide may contain some mismatches between bases. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also comprise substantially similar nucleotide sequences as well as separate nucleic acid fragments complementary to the entire sequence.

[0084] For example, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected by hybridization at a Tm value of 55°C. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art.

[0085] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., see above).

[0086] Another aspect of this disclosure provides a vector comprising the polynucleotides of this disclosure. The vector may be an expression vector for expressing the polynucleotides in microorganisms, but is not limited thereto.

[0087] As used herein, the term "vector" refers to a DNA construct used to deliver desired polynucleotides into a suitable host or host cell.

[0088] For example, the vector may contain a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operatively linked to a suitable expression regulatory region (regulatory sequence) to express the desired polypeptide in a suitable host cell. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a termination sequence for regulating transcription and translation. Once transformed into a suitable host cell (microorganism), the vector may replicate or function independently of the host genome, or it may integrate into the host genome to replicate or function.

[0089] For example, the vector disclosed herein may contain a sequence for inserting the desired polynucleotide into a chromosome. The polynucleotide can be inserted into the chromosome using any method known in the art, such as homologous recombination, but is not limited thereto.

[0090] There are no particular limitations on the vectors used in this disclosure, but any vector known in the art may be used. Examples of commonly used vectors may include natural or recombinant plasmids, granules, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., can be used as phage vectors or granule vectors, and the pDZ system, pDC system, pBR system, pUC system, pBluescriptII system, pGEM system, pTZ system, pCL system, pET system, etc., can be used as plasmid vectors. For example, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pCES208, pET-28a vectors, etc., may be used.

[0091] The vector may also contain selection markers to confirm transformation into host cells, or further, insertion into the chromosome of the host cells. Selection markers are used to select cells transformed with the vector, or to confirm the desired polynucleotide insertion into the chromosome, and may use markers that provide selectable phenotypes, such as drug resistance, auxotrophic phenotypes, resistance to cytotoxic agents, or expression of surface peptides. Only cells expressing the selection markers can survive or exhibit different phenotypes under conditions treated with the selection agent, thus allowing for the selection of transformed cells.

[0092] As used herein, the term "transformation" refers to the introduction of a desired polynucleotide or a vector containing it into a host cell (including microorganisms) to alter the genetic characteristics of the host cell. The transformed polynucleotide may be inserted into the host cell's chromosome or may be located extrachromosomally. Furthermore, the polynucleotide may comprise DNA or RNA. Depending on the purpose of the introduction, the polynucleotide may be introduced in a suitable form. For example, a polynucleotide for expressing a desired polypeptide may be introduced into a host cell (microorganism) in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. The expression cassette may often contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the coding sequence of the desired polypeptide. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into the host cell (microorganism) in its own form and operably linked to, but is not limited to, the desired sequence for expression in the host cell (microorganism).

[0093] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned such that the regulatory sequence directs the expression of a coding sequence. Therefore, the term "operably linked" encompasses the attachment or connection between a regulatory region (such as a promoter, terminator, signal sequence, or enhancer) with a functional domain having known or desired activity and a target (gene or polypeptide) such that the expression, secretion, or function of the target can be regulated according to known or desired activity. For example, the term implies the functional linking of a polynucleotide sequence to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the polypeptide.

[0094] As used in this article, the term “genetic recombination” refers to a natural or artificial process in which elements of a gene, such as DNA or RNA, are altered from their original sequence during the process of breakdown and recombination.

[0095] As used herein, the term "recombinant gene" refers to a gene having a novel genetic conformation produced by genetic recombination, such as chemical synthesis or genetic engineering techniques. In this disclosure, the terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" are used interchangeably. For example, a recombinant gene may comprise an artificial combination of nucleic acid fragments, such as regulatory sequences, that do not coexist in nature.

[0096] Another aspect of this disclosure provides a microorganism comprising any one or more of the variant polypeptide of this disclosure, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide.

[0097] The variant peptides, polynucleotides, and vectors are as described elsewhere.

[0098] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified prokaryotic or eukaryotic microorganisms, and can be a microorganism in which a particular mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and can be a microorganism containing genetic modifications for the production of desired polypeptides, proteins, or products. As used herein, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0099] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a genotype and / or phenotype different from that of a naturally occurring microorganism (e.g., when the genetic modification affects the nucleotide sequence encoding of the microorganism), and may contain any offspring or potential offspring of that microorganism. As used herein, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably.

[0100] For example, the microorganisms disclosed herein may be, but are not limited to, microorganisms modified to express variant peptides of the present disclosure, or recombinant microorganisms having the activity of variant peptides of the present disclosure.

[0101] In one embodiment, the microorganism disclosed herein may have the ability to produce any one or more of rebaudine D and rebaudine M.

[0102] As used herein, the term "microorganism capable of producing any one or more of rebaudine D and rebaudine M" refers to a prokaryotic or eukaryotic microorganism capable of producing rebaudine D and / or M in a living organism, and said microorganism may include both microorganisms inherently capable of producing rebaudine D and / or M and microorganisms not inherently capable of such production but conferred such production capacity. The ability to produce rebaudine D and / or M may be conferred or enhanced by expressing variant polypeptides of the present disclosure or by species modification. The term "microorganism capable of producing any one or more of rebaudine D and rebaudine M" may be used interchangeably with "microorganism capable of producing rebaudine D," "microorganism capable of producing rebaudine M," "microorganism capable of producing rebaudine D or M," and "microorganism capable of producing both rebaudine D and M."

