UDP-glucosyltransferase variant and use thereof for steviol glycoside production

Abstract

The present invention relates to a UDP-glucosyltransferase variant and a use thereof for producing steviol glycosides. The UDP-glucosyltransferase variant and the fusion enzyme comprising the variant and sucrose synthase according to the present invention exhibit improved thermal stability and enzyme activity, and thus can find a broad spectrum of applications in the field of steviol glycoside synthesis, offering excellent industrial value and significance by increasing production efficiency and lowering production costs.

Description

UDP-glucosyltransferase mutant and its use in producing steviol glycosides

[0001] The present invention relates to a UDP-glucosyltransferase variant and its use for producing steviol glycosides.

[0002] Stevia extract is a natural sweetener derived from the perennial shrub, Stevia rebaudiana. Stevia extracts, refined to varying degrees, are used as a high-potency flavoring in foods and blends, or are sold alone as a tabletop sweetener.

[0003] Stevia extracts contain rebaudioside and other steviol glycosides that contribute to sweetness, but existing commercial products primarily contain rebaudioside (Reb) A, with smaller amounts of other glycosides such as Reb C, D, and F. Stevia extracts derived from the plant may contain contaminants, such as derived compounds, that cause off-flavors. These off-flavors can be problematic, depending on the food system or intended use.

[0004] Furthermore, stevia extracts or compositions containing stevia extracts can vary greatly depending on the soil and climate in which the plant is grown. Depending on the source plant, climate conditions, and extraction process, the amount of Reb A in commercial production has been reported to vary from 20% to 97% of the total steviol glycoside content. Other steviol glycosides are also present in varying amounts in stevia extracts.

[0005] Stevia extracts produced from the stevia plant contain various steviol glycosides and off-flavor compounds, and their recovery and purification are labor-intensive and inefficient. Therefore, a recombinant production system capable of accumulating desired steviol glycosides, such as Reb M, at high yields remains a pressing need. The development of efficient enzymes for this purpose remains essential. Furthermore, improved production of Reb M precursors (e.g., Reb I) for commercial production in recombinant hosts remains a pressing need.

[0006] Furthermore, the activity of existing wild-type enzymes used in steviol glycoside production rapidly declines at temperatures above 50°C during enzymatic reaction, and continued reaction at low temperatures raises concerns about contamination with other viruses. Therefore, the development of enzymes with enhanced thermostability is urgently needed to prevent this.

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] (Patent Document 1) Korean Patent Publication No. 10-2021-0114899

[0010] One object of the present invention is to provide a UDP-glucosyltransferase (UGT) mutant.

[0011] Another object of the present invention is to provide a fusion protein comprising the UDP-glucosyltransferase variant and sucrose synthase.

[0012] Another object of the present invention is to provide a polynucleotide encoding the UDP-glucosyltransferase variant or the fusion protein.

[0013] Another object of the present invention is to provide a vector comprising the polynucleotide.

[0014] Another object of the present invention is to provide a microorganism comprising the polynucleotide or a vector comprising the same.

[0015] Another object of the present invention is to provide a composition for producing steviol glycosides, comprising at least one selected from the group consisting of the UDP-glucosyltransferase variant; the fusion protein; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0016] Another object of the present invention is to provide a method for producing steviol glycosides, comprising the step of reacting (i) stevioside, rebaudioside, or a mixture thereof, and (ii) at least one selected from the group consisting of UDP, UDP-glucose, and sucrose with the composition.

[0017] Another object of the present invention is to provide a use for producing steviol glycosides of a composition comprising at least one selected from the group consisting of the UDP-glucosyltransferase variant; the fusion protein; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0018] Another object of the present invention is to provide a use for producing a composition for producing steviol glycosides of the UDP-glucosyltransferase variant; the fusion protein; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; or an extract thereof.

[0019] One aspect of the present invention provides a UDP-glucosyltransferase (UGT) variant, wherein one or more (e.g., 1, 2, 3, 4, or 5) of the following amino acids are substituted:

[0020] From the N-terminus of the amino acid sequence of sequence number 1,

[0021] Substitution of the amino acid corresponding to position 264 with cysteine ​​(C);

[0022] Substitution of the amino acid corresponding to position 301 with asparagine (N);

[0023] Substitution of the amino acid corresponding to position 305 with proline (P);

[0024] Substitution of the amino acid corresponding to position 376 with proline (P); and

[0025] Substitution of the amino acid corresponding to position 392 with tryptophan (W).

[0026] The variant of the present invention exhibits improved thermal stability and enzyme activity, making it widely applicable to the field of steviol glycoside synthesis. In particular, it can play a key role in the production of Reb M, known to have the highest sweetness. Utilizing the variant of the present invention increases the productivity of steviol glycosides, ensuring economic feasibility. At the same time, it allows for increased reaction temperatures in the production process, thereby preventing, controlling, and eliminating potential contamination.

[0027] In the present invention, "UDP-glucosyltransferase (UGT)" is an enzyme having an activity of transferring glucose to a substrate in the presence of a glucose donor, and can perform a reaction of transferring glucose from a glucose donor (e.g., UDP-glucose) to a steviol glycoside.

[0028] In the present invention, steviol glycosides include, but are not limited to, stevioside, rebaudioside (Reb A, Reb B, Reb C, Reb D, Reb E, Reb F, Reb I, and Reb M) or mixtures thereof.

[0029] In one embodiment, the UDP-glucosyltransferase according to the present invention has an activity of converting stevioside to Reb A, an activity of converting Reb A to Reb I, an activity of converting Reb E to Reb D, and / or an activity of converting Reb D to Reb M. That is, the UDP-glucosyltransferase according to the present invention has a conversion activity for all of the single substrates or mixed substrates of stevioside, Reb A, Reb E, and Reb D.

[0030] In the present invention, the “UDP-glucosyltransferase variant” may be a variant in which ‘an amino acid corresponding to one or more of positions 264, 301, 305, 376, and 392 from the N-terminus of the amino acid sequence of SEQ ID NO: 1’ in the amino acid sequence of a polypeptide having UDP-glucosyltransferase activity is substituted with another amino acid.

