Sucrose synthase variant, UDP-glucosyltransferase variant, and uses thereof for producing steviol glycosides

Abstract

The present invention relates to a sucrose synthase variant, a UDP-glucosyltransferase variant, and uses thereof for producing steviol glycosides. The UDP-glucosyltransferase variant, the sucrose synthase variant, and a fusion enzyme using same according to the present invention have improved thermal stability and enzyme activity, and thus can be widely applied to the field of steviol glycoside synthesis, and have excellent industrial value and significance in being capable of increasing production efficiency and lowering production costs.

Description

Sucrose synthase mutants, UDP-glucosyltransferase mutants and their use in producing steviol glycosides

[0001] The present invention relates to a sucrose synthase mutant, a UDP-glucosyltransferase mutant, and their use in 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 D and Reb M, in high yields remains a pressing need. The development of efficient enzymes for this purpose remains essential. Furthermore, improved production of Reb D for the production of Reb M in recombinant hosts for commercial applications remains a pressing need.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] (Patent Document 1) Korean Patent Publication No. 10-2021-0114899

[0009] One object of the present invention is to provide a sucrose synthase variant.

[0010] Another object of the present invention is to provide a fusion protein comprising the sucrose synthase variant and UDP-glucosyl transferase (UGT).

[0011] Another object of the present invention is to provide a UDP-glucosyltransferase variant.

[0012] Another object of the present invention is to provide a fusion protein comprising the UDP-glucosyltransferase variant and sucrose synthase.

[0013] Another object of the present invention is to provide a polynucleotide encoding the sucrose synthase variant; the UDP-glucosyltransferase variant; or the fusion protein.

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

[0015] Another object of the present invention is to provide a microorganism comprising the polynucleotide or a vector comprising the same.

[0016] 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 fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0017] 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.

[0018] 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 fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0019] Another object of the present invention is to provide a use for producing a composition for producing steviol glycosides of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; or an extract thereof.

[0020] One aspect of the present invention provides a sucrose synthase variant comprising 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, with glutamic acid (E), and at least one substitution of an amino acid corresponding to position 456 with lysine (K).

[0021] In the present invention, "sucrose synthase (SUS)" is an enzyme that produces fructose and UDP-glucose from sucrose and UDP. Therefore, when sucrose and UDP are supplied together with SUS to a reaction in which UDP-glucosyltransferase transfers glucose from UDP-glucose to a steviol glycoside (e.g., a reaction in which Reb A is converted to Reb D or a reaction in which stevioside is converted to Reb E), SUS produces UDP-glucose from sucrose and UDP and supplies 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 above SUS can be provided on its own or in the form of a fusion protein with UDP-glucosyltransferase to produce steviol glycosides.

[0022] The "sucrose synthase variant" of the present invention refers to a variant in which, in the amino acid sequence of a polypeptide having sucrose synthase activity, 'an amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with glutamic acid (E), and / or 'an amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with lysine (K).

[0023] In the present invention, the sucrose synthase may be a polypeptide having sucrose synthase activity that is modified to produce the sucrose synthase 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 be the parent of the sucrose synthase variant of the present invention.

[0024] 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).

[0025] The sequence of the sucrose synthase of the present invention can be obtained from a known database such as NCBI's GenBank.

[0026] In addition, the sucrose synthase of the present invention may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 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: 3, and is included in the scope of sucrose synthase without limitation if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 3.

[0027] It is obvious that a polypeptide or protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application, provided that it has the homology or identity mentioned in the present invention and exhibits an activity corresponding to the polypeptide or protein. That is, even if the present application describes "a polypeptide or protein having or including an amino acid sequence described by a specific sequence number," it is obvious that a polypeptide or protein having (or including) an amino acid sequence in which some sequences are deleted, modified, substituted or added can also be used in the present application, provided that it has the same or corresponding activity as a polypeptide or protein consisting of the amino acid sequence of the corresponding sequence number.

[0028] 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, the terms "homology" and "identity" may be used interchangeably.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] In the present invention, the "corresponding amino acid at a position" refers to an amino acid residue at that 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: 3, 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: 3. For example, the position of the corresponding amino acid, or the position where a modification such as a substitution, insertion, or deletion occurs, can 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.

[0033] In one embodiment, the sucrose synthase variant 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 and has sucrose synthase activity, wherein 'the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with glutamic acid (E), and / or 'the amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with lysine (K).