[0103] As used herein, the term "unmodified microorganism" does not exclude the inclusion of microorganisms that may have naturally occurring mutations, and can be a wild-type microorganism or the natural microorganism itself, or a microorganism whose characteristics have not been altered due to genetic variation caused by natural or artificial factors. In this disclosure, the term "unmodified microorganism" may be used interchangeably with "pre-modified microorganism," "unmutated microorganism," "parental microorganism," "parental strain," "wild-type microorganism," "reference microorganism," or "standard microorganism." In this disclosure, an unmodified microorganism may refer to a microorganism in which the variant polypeptide of this disclosure has not been introduced or has not yet been introduced; or a microorganism containing a wild-type polypeptide, but is not limited thereto. Furthermore, in this disclosure, an unmodified microorganism may be a microorganism containing a polypeptide composed of SEQ ID NO: 1 or a polynucleotide encoding it (e.g., a polynucleotide composed of SEQ ID NO: 2), but is not limited thereto.

[0104] For example, the microorganisms of this disclosure include, but are not limited to, microorganisms that contain variant polypeptide sequences of this disclosure by mutating genes on chromosomes encoding polypeptides containing parental sequences of this disclosure; and / or microorganisms that contain variant polypeptide sequences of this disclosure by introducing vectors containing polynucleotides encoding variant polypeptide sequences of this disclosure.

[0105] The microorganisms disclosed herein may include any microorganism comprising any one or more of the following: a variant polypeptide of the present disclosure; a polynucleotide encoding the variant polypeptide; and a vector comprising the polynucleotide, and the microorganisms of the present disclosure are capable of producing any one or more of rebaudioside D and rebaudioside M. For example, the microorganisms of the present disclosure may be microorganisms transformed with a vector comprising a polynucleotide encoding the variant polypeptide of the present disclosure to express the variant polypeptide of the present disclosure, thereby having enhanced rebaudioside D and / or M production capabilities, but are not limited thereto.

[0106] Microorganisms having enhanced ability to produce any one or more of rebaudine D and rebaudine M can be, but are not limited to, microorganisms having enhanced rebaudine D and / or M production capacity compared to natural wild-type microorganisms or unmodified microorganisms (e.g., microorganisms expressing wild-type peptides or peptides containing parental sequences; or microorganisms not expressing variant peptides of the present disclosure).

[0107] For example, the microorganisms of this disclosure that have enhanced ability to produce any one or more of rebaudine D and rebaudine M can be, but are not limited to, microorganisms with enhanced rebaudine D and / or M production capacity compared to the unmodified parental microorganism (parental strain) or unmodified microorganisms (e.g., microorganisms expressing wild-type polypeptides containing the parental sequence; or the polypeptide of SEQ ID NO: 1; or microorganisms not expressing variant polypeptides of this disclosure). For example, the unmodified parental microorganism (parental strain) or the unmodified microorganism used to compare enhanced production capacity can be, but is not limited to, *Corynebacterium ammoniacum* (…). Corynebacterium ammoniagenes ATCC6872.

[0108] For example, a microorganism with enhanced production capacity may have an increased production capacity of rebaudioside D and / or M by about 1% or more, specifically about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 50% or more, about 70% or more, about 90% or more, or about 100% or more (there is no particular upper limit, but it can be, for example, about 500% or less) compared to the parental microorganism before modification or the unmodified microorganism. However, the amount of increase is not limited to this, as long as the production capacity has an increased + value amount compared to the production capacity of the parental microorganism before modification or the unmodified microorganism. In another instance, a recombinant microorganism with enhanced production capacity may have a production capacity that is about 1.1 times or more, about 1.15 times or more, about 1.2 times or more, about 1.3 times or more, about 1.5 times or more, about 1.7 times or more, about 1.9 times or more, or about 2.0 times or more (with no particular upper limit, but may be, for example, about 10 times or less) greater than that of the parent microorganism before modification (parent strain) or the unmodified microorganism, but is not limited thereto.

[0109] For example, the microorganisms disclosed herein can be prokaryotic or eukaryotic cells, specifically prokaryotic cells. The prokaryotic cells may include, for example, Escherichia coli, Erwinia spp. Erwinia sp.), Serratia spp. Serratia sp.), Providencia genus ( Providence sp.), Corynebacterium, Pseudomonas (sp.), Corynebacterium, Pseudom Pseudomonas sp.), Leptospira ( Leptospira sp.), Salmonella spp. Salmonella sp.), genus *Brugia* ( Brevibacteria sp.), genus *Firmionum* ( Hypomononas sp.), Chromobacterium genus ( Chromobacterium sp.) and Nocardia spp. North cardiaThe prokaryotic cells may be microorganisms of the genera *Escherichia*, *Corynebacterium*, *Leptospira*, and yeast, but are not limited thereto. Specifically, the prokaryotic cells may be microorganisms of the genera *Corynebacterium* or *Escherichia*. More specifically, the prokaryotic cells may be microorganisms of the genera *Corynebacterium* or *Escherichia*.

[0110] Regarding the microorganisms according to any of the foregoing specific embodiments, the Corynebacterium genus microorganisms disclosed herein may be Corynebacterium glutamicum (…). Corynebacterium glutamicum ), crude lactobacillus ( Corynebacterium raw milk ), Corynebacterium desertis ( Corynebacterium deserti ), High-efficiency Corynebacterium ( Corynebacterium efficiens ), Corynebacterium tumefaciens ( Corynebacterium callunae ), Corynebacterium stasis ( Corynebacterium stationis ), single rod-shaped bacteria ( Corynebacterium singular ), salt-tolerant Corynebacterium ( Corynebacterium halotolerans ), Corynebacterium styloides ( Corynebacterium striped ), ammonia-producing Corynebacterium, contaminating Corynebacterium ( Corynebacterium pollutisoli ), Corynebacterium ( Corynebacterium imitans Corynebacterium tectorum ( ) Corynebacterium testudinoris ) or Corynebacterium fulvidracosum ( Corynebacterium flavescens ).