[0031] In the present invention, the UDP-glucosyltransferase may be a polypeptide having UDP-glucosyltransferase activity that is modified to produce the UDP-glucosyltransferase variant of the present invention. Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, and may include a variant or functional fragment thereof, but is included without limitation as long as it can serve as a parent of the UDP-glucosyltransferase variant of the present invention.

[0032] In the present invention, the UDP-glucosyltransferase may be derived from a plant of the genus Triticum (e.g., Triticum aestivum, etc.), a plant of the genus Stevia (e.g., Stevia rebaudiana, etc.), a plant of the genus Oryza (e.g., Oryza sativa, etc.), or a plant of the genus Hordeum (e.g., Hordeum vulgare, etc.), but is not limited thereto.

[0033] The sequence of the UDP-glucosyltransferase of the present invention can be obtained from a known database such as NCBI's GenBank.

[0034] In addition, the UDP-glucosyltransferase of the present invention may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having sequence homology or identity of about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more with the amino acid sequence of SEQ ID NO: 1, and is included in the scope of UDP-glucosyltransferase without limitation if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0035] In addition, it is obvious that a polypeptide or protein having an amino acid sequence in which some of the sequences are deleted, modified, substituted or added is also included within the scope of the present application, provided that the amino acid sequence has such homology or identity and exhibits an activity corresponding to the polypeptide or protein.

[0036] In the present invention, "homology" or "identity" refers to the degree of relationship between two given amino acid sequences or base sequences, and may be expressed as a percentage. In the present invention, homology and identity may be used interchangeably.

[0037] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, sequences that are homologous or identical are generally capable of hybridizing under moderate or high stringency conditions, typically along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its full length. Hybridization is also contemplated for polynucleotides that contain degenerate codons in place of codons in the polynucleotide.

[0038] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using, for example, but not limited to, BLAST of the National Center for Biotechnology Information Database or ClustalW.

[0039] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the appropriate hybridization conditions defined are within the scope of the relevant technology and can be determined by methods well known to those skilled in the art.

[0040] In the present invention, the "corresponding amino acid" refers to an amino acid residue at a corresponding position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the amino acid residue at that position. Identifying the amino acid at the corresponding position may determine a specific amino acid of a sequence that references a specific sequence. In the present invention, the "corresponding position" generally refers to a similar or corresponding position in the amino acid sequence of a related protein or a reference sequence. For example, any amino acid sequence may be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence may be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the position of the corresponding amino acid, or the position where a modification such as a substitution, insertion, or deletion occurs, may be identified by comparing it with a query sequence (also referred to as a "reference sequence") using a sequence alignment algorithm known in the art.

[0041] In one embodiment, the UDP-glucosyltransferase variant comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity to the amino acid sequence of SEQ ID NO: 1 and has UDP-glucosyltransferase activity, wherein the variant may be a variant in which one or more (e.g., 1, 2, 3, 4, or 5) amino acids are substituted:

[0042] From the N-terminus of the amino acid sequence of sequence number 1,

[0043] Substitution of cysteine ​​for the amino acid corresponding to position 264 (C);

[0044] Substitution of the amino acid corresponding to position 301 with asparagine (N);

[0045] Substitution of the amino acid corresponding to position 305 with proline (P);

[0046] Substitution of the amino acid corresponding to position 376 with proline (P); and

[0047] Substitution of the amino acid corresponding to position 392 with tryptophan (W).

[0048] In one embodiment, the UDP-glucosyltransferase variant may be a variant in which 'an amino acid corresponding to one or more (e.g., 1, 2, 3, 4, or 5) positions 264, 301, 305, 376, and 392 from the N-terminus of the amino acid sequence of SEQ ID NO: 1' is substituted as described above in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity thereto.

[0049] For example, the UDP-glucosyltransferase variant may comprise substitutions of amino acids corresponding to the following two or more positions of the amino acid sequence of SEQ ID NO: 1:

[0050] No. 376 and No. 264;

[0051] 376 and 301;

[0052] No. 376 and No. 305;

[0053] No. 376 and No. 392;

[0054] No. 376, No. 301 and No. 264;

[0055] No. 376, No. 301, and No. 305;

[0056] Nos. 376, 301, and 392;

[0057] No. 376, No. 301, No. 264, and No. 305;

[0058] 376, 301, 264, and 392; or

[0059] Numbers 376, 301, 264, 305, and 392.

[0060] For example, from the N-terminus of the amino acid sequence of sequence number 1

[0061] The amino acid corresponding to position 264 is valine (V).

[0062] The amino acid corresponding to position 301 is aspartic acid (D).

[0063] The amino acid corresponding to position 305 is serine (S).

[0064] The amino acid corresponding to position 376 is aspartic acid (D).

[0065] The amino acid corresponding to position 392 may be leucine (L).

[0066] It is obvious that a variant of the present invention, if it has an amino acid sequence that exhibits an activity corresponding to the polypeptide or protein described herein and has homology or identity, also includes a polypeptide or protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added. Specifically, the variant of the present invention may include deletions or additions of amino acids that have minimal influence on the properties and secondary structure of the polypeptide, and for example, a signal (or leader) sequence that is involved in translocation of the protein co-translationally or post-translationally may be conjugated to the N-terminus of the variant.

[0067] Additionally, the variants of the present invention may be conjugated to other sequences or linkers so that they can be identified, purified, or synthesized.

[0068] In addition, if it has the same or corresponding activity as the variant of the present invention, it does not exclude meaningless sequence additions before and after the amino acid sequence of the corresponding sequence number, mutations that can occur naturally, or silent mutations thereof, in addition to the variant of the present invention, and it is clear that even if it has such sequence additions or mutations, it falls within the scope of the present invention.

[0069]

[0070] Another aspect of the present invention provides a fusion protein comprising a UDP-glucosyltransferase variant according to the present invention and sucrose synthase (SUS).