[0111] Specifically, the microorganisms disclosed herein may be ammonia-producing Corynebacterium, glutamate-producing Corynebacterium, or Escherichia coli, but are not limited thereto.

[0112] In one embodiment, the microorganisms of this disclosure capable of producing rebaudioside D and / or M may further comprise any one or more of a UGT-A polypeptide, a polynucleotide encoding the UGT-A polypeptide, and a carrier containing the polynucleotide. The polypeptide and polynucleotide may be wild-type or variants, and the polypeptide may include any polypeptide of any type, as long as it possesses UGT-A activity.

[0113] Specifically, the UGT-A of this disclosure may have the following amino acid sequence, contain the following amino acid sequence, consist of the following amino acid sequence, or consist substantially of the following amino acid sequence: the amino acid sequence of SEQ ID NO: 129; or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with it.

[0114] Specifically, the polynucleotide encoding the UGT-A of this disclosure may have the following polynucleotide sequence, contain the following polynucleotide sequence, consist of the following polynucleotide sequence, or consist substantially of the following polynucleotide sequence: the polynucleotide sequence of SEQ ID NO:130; or a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with it.

[0115] In one embodiment, the microorganisms of this disclosure having the ability to produce rebaudioside D and / or M may also contain any one or more of sucrose synthase, a polynucleotide encoding sucrose synthase, and a carrier containing the polynucleotide.

[0116] Specifically, the sucrose synthase of this disclosure may have the following amino acid sequence, contain the following amino acid sequence, consist of the following amino acid sequence, or consist substantially of the following amino acid sequence: the amino acid sequence of SEQ ID NO: 131; or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with it.

[0117] Specifically, the polynucleotide encoding the sucrose synthase disclosed herein may have the following polynucleotide sequence, contain the following polynucleotide sequence, consist of the following polynucleotide sequence, or consist substantially of the following polynucleotide sequence: the polynucleotide sequence of SEQ ID NO: 132; or a polynucleotide sequence having at least 70%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with it.

[0118] Another aspect of this disclosure provides a method for producing steviol glycosides, the method comprising culturing microorganisms in a culture medium containing any one or more of the variant polypeptides of this disclosure, polynucleotides encoding the variant polypeptides, and vectors containing the polynucleotides.

[0119] Specifically, the steviol glycoside can be any one or more of rebaudioside D and rebaudioside M.

[0120] The variant peptides, polynucleotides, vectors, and microorganisms are as described elsewhere.

[0121] As used herein, the term "culture" refers to the cultivation of microorganisms under appropriately regulated environmental conditions. Cultivation procedures can be performed using suitable culture media or cultivation conditions known in the art. Those skilled in the art can readily adapt and use such cultivation methods depending on the selected microorganisms. Specifically, cultivation can be batch, continuous, and / or fed-batch, but is not limited to these.

[0122] As used herein, the term "culture medium" refers to a mixture containing nutrients essential for the cultivation of microorganisms as its main components, wherein the culture medium provides nutrients, including water, growth factors, etc., which are indispensable for survival and growth. Specifically, for the culture medium and other culture conditions used to cultivate the microorganisms of this disclosure, any culture medium commonly used for culturing microorganisms may be used without particular limitation. For example, the microorganisms of this disclosure may be cultured under aerobic conditions, with controlled temperature, pH, etc., in a general-purpose culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins. For example, culture media for Corynebacterium spp. can be found in the literature ["Manual of Methods for General Bacteriology", American Bacteriological Society (Washington DC, USA, 1981)].

[0123] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, brown molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and various other carbon sources may be used without limitation in appropriate amounts. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0124] As for nitrogen sources, inorganic nitrogen sources can be used, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; amino acids, such as glutamic acid, methionine, and glutamine; and organic nitrogen sources, such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or its decomposition products, defatted soybean meal or its degradation products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0125] The phosphate source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. As for inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously. However, this disclosure is not limited thereto.

[0126] Furthermore, during the cultivation of the microorganisms disclosed herein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., in an appropriate manner. Additionally, antifoaming agents, such as polyethylene glycol esters of fatty acids, can be added to inhibit foam formation during cultivation. Furthermore, oxygen or oxygen-containing gases can be injected into the culture medium to maintain an aerobic state, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected to maintain an anaerobic or non-anaerobic state, but these are not limited to these methods.

[0127] In one embodiment, the culture medium for the microorganisms of this disclosure may contain substrates for the synthesis of rebaudine D and / or rebaudine M. The culture medium may contain any one or more of rebaudine A, rebaudine D, steviol, nucleotide diphosphate-linked glucose, sucrose, and nucleotide diphosphates. Specifically, the culture medium for producing rebaudine D may contain rebaudine A and nucleotide diphosphate-linked glucose; rebaudine A, sucrose, and nucleotide diphosphates; or rebaudine A, nucleotide diphosphate-linked glucose, sucrose, and nucleotide diphosphates. The culture medium may contain steviol and nucleotide diphosphate-linked glucose to replace or supplement rebaudine A, and may contain sucrose and nucleotide diphosphates to replace or supplement nucleotide diphosphate-linked glucose. The culture medium for producing rebaudine M may contain rebaudine D and nucleotide diphosphate-linked glucose; or rebaudine D, sucrose, and nucleotide diphosphates. A culture medium for producing rebaudine D may be included to replace rebaudine D. In one embodiment, the nucleotide diphosphate may be uridine diphosphate, but is not limited thereto.

[0128] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, and the cultivation can be carried out for approximately 10 hours to 160 hours, but is not limited thereto.