[0071] The above "sucrose synthase (SUS)" is an enzyme that produces fructose and UDP-glucose from sucrose and UDP. Therefore, when sucrose and UDP are supplied to SUS together with a reaction in which UDP-glucosyltransferase transfers glucose from UDP-glucose to a steviol glycoside (e.g., a reaction in which stevioside is converted to Reb A, a reaction in which Reb A is converted to Reb I, a reaction in which Reb E is converted to Reb D, and / or a reaction in which Reb D is converted to Reb M), SUS produces UDP-glucose from sucrose and UDP and provides it to UDP-glucosyltransferase (at this time, UDP-glucose may be supplied instead of or together with UDP). Therefore, using SUS in the conversion reaction of the UDP-glucosyltransferase is more economical than supplying UDP-glucose separately. The SUS can be provided on its own or in the form of a fusion protein with UDP-glucosyltransferase to produce steviol glycosides. In addition, when UDP-glucosyltransferase and sucrose synthase are prepared as a fusion protein and the conversion reaction of the steviol glycosides is performed, the two enzymatic reactions can form a single channel and occur simultaneously, thereby causing an efficient conversion reaction. In other words, when the fusion enzyme is used, economical substrate conversion is possible, and steviol glycosides can be produced through a high-concentration substrate reaction.

[0072] In the present invention, the sucrose synthase includes a naturally occurring polypeptide, a wild-type polypeptide, or a variant or functional fragment thereof, as long as it has the activity of producing fructose and UDP-glucose from sucrose and UDP.

[0073] The sucrose synthase of the present invention may be, but is not limited to, a sucrose synthase derived from a plant of the genus Glycine (e.g., Glycine max), a plant of the genus Arabidopsis (e.g., Arabidopsis thaliana), a plant of the genus Solanum (e.g., Solanum lycopersicum), a plant of the genus Nicotiana (e.g., Nicotiana tabacum), or a microorganism of the genus Thermosynechococcus (e.g., Thermosynechococcus elongatus).

[0074] The sequence of the sucrose synthase of the present invention can be obtained from a known database such as NCBI's GenBank.

[0075] In one embodiment, the sucrose synthase may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or identity thereto, and is included within the scope of sucrose synthase without limitation as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

[0076] In one embodiment, the sucrose synthase is a variant having sucrose synthase activity, which comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity with the amino acid sequence of SEQ ID NO: 3, wherein the variant comprises at least one substitution of 'an amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' to glutamic acid (E) and 'an amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' to lysine (K).

[0077] In one embodiment, the sucrose synthase may have at least one of a substitution of 'the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' with glutamic acid (E) and a substitution of 'the amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' with lysine (K) in the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or homology thereto.

[0078] In one embodiment, the sucrose synthase may have at least one of a substitution of serine (S) at position 11 to glutamic acid (E) and a substitution of arginine (R) at position 456 to lysine (K) in the amino acid sequence of SEQ ID NO: 3.

[0079] In the present invention, the UDP-glucosyltransferase variant can be fused with sucrose synthase directly or via a linker. The linker may be a peptide consisting of 4 to 15 amino acids, and specifically may include 4 to 15 amino acids selected from the group consisting of G (Gly), S (Ser), E (Glu), A (Ala), K (Lys), and P (Pro), but is not limited thereto. For example, it may be GGGS (SEQ ID NO: 10), GGGGS (SEQ ID NO: 11), GGGSGGGGS (SEQ ID NO: 12), GGGGPSPGGGGS (SEQ ID NO: 13), EAAAK (SEQ ID NO: 34), EAAAKEAAAK (SEQ ID NO: 35), EAAAKEAAAKEAAAK (SEQ ID NO: 36), or GGGGSGGGGSGGGGS (SEQ ID NO: 9).

[0080] In one embodiment, the fusion protein of the present invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or identity with the amino acid sequence of SEQ ID NO: 5 (a total of 1278 amino acids; UGT enzyme of SEQ ID NO: 1 at positions 1-458 + linker of SEQ ID NO: 9 at positions 459-473 + SUS of SEQ ID NO: 3 at positions 474-1278), and is a fusion protein having UDP-glucosyltransferase and sucrose synthase activities,

[0081] (a) comprising one or more (e.g., 1, 2, 3, 4, or 5) substitutions within the UDP-glucosyltransferase moiety:

[0082] From the N-terminus of the amino acid sequence of sequence number 5,

[0083] Substitution of the amino acid corresponding to position 264 with cysteine ​​(S);

[0084] Substitution of the amino acid corresponding to position 301 with asparagine (N);

[0085] Substitution of the amino acid corresponding to position 305 with proline (P);

[0086] Substitution of the amino acid corresponding to position 376 with proline (P); and

[0087] Substitution of the amino acid corresponding to position 392 with tryptophan (W); or

[0088] (b) In addition to the substitution of (a), the sucrose synthase moiety may further comprise one or more of the following substitutions:

[0089] From the N-terminus of the amino acid sequence of sequence number 5,

[0090] Substitution of the amino acid corresponding to position 484 (i.e., position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3) with glutamic acid (E); and

[0091] Substitution of the amino acid corresponding to position 929 (i.e., position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3) with lysine (K).

[0092] In one embodiment, the fusion protein of the present invention may comprise a substitution of (a) or (b) in the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity thereto.

[0093] For example, the fusion protein of the present invention may comprise substitutions of amino acids corresponding to the following two or more positions of the amino acid sequence of SEQ ID NO: 5:

[0094] No. 376 and No. 264;

[0095] 376 and 301;

[0096] No. 376 and No. 305;

[0097] No. 376 and No. 392;

[0098] No. 484 and No. 929;

[0099] No. 376, No. 301 and No. 264;

[0100] No. 376, No. 301, and No. 305;

[0101] Nos. 376, 301, and 392;

[0102] No. 376, No. 301, No. 264, and No. 305;

[0103] 376, 301, 264, and 392; or

[0104] Numbers 376, 301, 264, 305, and 392.

[0105] For example, from the N-terminus of the amino acid sequence of sequence number 5

[0106] The amino acid corresponding to position 264 is valine (V).

[0107] The amino acid corresponding to position 301 is aspartic acid (D).

[0108] The amino acid corresponding to position 305 is serine (S).

[0109] The amino acid corresponding to position 376 is aspartic acid (D).

[0110] The amino acid corresponding to position 392 is leucine (L).

[0111] The amino acid corresponding to position 484 is serine (S).

[0112] The amino acid corresponding to position 929 is arginine (R).

[0113]

[0114] As used herein, "amino acid" and "amino acid residue" refer to a natural amino acid, an unnatural amino acid, or a modified amino acid. Unless otherwise stated, all references to amino acids, either generically or by name, include reference to both the D and L stereoisomers (where the structure permits such stereoisomeric forms). Natural amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Non-natural amino acids include modified amino acid residues that are chemically modified, or reversibly or irreversibly chemically blocked, at the N-terminal amino group or side chain, such as N-methylated D and L amino acids or residues in which a side chain functional group is chemically modified with another functional group.

[0115] It is obvious that a polypeptide or protein having an amino acid sequence having homology or identity as mentioned in the present invention and exhibiting an activity corresponding to the polypeptide or protein is also included within the scope of the present application, including a polypeptide or protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted (e.g., conservatively substituted) or added.

[0116] Even if the present invention describes "a polypeptide or protein having or including an amino acid sequence of a specific sequence number," it is clear that a polypeptide or protein having or including an amino acid sequence in which a part of the amino acid sequence of the sequence number is deleted, modified, substituted (e.g., conservatively substituted), or added may also be used in the present invention, provided that it has the same or corresponding activity as a polypeptide or protein consisting of the amino acid sequence of the sequence number.

[0117] As used herein, "conservative substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain without causing a loss of the biological or biochemical function of the polypeptide or protein. Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0118]

[0119] Another aspect of the present invention provides a polynucleotide encoding a UDP-glucosyltransferase variant according to the present invention; or a fusion protein according to the present invention.

[0120] The polynucleotide of the present invention can have various modifications made to the coding region within a range that does not change the amino acid sequence of the variant and fusion protein of the present invention, taking into account the degeneracy of the codon or the codon preferred in the organism to which the variant and fusion protein of the present application is to be expressed.

[0121] The polynucleotide of the present invention may be DNA or RNA.

[0122]

[0123] Another aspect of the present invention provides a vector comprising a polynucleotide according to the present invention.

[0124] The vector of the present invention may comprise a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. The vector may be capable of replicating or functioning independently of the host genome after being transformed into a suitable host cell, or may be integrated into the genome itself.

[0125] The term "transformation" in the present invention refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell so that the polypeptide encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or outside the chromosome, as long as it can be expressed within the host cell. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of autonomous replication. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0126] Additionally, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target polypeptide of the present invention.

[0127] The vector of the present invention is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include, but are not limited to, plasmids, cosmids, viruses, and bacteriophages, either in a natural or recombinant state.

[0128] The vector of the present invention may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0129] The vector of the present invention may be a vector for intracellular chromosomal insertion to insert the polynucleotide into a chromosome, but is not limited thereto. A polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming whether the chromosome has been inserted may be additionally included.

[0130]

[0131] Another aspect of the present invention provides a microorganism comprising a polynucleotide according to the present invention or a vector comprising the same.

[0132] In the present invention, “microorganism” or “strain” includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and specifically, microorganisms whose specific mechanisms have been weakened or strengthened due to causes such as insertion of an external gene or enhanced or inactivated activity of an endogenous gene, and may be microorganisms that have genetic modification for the production of a desired polypeptide, protein or product, but are not limited thereto.

[0133] In one embodiment, the microorganism of the present invention includes, but is not limited to, one or more selected from the group consisting of microorganisms of the genus Escherichia (e.g., Escherichia coli), microorganisms of the genus Saccharomyces (e.g., Saccharomyces cerevisiae), microorganisms of the genus Bacillus (e.g., Bacillus subtilis), microorganisms of the genus Pichia (e.g., Pichia pastoris), and microorganisms of the genus Corynebacterium (e.g., Corynebacterium glutamicum).

[0134]

[0135] Another aspect of the present invention provides a composition for producing steviol glycosides, comprising at least one selected from the group consisting of a UDP-glucosyltransferase variant according to the present invention; a fusion protein according to the present invention; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; and extracts thereof. The steviol glycosides are as described above.

[0136] The above microorganism comprises a polynucleotide encoding a fusion protein or UDP-glucosyltransferase variant according to the present invention, and may be a microorganism capable of expressing or having expressed the fusion protein or UDP-glucosyltransferase variant, but is not limited thereto.

[0137] The above culture means a medium obtained by culturing the microorganism in an appropriate medium, and containing an enzyme produced from the microorganism, and may include the microorganism or may be in a cell-free form that does not contain the microorganism. The lysate includes a lysate obtained by crushing the cells of the microorganism or a supernatant obtained by centrifuging the lysate, and contains an enzyme produced from the microorganism. The extract is obtained by extracting the microorganism, its culture, or its lysate using an appropriate solvent or method, and contains an enzyme produced from the microorganism.

[0138] In this specification, unless otherwise stated, the microorganism according to the present invention means at least one selected from the group consisting of cells of the microorganism, freeze-dried cells of the microorganism, cultures of the microorganism, lysates of the microorganism, supernatants of the lysates, and extracts thereof.

[0139] The composition of the present invention may further comprise, as a substrate, at least one selected from the group consisting of (i) a steviol glycoside (e.g., stevioside, rebaudioside, or a mixture thereof) and (ii) UDP, UDP-glucose, and sucrose, but is not limited thereto. For example, the composition of the present invention may have an activity of converting stevioside to Reb A, an activity of converting Reb A to Reb I, an activity of converting Reb E to Reb D, and / or an activity of converting Reb D to Reb M.

[0140] The composition or microorganism of the present invention may additionally comprise a sucrose synthase, which is as described above. When the microorganism comprises a sucrose synthase, the microorganism comprises a polynucleotide encoding the sucrose synthase, and is thus capable of expressing the enzyme, but is not limited thereto.

[0141] The composition or microorganism of the present invention may further comprise an 'enzyme that produces Reb D or Reb E from steviol glycosides (e.g., an enzyme that converts Reb A or stevioside into Reb D or Reb E, respectively)' to produce Reb M. For example, the composition or microorganism of the present invention may comprise a second UDP-glucosyltransferase, such as UGT91D2. That is, Reb M may be produced by sequentially or together reacting (i) 'an enzyme that produces Reb D or Reb E from steviol glycosides' with (ii) a UDP-glucosyltransferase variant according to the present invention; a fusion protein according to the present invention; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; and / or extracts thereof, but is not limited thereto. In this case, the enzyme of (i) can produce Reb D or Reb E, and the enzyme included in (ii) can ultimately produce Reb M from the produced Reb D or Reb E. When the microorganism further includes an 'enzyme that produces Reb D or Reb E from steviol glycosides', the microorganism includes a polynucleotide encoding the 'enzyme that produces Reb D or Reb E from steviol glycosides', and can thus express the enzyme, but is not limited thereto.