[0129] As used herein, the term "culture" refers to a culture medium, a concentrated culture medium, a dried product of a culture medium, a culture filtrate, a concentrated culture filtrate, or a dried product of a culture filtrate obtained by culturing a specific microorganism in a culture medium. A culture medium refers to liquids containing a specific microorganism, and a culture filtrate refers to liquids that substantially do not contain a specific microorganism (meaning that the specific microorganism isolated by filtration, etc., is substantially excluded, but this does not mean that the microorganism is completely excluded from the filtrate). The dosage form of a culture is not limited, but it can be, for example, a liquid, an emulsion, or a solid.

[0130] As used in this article, the term "fermentation" refers to the process by which microorganisms use their own enzymes to break down organic matter, excluding putrefaction. Fermentation and putrefaction occur through similar processes, but when the result of decomposition is the production of useful substances, it is called fermentation, while when it produces unpleasant odors or harmful substances, it is called putrefaction.

[0131] In this disclosure, the methods for obtaining fermentation products from microorganisms are not particularly limited, and the fermentation products can be obtained according to methods commonly used in the art or similar fields.

[0132] As used herein, the term "fermentation product" includes not only the fermentation material itself, but also all types of substances containing fermentation products produced by microorganisms, such as substances containing fermenting microorganisms, cultures produced by fermenting microorganisms, fermentation products of cultures, concentrated fermentation products, dried products of fermentation products, filtrates of fermentation products, filtrates of concentrated fermentation products or dried products of filtrates of fermentation products, extracts of fermentation products or dilutions of fermentation products, etc.

[0133] In the methods disclosed herein, any culture conditions and methods known in the art can be used to culture microorganisms. Those skilled in the art can easily adjust and use this culture method according to the selected microorganism.

[0134] Rebaudioside D and / or M produced by the culture of this disclosure can be released into the culture medium or can be retained in the cells.

[0135] Another aspect of this disclosure provides a method for producing steviol glycosides, the method comprising the step of transferring glucose to rebaudioside A by reacting rebaudioside A with glucose linked to a nucleotide diphosphate in the presence of a variant polypeptide of this disclosure and / or a microorganism of this disclosure.

[0136] The stevioside can be any one or more of rebaudioside D and rebaudioside M.

[0137] The variant polypeptide may be contained in or obtained from the microorganism disclosed herein (e.g., purified); and the reaction steps may include culturing any one or more microorganisms containing the variant polypeptide, the polynucleotide encoding the variant polypeptide, and the vector containing the polynucleotide in a medium containing nucleotide diphosphate-linked glucose and rebaudioside A.

[0138] The variant peptides, polynucleotides, vectors, microorganisms, cultures, and culture media are as described elsewhere.

[0139] In one embodiment, the method for producing steviol glycosides may also include, but is not limited to, the step of producing nucleotide diphosphate-linked glucose by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase.

[0140] As used herein, the term "sucrose synthase" plays a role in sucrose production by reversibly transferring glucose bound to nucleotide diphosphates to fructose in plant metabolism, and in this disclosure, it exhibits activity in separating glucose and fructose linked to nucleotide diphosphates by reacting sucrose with nucleotide diphosphates (e.g., in a pH range of 5 to 10).

[0141] Sucrose can be used without restriction, as long as it can serve as a substrate for sucrase synthase to provide glucose to nucleotide diphosphates. For example, raw sugar or sugar can be used, but it is not limited to these.

[0142] In this disclosure, the nucleotide diphosphate can be a purine nucleotide or a pyrimidine nucleotide. Specifically, uridine diphosphate (UDP) can be used, but it is not limited thereto.

[0143] In one embodiment, the method for producing steviol glycosides may further include the step of reacting ribavirin A with nucleotide diphosphate-linked glucose in the presence of glycosyltransferase A (UGT-A); and / or a microorganism containing glycosyltransferase A, a polynucleotide encoding glycosyltransferase A, and a carrier containing said polynucleotide.

[0144] In one embodiment of the foregoing implementation, the step of producing rebaudioside A may be, but is not limited to, culturing any one or more microorganisms containing a glycosyltransferase A (UGT-A) polypeptide, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0145] In one embodiment of the aforementioned implementation scheme, glycosyltransferase A (UGT-A) may be derived from rice ( White rice ), Stevia ( Stevia rebaudiana Bertoni ), green bamboo ( Bamboo oldhamii ), two spikes short-stalked grass ( Brachypodium distachyon ),barley( Barley ), two-color sorghum ( Sorghum two-colored ),corn( Corn Arabidopsis thaliana Arabidopsis thaliana The glycosyltransferase A (UGT-A) is not limited to its source, as long as it has glycosyltransferase A (UGT-A) activity, such as those produced from recombinant microorganisms (e.g., Escherichia coli, Bacillus, yeast, Corynebacterium, or Agrobacterium) transformed with a polynucleotide containing the glycosyltransferase gene and / or a vector containing the gene, those obtained after microbial production (e.g., purified), those known, those commercially available, etc.

[0146] In one embodiment, the method for producing steviol glycosides and / or the step of producing rebaudioside A (or the method for producing rebaudioside A) may further include, but is not limited to, the step of producing nucleotide diphosphate-linked glucose by reacting sucrose with nucleotide diphosphate in the presence of sucrose synthase.

[0147] In one embodiment, the production of rebaudiosides A, D and / or M can be carried out continuously or discontinuously in the same reaction system, and can be carried out in one pot, but is not limited thereto.

[0148] In one embodiment, the method for producing steviol glycosides may further include the step of producing rebaudioside M by reacting rebaudioside D with glucose linked to a nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A); and / or a microorganism containing glycosyltransferase A, a polynucleotide encoding glycosyltransferase A, and a carrier containing said polynucleotide.

[0149] In one specific embodiment, the method for producing steviol glycosides disclosed herein may further include the steps of preparing the microorganisms of the present disclosure, preparing a culture medium for culturing the microorganisms, or a combination thereof (in any order), for example, prior to the culturing step.

[0150] The production method disclosed herein may further include the step of recovering any one or more of rebaudine D and rebaudine M from cultured microorganisms, cultures of said microorganisms, fermentation products of said microorganisms, culture media, or reaction solutions in which enzymatic reactions have occurred. A recovery step may also be included after the culturing step.

[0151] According to the methods for culturing microorganisms disclosed herein, such as batch, continuous, or fed-batch cultures, the recovery can be achieved by collecting any one or more of rebaudine D and rebaudine M using appropriate methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography), HPLC, and combinations of these methods can be used, and rebaudine D and / or M can be recovered from the culture medium, microorganisms, or reaction solution using appropriate methods known in the art.

[0152] Furthermore, the method for producing steviol glycosides disclosed herein may also include a purification step. Purification can be performed using suitable methods known in the art. In exemplary embodiments, when the method for producing steviol glycosides disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously regardless of the order, or may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0153] Another aspect of this disclosure provides a method for producing rebaudine D, the method comprising the step of reacting rebaudine D with nucleotide diphosphate-linked glucose in the presence of a variant polypeptide of this disclosure and / or a microorganism of this disclosure.

[0154] The method for producing rebaudioside D may include the method (steps) for producing rebaudioside A provided above, and glycosyltransferase A (UGT-A) as described in other aspects.

[0155] In one embodiment, the method for producing rebaudioside D may also include a step of recovering rebaudioside D.

[0156] In one implementation, the production of rebaudioside A and / or D can be carried out continuously or discontinuously in the same reaction system, but is not limited thereto.

[0157] Another aspect of this disclosure provides a method for producing rebaudine M, the method comprising the step of reacting rebaudine D with glucose linked to a nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A); and / or a microorganism containing glycosyltransferase A, a polynucleotide encoding glycosyltransferase A, and a carrier containing said polynucleotide.

[0158] The method for producing rebaudioside M may include the method (steps) for producing rebaudioside A provided above and / or the method (steps for producing rebaudioside D) for producing rebaudioside D.

[0159] Methods for producing rebaudioside A, methods for producing rebaudioside D, and glycosyltransferase A (UGT-A) are described elsewhere.

[0160] In one embodiment, the method for producing rebaudioside M may further include a step of recovering rebaudioside M.

[0161] In one implementation, the production of rebaudiosides A, D and / or M can be carried out continuously or discontinuously in the same reaction system, but is not limited thereto.

[0162] Another aspect of this disclosure provides a composition for producing any one or more of rebaudioside D and rebaudioside M, the composition comprising: a variant polypeptide of this disclosure; a polynucleotide encoding the variant polypeptide; a microorganism comprising any one or more of the variant polypeptide, the polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide; a culture of the microorganism; or any one or more of these.

[0163] The variant peptides, polynucleotides, vectors, microorganisms, and cultures are as described elsewhere.

[0164] The compositions disclosed herein may further comprise any suitable excipients commonly used in compositions for the production of rebaudioside D and / or M, and examples of excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc.

[0165] In one specific embodiment, each component present in the compositions of this disclosure may be contained in a microbially effective amount or in an amount that may be suitably present in the compositions used for production.

[0166] Another aspect of this disclosure provides the use of the variant polypeptides of this disclosure or the microorganisms of this disclosure in the production of rebaudioside D and / or M. Specifically, this aspect provides the use of microorganisms in the production of any one or more of rebaudioside D and rebaudioside M, said microorganisms comprising any one or more of the variant polypeptides of this disclosure, polynucleotides encoding said variant polypeptides, and vectors comprising said polynucleotides.

[0167] The variant peptides and microorganisms, etc., are as described in other aspects.

[0168] Another aspect of this disclosure provides the use of the composition in the production of any one or more of rebaudioside D and rebaudioside M, the composition comprising: a variant polypeptide of this disclosure; a polynucleotide encoding the variant polypeptide; a microorganism comprising any one or more of the variant polypeptide, the polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide; a culture of the microorganism; or any one or more of these.

[0169] The variant peptides, polynucleotides, microorganisms, and cultures, etc., are as described in other aspects.

[0170] [Modes for Implementing the Invention] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are merely preferred embodiments for illustrating the present disclosure and are therefore not intended to limit the scope of the disclosure thereto. Furthermore, technical matters not described in this specification can be fully understood and readily implemented by those skilled in the art or similar art.

[0171] Example 1. Design and preparation of UGT-B modified enzyme 1-1. Design of UGT-B Modified Enzyme To increase the activity of the UGT-B enzyme, a total of 26 variants were designed based on the amino acid sequence of SEQ ID NO: 1, as shown in Table 1 below.

[0172] [Table 1] 1-2. Preparation of UGT-B modified enzyme The modified enzyme designed in Example 1-1 was prepared by site-directed mutagenesis. In the site-directed mutagenesis PCR reaction, AccuPower® Pfu PCR premix (Bioneer), 10 ng template plasmid (E. coli-Corynebacterium shuttle vector pCES208; or pET-28a vector for expression in E. coli) and 0.5 μM of each forward and reverse primer (primers as shown in Table 2 below were used depending on the type of modified enzyme). Furthermore, the above PCR was performed using Mastercycler® X50s (Eppendorf) under the following conditions: pre-denaturation at 94°C for 3 min; the following 20 cycles: denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min / kb; and a final extension at 72°C for 5 min.