[0142]

[0143] Another aspect of the present invention provides a method for producing a steviol glycoside, comprising the step [step (a)] of reacting (i) a steviol glycoside (e.g., stevioside, rebaudioside, or a mixture thereof) and (ii) at least one selected from the group consisting of UDP, UDP-glucose, and sucrose, with a composition for producing a steviol glycoside according to the present invention.

[0144] At this time, the produced steviol glycoside is characterized in that it is a different type from the steviol glycoside (stevioside or rebaudioside, etc.) of the above (i). More preferably, the produced steviol glycoside is characterized in that one or more glucoses are further added to the stevioside or rebaudioside. For example, the rebaudioside may include at least one selected from the group consisting of Reb A, Reb B, Reb C, Reb D, Reb E, Reb F, and Reb M, and the produced steviol glycoside may include at least one selected from the group consisting of Reb A, Reb B, Reb C, Reb D, Reb E, Reb F, and Reb M.

[0145] In the present invention, “reaction” includes contacting a composition for producing steviol glycosides according to the present invention with a substrate or medium comprising (i) a steviol glycoside (e.g., stevioside, rebaudioside, or a mixture thereof) and (ii) at least one selected from the group consisting of UDP, UDP-glucose, and sucrose. The steviol glycoside, UDP, UDP-glucose, or sucrose may be included in the substrate or medium in any form they may have (e.g., solid, solution, etc.), but are not limited thereto.

[0146] In one embodiment, the method of the present invention can produce Reb A from stevioside, Reb I from Reb A, Reb D from Reb E, and / or Reb M from Reb D.

[0147] In one embodiment, when the composition for producing steviol glycosides according to the present invention has sucrose synthase activity together with UDP-glucosyltransferase activity, (i) steviol glycosides and (ii) UDP, UDP-glucose, or a mixture thereof and sucrose can be used to produce steviol glycosides further containing one or more glucoses, but is not limited thereto.

[0148] In addition, the method of the present invention may further include a step [step (b)] of reacting an enzyme that produces Reb D or Reb E from a steviol glycoside (e.g., an enzyme that converts Reb A or stevioside into Reb D or Reb E, respectively)) with a steviol glycoside to produce Reb M. The enzyme that produces Reb D or Reb E from a steviol glycoside is as described above. In the present invention, step (b) may be performed before step (a) or simultaneously with step (a).

[0149] The reaction of the present invention is carried out at a temperature and pH at which the enzyme according to the present invention is active. Such temperature and pH can be appropriately selected by those skilled in the art, and for example, the reaction temperature can be 10 to 90°C, 20 to 80°C, 30 to 70°C, 40 to 65°C, 45 to 60°C, or 50 to 60°C, and the reaction pH can be pH 5.0 to 9.0, pH 5.5 to 8.5, pH 6.0 to 8.0, pH 6.5 to 8.0, or pH 6.5 to 7.5.

[0150] The method of the present invention may further include a step of recovering the steviol glycoside produced from the reactant.

[0151] The above recovery can be performed using any suitable method known in the art, for example, centrifugation, filtration, precipitation, extraction, cell disruption, chromatography, or a combination thereof.

[0152]

[0153] Another aspect of the present invention is a use for producing steviol glycosides of a composition comprising at least one selected from the group consisting of the UDP-glucosyltransferase variant; the fusion protein; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0154] Another aspect of the present invention is the use of the UDP-glucosyltransferase variant; the fusion protein; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; or an extract thereof for producing a composition for producing steviol glycosides.

[0155] The UDP-glucosyltransferase mutant, fusion protein, microorganism, culture, lysate, extract, steviol glycoside production, etc. are as described above.

[0156] The UDP-glucosyltransferase variant according to the present invention and the fusion enzyme comprising the variant and sucrose synthase have improved thermal stability and enzyme activity, and thus can be widely applied in the field of steviol glycoside synthesis, and have excellent industrial value and significance in that they can increase production efficiency and reduce production costs.

[0157] Hereinafter, the present invention will be described in more detail through examples. These examples are intended merely to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

[0158]

[0159] Example 1. Production and cultivation of microorganisms expressing the fusion enzyme, SrUGT-GmSUS.

[0160]

[0161] 1-1: Construction of a vector expressing the fusion enzyme, SrUGT-GmSUS

[0162] A gene encoding a wild-type enzyme protein (SrUGT) having an amino acid sequence of SEQ ID NO: 1 derived from Stevia rebaudiana and a gene encoding a wild-type enzyme protein (GmSUS) having an amino acid sequence of SEQ ID NO: 3 derived from Glycine max were synthesized by codon optimization (IDT, Gblock synthesis) so that they can be optimally expressed in yeast, and the polynucleotide sequences of the synthesized genes are shown in SEQ ID NOs: 2 and 4. In addition, a gene encoding a GmSUS mutant (SEQ ID NO: 7) in which S11E and R456K mutations were introduced into GmSUS of SEQ ID NO: 3 was synthesized. GGGGSGGGGSGGGGS (SEQ ID NO: 9) was used as a linker to fuse the synthesized genes. The enzyme gene was amplified by PCR and subcloned by fusion of the genes using Gibson assembly (2X HiFi DNA master mix, NEW ENGLAND BIOLABS). The amino acid sequence of the fused SrUGT-GmSUS is shown in SEQ ID NO: 5 (including GmSUS wild type) or SEQ ID NO: 8 (including GmSUS mutant), and the polynucleotide sequence encoding SEQ ID NO: 5 is shown in SEQ ID NO: 6. Specifically, the vector manufactured to confirm the activity of the protein was the pRS423 vector containing the His3 auxotrophic marker (selectable marker) that can be selected in S. cerevisiae, and the pRS423_SrUGT-GmSUS recombinant vector was constructed. The pRS423 vector was cut using restriction enzymes NotI and EcoRI, and the amplified genes and the restriction enzyme-treated pRS423 vector were inserted into the restriction enzyme sites so as to be ligated using 2X HiFi DNA master mix (NEB). The gene in the form of a subcloned plasmid was transformed into E. coli (DH5a), selected and amplified, and the sequence of the amplified plasmid was analyzed to confirm that the expression gene of the corresponding enzyme was accurately subcloned.