[0173] Subsequently, the template plasmid was digested with 1 μl DpnI (NEB, R0176L), and 5 μl of PCR reaction solution was injected into Escherichia coli DH5α strain using a heat shock method.

[0174] Each transformed E. coli strain was cultured in LB medium containing 50 mg / L kanamycin. After colony selection, mutations were identified by plasmid DNA sequencing (Bioneer).

[0175] Two types of plasmid vectors were used to prepare the modified enzyme. One is the *E. coli*-*Corynebacterium* shuttle vector (pCES208), which contains a codon-optimized UGT-B gene sequence (SEQ ID NO: 2) for expression in *Corynebacterium* strains and is designated pCES_UGT-B. The other vector is the pET-28a vector for expression in *E. coli*, which contains a codon-optimized UGT-B gene sequence (SEQ ID NO: 127) for expression in *E. coli* strains and a 6x his tag for purification. Both vectors contain a kanamycin resistance gene.

[0176] [Table 2] Example 2. Preparation of microorganisms expressing modified enzymes and comparison of RebD synthesis capabilities of 26 modified enzymes. In ammonia-producing Corynebacterium (hereinafter referred to as) C.amm The 26 modified enzymes prepared in Example 1 were expressed in cells, and cell reactions were performed to compare the conversion rates of rebaudioside A (hereinafter referred to as RebA) to rebaudioside D (hereinafter referred to as RebD).

[0177] Therefore, electroporation was first used to insert the expression vector of Corynebacterium spp., namely the modified enzyme plasmid vector prepared in Example 1, into the wild-type... C.amm strain ATCC6872.

[0178] Transformed using plasmid vectors C.amm The strain was cultured on solid medium containing 50 μg / ml kanamycin (80 g / L glucose, 20 g / L soybean peptone, 10 g / L (NH4)2SO4, 1.2 g / L KH2PO4, 1.4 g / L MgSO4, 15 g / L agar). To express the modified enzyme, colonies grown on solid medium were inoculated into 400 μl of Corynebacterium spp. microbial medium containing 50 μg / ml kanamycin (80 g / L glucose, 20 g / L soybean peptone, 10 g / L (NH4)2SO4, 1.2 g / L KH2PO4, 1.4 g / L MgSO4) and pre-cultured in 96-well plates. Pre-cultured for 24 hours at 30°C and 800 rpm using an Infortreller (Infors HT Multitron Incubator Shaker), then 10% (v / v) of the total volume of the pre-culture was inoculated into fresh medium for Corynebacterium (containing 50 μg / ml kanamycin) (80 g / L glucose, 20 g / L soy protein, 10 g / L (NH4)2SO4, 1.2 g / L KH2PO4, 1.4 g / L MgSO4) and cultured under the same conditions for 24 hours.

[0179] To confirm reproducibility, two colonies were cultured for each modified enzyme in strains containing each modified enzyme, and four colonies were cultured for comparison in strains containing wild-type UGT-B.

[0180] For the enzyme activity assay based on RebD synthesis capacity, 200 μl of culture from each strain containing the modified enzyme or wild-type UGT-B was placed in a 96-well plate and centrifuged, discarding the supernatant. A total of 400 μl of reaction solution containing 10 mM uridine-5'-bisphosphate-glucose (UDPG; Carbosynth), 10% (w / v) RebA (Daepyung), 0.8% POESA (v / v), and 50 mM potassium phosphate (pH 7.0) was added to the remaining cells, and the reaction was allowed to proceed at 40 °C for 24 h. The reaction solution was then diluted 1:10 with triple-distilled water, the enzyme was inactivated at 100 °C for 5 min, and the solution was pretreated with a filter. Transformed RebD was analyzed from the pretreated reaction solution using HPLC (Agilent). The HPLC analytical conditions are as follows.

[0181] - Detector wavelength: 210 nm - Flow rate: 1 ml / min - Sample injection volume: 10 μl - Column: Capcell pak C18 MG II (Shiseido, 250 x 4.6 mm, particle size: 5 μm) Solvent: 30% acetonitrile Analysis results as follows Figure 1 and Figure 2 As shown.

[0182] As a result, Figure 1 As shown, all prepared monovariates (M1, M2, M3, M4, M5, M6, M7, M8, M9 and M10) were confirmed to have higher RebD conversion activity compared with wild-type UGT-B.

[0183] In addition, such as Figure 2 As shown, all bivariates (M11-M26) of F380X-G381X prepared based on the activity results of the monovariate were confirmed to have higher activity compared with wild-type UGT-B and each monovariate on which the bivariate was based.

[0184] Example 3. Comparison of activities between UGT-B (WT) and modified enzymes 3-1. Evaluation of RebD Synthetic Activity of UGT-B Modified Enzyme Based on the results of Example 2, seven types (M1, M2, M14, M19, M20, M21, M26) were selected from the modified enzymes that had higher activity compared with wild-type UGT-B, and their activity against RebA was evaluated.

[0185] Therefore, the recombinant Corynebacterium spp. prepared in Example 2 was inoculated into a medium containing 50 μg / ml kanamycin at an initial concentration of OD 600 = 0.1 and pre-cultured at 30°C for 24 hours. Then, 5 ml of the obtained pre-culture solution was inoculated into 50 ml of a medium containing 50 μg / ml kanamycin and cultured at 30°C for 24 hours to induce enzyme expression. Subsequently, cells were isolated from the culture using centrifugation.