[0163] The transformation method for introducing genes into S. cerevisiae CENPK2-1c (Euroscarf) was through heat shock treatment with 0.1 M LiAc (Lithium Acetate). The introduced strain was selected on SC-Ura medium, and the selected colonies were cultured to confirm enzyme activity.

[0164] The prepared recombinant plasmid was transformed into S. cerevisiae CENPK2-1c (Euroscarf) strain. Recombinant strains with inserted gene cassettes were selected through selection markers on SC-His solid medium (SC-His medium + 2% agar). The composition of SC-His solid medium was YNB (Difco TM ) 6.7 g / L, CSM-His powder (MP Biomedicals TM ) 0.7 g / L, Glucose 20 g / L and agar 20 g / L, and the pH was adjusted to 6.0.

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] - In sequence numbers 5 to 8, bold indicates the sequence of SrUGT, ​​underline indicates the sequence of the linker, the remaining part indicates the sequence of GmSUS, and a combination of bold and underline indicates a mutant residue.

[0174]

[0175] 1-2: Mutation of the fusion enzyme, SrUGT-GmSUS gene

[0176] Based on the amino acid sequence analysis and the analysis of the 3D structure model of the active site and metal binding site between homologous genes, the selected amino acids were substitutionally mutated into other amino acids. After producing these recombinant mutant enzymes in yeast, the thermostability and activity changes of the SrUGT-GmSUS enzyme were analyzed. The amino acid candidates identified from the analysis are V264C, D301N, S305P, D376P, and L392W of SrUGT. To perform substitutional mutations in SrUGT-GmSUS (SEQ ID NO: 8), pRS423_SrUGT-GmSUS of Example 1-1 was used as a template DNA. To substitute each amino acid residue (V264C, D301N, S305P, D376P, L392W), two PCR fragments overlappingly containing the substituted amino acids were constructed. Specifically, to construct mutant Fragment 1, a polymerase chain reaction (PCR) reaction solution containing 10 ng of pRS426_SrUGT-GmSUS, 10 pmol of forward primer, 10 pmol of reverse primer, 0.5 μl of Q5 polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP (final volume of 50 μl) was prepared and amplified by PCR. Then, to construct mutant Fragment 2, the sequences of the primers were changed and amplified by PCR. The primer information used is shown in Table 3 below. After cutting the pRS423 vector using restriction enzymes NotI and EcoRI, the amplified Fragment 1, Fragment 2 were inserted into the 2X HiFi DNA master mix (NEB) so as to be ligated to construct the pRS423 / SrUGT-GmSUS mutant vector. And in the case of combinations of two or more mutations, they were constructed in the same way as above but with different template DNAs. For sequence verification and transformation, it is the same as Example 1-1.

[0177] 종류염기서열 (5' -> 3')서열번호V264C_1_fwdCGCTGTACTAATCCAAGG14V264C_1_revTGATCCAACCATTGGAAACATGTTCTATCATGATCTAACAAAG15V264C_2_fwdTGTTAGATCATGATAGAACATGTTTCCAATGGTTGGATCAG16V264C_2_revTTTGAAAGATGATACTCTTTATTTC17D301N_1_fwdCGCTGTACTAATCCAAGG18D301N_1_revAAGAAAGATTGTTTAGAATTAACCAAACCTCTAGCAATTTC19D301N_2_fwdAAATTGCTAGAGGTTTGGTTAATTCTAAACAATCTTTCTTATGGG20D301N_2_revTTTGAAAGATGATACTCTTTATTTC21S305P_1_fwdCGCTGTACTAATCCAAGG22S305P_1_revCTAACAACCCATAAGAATGGTTGTTTAGAATCAACCAAACC23S305P_2_fwdGTTTGGTTGATTCTAAACAACCATTCTTATGGGTTGTTAGAC24S305P_2_revTTTGAAAGATGATACTCTTTATTTC25D376P_1_fwdCGCTGTACTAATCCAAGG26D376P_1_revGGTTGATCCAAACCGAATGGAGAGAAAATCATTGGAACAC27D376P_2_fwdGTGTTCCAATGATTTTCTCTCCATTCGGTTTGGATCAACCATTG28D376P_2_revTTTGAAAGATGATACTCTTTATTTC29L392W_1_fwdCGCTGTACTAATCCAAGG30L392W_1_revAAATAAACACCAACTTTCCAGACATCAGACATATATCTAGC31L392W_2_fwdCTAGATATATGTCTGATGTCTGGAAAGTTGGTGTTTATTTGGAAAATG32L392W_2_revTTTGAAAGATGATACTCTTTATTTC33

[0178]

[0179] 1-3: 미생물 배양

[0180] The yeast selected through transformation in Examples 1-1 and 1-2 were induced to express enzymes through liquid culture. Specifically, the colonies selected from the SC-His solid medium were inoculated into test tubes containing 3 mL of Minimal medium and cultured at 30°C for 24 hours. The concentration of the cultured cells was confirmed by measuring the OD600 absorbance, and the cells were inoculated into a 250 mL flask containing 50 mL of Minimal medium until the OD600 absorbance reached 0.1 (final) and cultured for 48 hours (30°C, 240 rpm). The cultured cells were collected by centrifugation (4,000 rpm, 10 min). The composition of minimal medium included 7.5 g / L ammonium sulfate, 14.4 g / L potassium phosphate, 0.5 g / L magnesium sulfate, 50 mM MES, 2 mL / L trace metals, and 1 mL / L vitamins, and the pH was adjusted to 6.0.

[0181]

[0182] Example 2. Measurement of enzyme activity of SrUGT-GmSUS and mutants against RA60 raw material.

[0183]

[0184] 2-1: Freeze-drying process of microorganisms expressing SrUGT-GmSUS and mutant enzymes

[0185] To perform whole-cell reactions using yeast expressing SrUGT-GmSUS and mutant enzymes, cells harvested after culture in Example 1-3 were freeze-dried. The freeze-drying process was performed under vacuum, and the temperature changes are shown below.