[0186] The enzymatic reaction was carried out at 30°C for 16 hours by mixing 100 mg / ml of Corynebacterium cells in a reaction solution containing 10 mM UDPG (Carbosynth), RebA (Daepyung), 0.8% POESA (v / v), and 50 mM potassium phosphate (pH 7.0). After the enzymatic reaction was completed, HPLC analysis was performed in the same manner as in Example 2. The results, relative to wild-type UGT-B, of the conversion to RebD are shown in Table 3 below.

[0187] [Table 3] As shown in Table 3, it was confirmed that the conversion rate of all tested modified enzymes to RebD was significantly higher compared with wild-type UGT-B.

[0188] 3-2. Evaluation of RebDM synthetic activity of UGT-B modified enzyme (1) When using glycosyltransferase UGT-A, sucrose synthase (SS), and glycosyltransferase UGT-B, sucrose and uridine-5'-bisphosphate (UDP) can be used instead of uridine-5'-bisphosphate-glucose (UDPG) to convert raw materials such as steviol glycosides into RebD and rebaudioside M (hereinafter referred to as RebM).

[0189] Therefore, in this embodiment, a mixture of recombinant Corynebacterium microorganisms expressing UGT-A and SS and recombinant Corynebacterium microorganisms expressing a modified UGT-B enzyme was used to evaluate the reaction of the raw material SG90 (Haigen), in which stevioside and RebA were mixed, to convert to RebD and RebM. For this purpose, the gene encoding UGT-A (SEQ ID NO: 129) (SEQ ID NO: 130) and the gene encoding SS (SEQ ID NO: 131) (SEQ ID NO: 132) were cloned into the Escherichia coli-Corynebacterium shuttle vector (pCES208). This vector was transformed into Corynebacterium microorganisms in the same manner as described in Example 2 to produce strains simultaneously expressing UGT-A and SS. The vector prepared and used for transformation was named pCES_SS_UGT-A. The method for culturing Corynebacterium microorganisms expressing UGT-B modified enzymes (representatively M20, M21, and M26) for enzyme expression was carried out in the same manner as described in Example 3-1.

[0190] Cells expressing UGT-A and SS, as well as cells expressing UGT-B, were mixed at 100 mg / ml in a reaction solution containing 10 mM UDP (Carbosynth), 1% (w / v) SG90 (Haigen), 10% (w / v) 0.8% POESA (v / v), and 50 mM potassium phosphate (pH 8.0), and the enzymatic reaction was carried out at 30°C for 24 hours. After the enzymatic reaction was completed, HPLC analysis was performed in the same manner as in Example 2. The conversion rates to a mixture of RebD and RebM (RebDM) relative to wild-type UGT-B are shown in Table 4 below.

[0191] [Table 4] As shown in Table 4, it was confirmed that the conversion rate of the modified enzyme to a mixture of RebD and RebM (RebDM) was significantly higher than that of wild-type UGT-B.

[0192] Example 4. Comparison of the activities of UGT-B (WT) and the compound modified enzyme 4-1. Preparation of UGT-B complex modified enzyme To analyze the synergistic effect among mutations that demonstrated increased activity of the modified enzyme compared to UGT-B, combined mutants containing two or more mutations were designed as shown in Table 5 below.

[0193] [Table 5] The above-designed composite modified enzyme was prepared using the site-directed mutagenesis PCR method as described in Examples 1-2.

[0194] In detail, each plasmid containing the sequence of the modified enzyme variant prepared in Examples 1-2 was used as a template, and one or two pairs of primers for additional mutations were used to prepare additional residual mutations by site-directed mutagenesis PCR. For example, in the case of the MM1 variant, a plasmid containing the polynucleotide sequence of the Y208A (M2) variant was used as a template, and primers for M26 were used to additionally prepare F380A-G381A. In another example, in the case of the MM10 variant, a plasmid containing the polynucleotide sequence of the Y92W (M1) variant was used as a template, and primers for M2 and M31 were used to contain Y208A and additional F380T-G381S mutations.

[0195] 4-2. Evaluation of RebD Synthetic Activity of the Compound Modified Enzyme Recombinant Corynebacterium strains expressing each of the modified enzymes prepared in Example 4-1 were prepared using the same transformation method as in Example 2, and the activity of these strains was evaluated in the same manner as in Example 3-1. The results showed... Figure 3 middle.

[0196] As a result, Figure 3 As shown, all prepared composite modified enzymes exhibited significantly higher enzyme activity compared to wild-type UGT-B.

[0197] Example 5. Evaluation of the RebDM synthetic activity of the modified enzyme (2) Furthermore, the RebDM synthetic activity of the modified enzymes was evaluated in different ways. Four types of *E. coli* BL21(DE3) strains were prepared, with each vector prepared by cloning pET28-α using the gene encoding the enzyme (SEQ ID NO: 130, 127, 128, or 132) introduced to express the four types of enzymes (UGT-A, UGT-B_WT, UGT-B_M21, and RSS). Each strain was inoculated into 5 ml LB medium and cultured at 37 °C and 200 rpm for 8 hours, followed by master culture in 2 L LB medium. After culturing at 37 °C and 200 rpm until the absorbance at 600 mm reached 0.4, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce high expression of the enzymes. After the addition of IPTG, the culture was incubated at 20 °C and 120 rpm for approximately 16 hours. The culture was centrifuged at 6000×g for 20 minutes at 4°C to obtain cells, and the soluble proteins of the four enzymes were extracted by sonication. Each enzyme was then purified by Ni-NTA affinity chromatography.

[0198] The purified enzyme and the raw material (SG90, Haigen) containing steviol glycoside and RebA as the main components were used to carry out the enzymatic reaction under conditions 1 to 3 at 40°C and pH 8.0 to 9.0, as shown in Table 6 below.

[0199] [Table 6] Then, after inactivating the enzyme at 100°C for 20 minutes, the production of RebD, RebM and RebDM (a mixture of RebD and RebM) with the addition of UGT-B_WT and UGT-B_M21 was compared by HPLC analysis in the same manner as in Example 2.

[0200] When the reaction solution of UGT-B_M21 enzyme was added under conditions 1 to 3, after 72 hours of reaction, the amount of RebDM was 100%, 100%, and 90.8%, respectively, while the amount of RebM was 89.5%, 83.8%, and 52.1%, respectively, and the results are shown in Table 7 below.

[0201] [Table 7] When the results of the reaction solution containing the UGT-B_M21 enzyme were compared with those containing the UGT-B_WT enzyme, both the amounts of RebDM and RebM were approximately 2.2 to 2.8 times higher. This indicates that the enzyme’s ability to convert RebA to RebD is significantly enhanced, and that the modified enzyme of this disclosure is advantageous for RebM conversion via a complex reaction.

[0202] The results above confirm that the production of RebDM can be increased by using the UGT-B single and complex variant enzymes disclosed herein.

[0203] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by the description that follows them; therefore, all changes and modifications falling within the boundaries and scope of the claims, or equivalents of such boundaries and scope, are included in the claims.

Claims

1. A variant polypeptide having glycosyltransferase activity, said variant polypeptide comprising any one or more of the following: the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 being replaced by tryptophan; the amino acid at position 208 being replaced by alanine or methionine; the amino acid at position 380 being replaced by histidine, threonine, alanine or glycine; and the amino acid at position 381 being replaced by phenylalanine, serine, alanine or asparagine.

2. The variant polypeptide of claim 1, wherein the variant polypeptide comprises: The amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced with tryptophan; The amino acid corresponding to position 208 is replaced by alanine or methionine. The amino acid corresponding to position 380 is replaced by histidine, threonine, or alanine; The amino acid corresponding to position 381 is replaced by phenylalanine, serine, alanine, or asparagine; or The amino acid at position 380 is replaced by histidine, threonine, or glycine, and the amino acid at position 381 is replaced by phenylalanine, serine, alanine, or asparagine.

3. The variant polypeptide of claim 1, wherein the variant polypeptide comprises: The amino acid corresponding to position 208 is replaced by alanine, the amino acid corresponding to position 380 is replaced by threonine or alanine, and the amino acid corresponding to position 381 is replaced by phenylalanine, serine, or alanine. The amino acid corresponding to position 92 is replaced by tryptophan, the amino acid corresponding to position 380 is replaced by threonine or alanine, and the amino acid corresponding to position 381 is replaced by phenylalanine, serine, or alanine. The amino acid at position 92 is replaced by tryptophan, and the amino acid at position 208 is replaced by alanine; or The amino acid at position 92 is replaced by tryptophan, the amino acid at position 208 is replaced by alanine, the amino acid at position 380 is replaced by threonine or alanine, and the amino acid at position 381 is replaced by phenylalanine, serine, or alanine.

4. The variant polypeptide of claim 1, wherein the variant polypeptide has 80% or more homology or identity with SEQ ID NO:

1.

5. A polynucleotide encoding a variant polypeptide according to any one of claims 1 to 4.

6. A vector comprising the polynucleotide according to claim 5.

7. A microorganism comprising any one or more of a variant polypeptide according to any one of claims 1 to 4, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide.

8. The microorganism of claim 7, wherein the microorganism has an enhanced ability to produce any one or more of rebaudioside D and rebaudioside M compared to a microorganism comprising the amino acid sequence of SEQ ID NO: 1 or encoding the polynucleotide therein.

9. The microorganism according to claim 7, wherein the microorganism is a member of the genus Corynebacterium or Escherichia.

10. The microorganism according to claim 9, wherein the microorganism is ammonia-producing Corynebacterium, glutamate-producing Corynebacterium, or Escherichia coli.

11. A method for producing steviol glycosides, the method comprising the step of transferring glucose to rebaudioside A by reacting rebaudioside A with glucose linked to a nucleotide diphosphate in the presence of the variant polypeptide of claim 1 or the microorganism of claim 7, wherein the steviol glycoside is any one or more of rebaudioside D and rebaudioside M.

12. The method of claim 11, further comprising the step of recovering any one or more of rebaudine D and rebaudine M.

13. The method of claim 11, further comprising the step of producing rebaudioside M by reacting rebaudioside D with glucose linked to a nucleotide diphosphate in the presence of glycosyltransferase A (UGT-A); or a microorganism comprising glycosyltransferase A, a polynucleotide encoding glycosyltransferase A, and a carrier comprising said polynucleotide.

14. A composition for producing any one or more of rebaudioside D and rebaudioside M, said composition comprising any one or more of the following: a variant polypeptide according to any one of claims 1 to 4; a polynucleotide encoding said variant polypeptide; a microorganism comprising said variant polypeptide, the polynucleotide encoding said variant polypeptide, and a vector comprising said polynucleotide; a culture of said microorganism; or any one or more thereof.

15. Use of a microorganism in the production of any one or more of rebaudioside D and rebaudioside M, said microorganism comprising any one or more of a variant polypeptide according to any one of claims 1 to 4, a polynucleotide encoding said variant polypeptide, and a vector comprising said polynucleotide.

16. Use of the composition in the production of any one or more of rebaudioside D and rebaudioside M, said composition comprising: a variant polypeptide according to any one of claims 1 to 4; a polynucleotide encoding said variant polypeptide; a microorganism comprising said variant polypeptide, the polynucleotide encoding said variant polypeptide, and a vector comprising said polynucleotide; a culture of said microorganism; or any one or more thereof.

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