[0186] ① Change the temperature from 20℃ to -40℃ for 0 to 30 minutes

[0187] ② Maintain at -40℃ for 30 to 270 minutes

[0188] ③ Change the temperature to -30℃ for 270 to 510 minutes

[0189] ④ Change the temperature to -20℃ for 510 to 750 minutes

[0190] ⑤ Change the temperature to -10℃ for 750 to 990 minutes

[0191] ⑥ Change the temperature to 0℃ for 990 to 1230 minutes

[0192] ⑦ Change the temperature to 10℃ for 1230 to 1470 minutes

[0193] ⑧ Temperature change of 25℃ for 1470 to 1710 minutes

[0194] ⑨ Maintain 25℃ for 1710 to 1950 minutes

[0195]

[0196] 2-2: Measurement of enzyme activity and thermostability of lyophilized strains expressing SrUGT-GmSUS and enzymes with one mutation introduced into SrUGT.

[0197] In order to measure the enzyme activity of the SrUGT-GmSUS obtained in Example 2-1 and the freeze-dried strain expressing the mutant enzyme, the productivity of Rebaudioside I and Rebaudioside A was compared and confirmed through whole-cell reaction.

[0198] The reaction solution for enzyme activity evaluation was prepared by dissolving substrate RA60 (95%, Sinochem) containing 60% Rebaudioside A and 40% Stevioside, UDP, Sucrose, EDTA, and freeze-dried cells in 100 mM phosphate buffer. A total reaction volume of 1 mL was prepared. The final reaction solution contained 5 g / L of substrate RA60 (95%, Sinochem), 0.3 mM UDP, 750 mM Sucrose, 5 mM EDTA, and 9.3 g / L of freeze-dried cells. Enzyme reaction was performed for the prepared reaction solution at 45°C, pH 7.2, and 150 rpm, and the degree of reaction was confirmed 1 hour after the start of the reaction.

[0199] The whole-cell reaction solution was boiled at 100°C for 5 minutes to terminate the whole-cell reaction, centrifuged (13,000 rpm, 10 min), and the obtained supernatant was analyzed to identify the whole-cell reaction products. Specifically, the whole-cell reaction products were analyzed using HPLC-Chromatography to confirm the conversion ratio of the products. Since the cells of the recombinant strain convert the stevioside substrate into Reb A and Reb A into Reb I, the enzyme activity was measured using the Reb I production ratio and the stevioside substrate reduction ratio.

[0200] Specifically, the column used for HPLC analysis of the product was UG120 (C 18 The HPLC analysis was performed using a 250 mm x 46 mm, 5 um 110 A particle, Shiseido, and the analysis was confirmed at 210 nm. The column temperature was maintained at 55℃. The mobile phase was analyzed as a gradient using water containing 0.01% TFA (Trifluoroacetic acid, Sigma) and 100% acetonitrile, respectively. The mobile phase flowed at 0.8 mL / min and the analysis was confirmed through a total of 34 minutes. The HPLC analysis conditions are shown in Table 3 below.

[0201] Time (min)0.01% TFA(%)Acetonitrile (%)0802012643617643622010027010027.18020348020

[0202] The results of the above analysis are shown in Tables 4 and 5 below. In Table 4 below, the substrate conversion activity is expressed as a relative conversion activity based on 100% of the substrate conversion activity of the strain producing the template enzyme SrUGT-GmSUS (SEQ ID NO: 8). In Table 5 below, the substrate conversion activity is expressed as a relative conversion activity based on 100% of the substrate conversion activity of the strain producing the template enzyme and the mutant enzyme at 45°C.

[0203] Genetic relative conversion activity (%) SrUGT-GmSUS (template) 100 SrUGT-GmSUS (V264C) 99 SrUGT-GmSUS (D301N) 93 SrUGT-GmSUS (S305P) 108 SrUGT-GmSUS (D376P) 119 SrUGT-GmSUS (L392W) 126

[0204] Gene Relative conversion activity 45℃ (%) Relative conversion activity 50℃ (%) SrUGT-GmSUS (template) 10093 SrUGT-GmSUS (V264C) 10098 SrUGT-GmSUS (D301N) 10085 SrUGT-GmSUS (S305P) 10085 SrUGT-GmSUS (D376P) 100106 SrUGT-GmSUS (L392W) 100106

[0205] The substrate conversion activity of the enzyme was confirmed using a strain producing the above enzyme, and the recombinant strain expressing the L392W mutant enzyme showed an increased activity of up to 126% or more compared to the recombinant strain expressing the template enzyme. In terms of thermostability, the recombinant strains expressing the D376P and L392W mutant enzymes showed an increased thermostability of up to 106% compared to the conversion activity at 45°C.

[0206]

[0207] 2-3: Measurement of enzyme activity and thermostability of a freeze-dried strain expressing an enzyme with SrUGT-GmSUS and two mutations.

[0208] In order to test the increase in thermostability of a strain producing an enzyme into which two or more mutations among the selected mutant residues were introduced, a plasmid into which two or more mutations were introduced was constructed using the same method as in Example 1-2, and then a strain producing an enzyme into which two or more mutations were introduced into SrUGT-GmSUS (SEQ ID NO: 8) was produced using the same method as in Example 1-1. In addition, the whole-cell reaction was performed at a temperature higher than 45°C in Example 2-2. Specifically, the reaction temperature was increased from 45°C to 50°C and 55°C, and the relative conversion activity of the enzyme was compared.

[0209] A reaction solution substantially identical to that in Example 2-2 was prepared, and an enzymatic reaction was performed on the prepared reaction solution under the conditions of 50°C and 55°C, pH 7.2, and 150 rpm, and the degree of reaction was confirmed 1 hour after the start of the reaction. The whole-cell reaction solution was boiled at 100°C for 5 minutes to terminate the whole-cell reaction, centrifuged (13,000 rpm, 10 min), and the obtained supernatant was analyzed to confirm the whole-cell reaction product. Analysis of the whole-cell reaction product was performed in the same manner as in Example 2-2.

[0210] The analysis results are shown in Tables 6 and 7 below. In Table 6 below, the substrate conversion activity was set as 100% based on the substrate conversion activity at 50°C and 55°C of the strain producing the template enzyme SrUGT-GmSUS (SEQ ID NO: 8), and the relative conversion activity of each mutant enzyme was shown with respect to this. In Table 7 below, the substrate conversion activity was set as 100% based on the substrate conversion activity at 50°C of the strain producing each mutant enzyme, and the substrate conversion activity at 55°C was shown as the relative conversion activity.

[0211] Genetic wild type compared to relative conversion activity 50℃ (%) 55℃ (%) SrUGT-GmSUS (template) 100 100 SrUGT-GmSUS (D376P) 126 162 SrUGT-GmSUS (D376P-D301N) 200 281 SrUGT-GmSUS (D376P-L392W) 173 258 SrUGT-GmSUS (D376P-S305P) 163 197 SrUGT-GmSUS (D376P-V264C) 162 250

[0212] Gene-identical enzyme comparison relative conversion activity 55℃ (%) SrUGT-GmSUS (template) 69 SrUGT-GmSUS (D376P) 89 SrUGT-GmSUS (D376P-D301N) 97 SrUGT-GmSUS (D376P-L392W) 103 SrUGT-GmSUS (D376P-S305P) 84 SrUGT-GmSUS (D376P-V264C) 106

[0213] As a result of confirming the substrate conversion activity of the enzyme according to temperature using the strain producing the above enzyme, the recombinant strain expressing the D376P, D376P-D301N, D376P-L392W, D376P-S305P, and D376P-V264C mutant enzymes showed an activity that was increased by at least 20% and up to 100% at 50℃ compared to the recombinant strain expressing the template enzyme. In addition, an activity increase of up to 150% was confirmed at 55℃. In addition, in the case of thermostability, it was confirmed that the recombinant strain expressing the enzyme introduced with two mutations maintained at least 80% of the activity at 50℃ even at 55℃. In particular, D376P-D301N showed a conversion activity of up to 281% compared to the template enzyme, and showed 97% of the conversion activity at 55℃ compared to 50℃, confirming thermostability at high temperatures.

[0214]

[0215] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A UDP-glucosyltransferase variant comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of sequence number 1, wherein at least one of the following amino acids is substituted: From the N-terminus of the amino acid sequence of sequence number 1, Substitution of the amino acid corresponding to position 264 with cysteine ​​(C); Substitution of the amino acid corresponding to position 301 with asparagine (N); Substitution of the amino acid corresponding to position 305 with proline (P); Substitution of the amino acid corresponding to position 376 with proline (P); and Substitution of the amino acid corresponding to position 392 with tryptophan (W).

2. A fusion protein comprising a UDP-glucosyltransferase variant according to paragraph 1 and sucrose synthase.

3. In the second paragraph, the sucrose synthase is a fusion protein derived from a plant of the genus Glycine, a plant of the genus Arabidopsis, a plant of the genus Solanum, a plant of the genus Nicotiana, or a microorganism of the genus Thermosynechococcus.

4. In the second paragraph, the sucrose synthase (i) containing the amino acid sequence of sequence number 3, or (ii) A fusion protein comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 3, and comprising at least one substitution of the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3 to glutamic acid (E) and at least one substitution of the amino acid corresponding to position 456 to lysine (K).

5. In the second paragraph, a fusion protein in which the UDP-glucosyltransferase mutant and sucrose synthase are connected via a linker.

6. In the second paragraph, the fusion protein comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 5, and comprises at least one of the following substitutions: From the N-terminus of the amino acid sequence of sequence number 5, Substitution of the amino acid corresponding to position 264 with cysteine ​​(C); Substitution of the amino acid corresponding to position 301 with asparagine (N); Substitution of the amino acid corresponding to position 305 with proline (P); Substitution of the amino acid corresponding to position 376 with proline (P); and Substitution of the amino acid corresponding to position 392 with tryptophan (W).

7. In claim 6, the fusion protein further comprises at least one substitution of an amino acid corresponding to position 484 from the N-terminus of the amino acid sequence of SEQ ID NO: 5 with glutamic acid (E) and a substitution of an amino acid corresponding to position 929 with lysine (K).

8. A polynucleotide encoding a UDP-glucosyltransferase variant according to claim 1; or a fusion protein according to any one of claims 2 to 7.

9. A vector comprising a polynucleotide according to Article 8.

10. A microorganism comprising a polynucleotide according to Article 8 or a vector comprising the same.

11. In the 10th paragraph, the microorganism comprises at least one selected from the group consisting of a microorganism of the genus Escherichia, a microorganism of the genus Saccharomyces, a microorganism of the genus Bacillus, a microorganism of the genus Pichia, and a microorganism of the genus Corynebacterium.

12. A composition for producing steviol glycosides, comprising at least one selected from the group consisting of a UDP-glucosyltransferase variant according to claim 1; a fusion protein according to any one of claims 2 to 7; a microorganism comprising the variant or fusion protein; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

13. A composition in claim 12, wherein the composition or microorganism further comprises sucrose synthase.

14. In paragraph 13, the sucrose synthase (i) containing the amino acid sequence of sequence number 3, or (ii) A composition comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 3, and comprising at least one substitution of the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3 with glutamic acid (E) and the amino acid corresponding to position 456 with lysine (K).

15. In the 12th paragraph, the composition (i) stevioside, rebaudioside, or mixtures thereof, and (ii) A composition further comprising at least one selected from the group consisting of UDP, UDP-glucose, and sucrose.

16. A composition according to claim 12, wherein the composition or microorganism further comprises an enzyme that converts rebaudioside A or stevioside into rebaudioside D or rebaudioside E, respectively. 17.(i) Stevioside, rebaudioside, or mixtures thereof, and (ii) A method for producing a steviol glycoside, comprising the step of reacting at least one selected from the group consisting of UDP, UDP-glucose, and sucrose with the composition of claim 12.

18. A method according to claim 17, wherein the composition or microorganism further comprises sucrose synthase.

19. In paragraph 18, the sucrose synthase (i) containing the amino acid sequence of sequence number 3, or (ii) A method comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 3, and comprising at least one substitution of the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3 with glutamic acid (E) and at least one substitution of the amino acid corresponding to position 456 with lysine (K).

20. A method according to claim 17, wherein the composition or microorganism further comprises an enzyme that converts rebaudioside A or stevioside into rebaudioside D or rebaudioside E, respectively.

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