[0034] In one embodiment, the sucrose synthase variant comprises an 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, wherein 'the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with glutamic acid (E), and / or 'the amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with lysine (K).

[0035] In one embodiment, the sucrose synthase variant may be, but is not limited to, one in which serine (S) at position 11 in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamic acid (E), and / or arginine (R) at position 456 is substituted with lysine (K).

[0036]

[0037] 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 specified, 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.

[0038] It is clear that a polypeptide or protein having an amino acid sequence having a deletion, modification, substitution (e.g., conservative substitution) or addition of a portion of the sequence is also included within the scope of the present invention, provided that the amino acid sequence has homology or identity as described herein and exhibits an activity corresponding to the polypeptide or protein.

[0039] 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).

[0040] Additionally, the variants of the present invention may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide, and for example, the N-terminus of the variant may be conjugated with a signal (or leader) sequence that is involved in translocation of the protein co-translationally or post-translationally.

[0041] Additionally, the variants of the present invention may be conjugated to other sequences or linkers so that they can be identified, purified, or synthesized.

[0042] 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.

[0043]

[0044] Another aspect of the present invention provides a UDP-glucosyltransferase (UGT) variant comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) substitutions of:

[0045] From the N-terminus of the amino acid sequence of sequence number 1,

[0046] Substitution of the amino acid corresponding to position 31 with leucine (L);

[0047] Substitution of the amino acid corresponding to position 97 with glutamine (Q);

[0048] Substitution of the amino acid corresponding to position 336 with leucine (L);

[0049] Substitution of the amino acid corresponding to position 362 with isoleucine (I);

[0050] Substitution of the amino acid corresponding to position 378 with methionine (M);

[0051] Substitution of the amino acid corresponding to position 344 with isoleucine (I);

[0052] Substitution of the amino acid corresponding to position 434 with lysine (K); and

[0053] Substitution of the amino acid corresponding to position 444 with leucine (L).

[0054] In the present invention, the "UDP-glucosyltransferase (UGT)" is not limited as long as it has an activity of transferring glucose to a substrate in the presence of a glucose donor, and may be a naturally occurring polypeptide or a wild-type polypeptide, and includes a mutant or functional fragment thereof. The UDP-glucosyltransferase can transfer glucose to a steviol glycoside to produce another steviol glycoside.

[0055] 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, and Reb M, etc.), or mixtures thereof.

[0056] In one embodiment, the UDP-glucosyltransferase according to the present invention has an activity of converting a substrate comprising stevioside, Reb A, or a mixture thereof to at least one selected from the group consisting of Reb D and Reb E, and specifically, can convert glucose to Reb A to Reb D, and can convert glucose to stevioside to Reb E. That is, the UDP-glucosyltransferase has a conversion activity for both single substrates of Reb A and stevioside, or mixed substrates.

[0057] In the present invention, the "UDP-glucosyltransferase variant" may be a variant in which 'an amino acid corresponding to one or more of positions 31, 97, 336, 362, 378, 344, 434 and 444 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. The other substituted amino acid may be, for example, leucine, glutamine, isoleucine, methionine and lysine.

[0058] 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.

[0059] 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.

[0060] The sequence of the UDP-glucosyltransferase of the present invention can be obtained from a known database such as NCBI's GenBank.

[0061] 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.

[0062] 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.

[0063]

[0064] 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% homology or identity with the amino acid sequence of SEQ ID NO: 1 and has UDP-glucosyltransferase activity, wherein 'an amino acid corresponding to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) positions from the N-terminus of the amino acid sequence of SEQ ID NO: 1' is substituted as follows:

[0065] From the N-terminus of the amino acid sequence of sequence number 1,

[0066] Substitution of the amino acid corresponding to position 31 with leucine (L);

[0067] Substitution of the amino acid corresponding to position 97 with glutamine (Q);

[0068] Substitution of the amino acid corresponding to position 336 with leucine (L);

[0069] Substitution of the amino acid corresponding to position 362 with isoleucine (I);

[0070] Substitution of the amino acid corresponding to position 378 with methionine (M);

[0071] Substitution of the amino acid corresponding to position 344 with isoleucine (I);

[0072] Substitution of the amino acid corresponding to position 434 with lysine; and

[0073] Substitution of the amino acid corresponding to position 444 with leucine (L).

[0074] In one embodiment, the UDP-glucosyltransferase variant may be a variant in which 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, wherein 'an amino acid corresponding to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) positions from the N-terminus of the amino acid sequence of SEQ ID NO: 1' is substituted as described above.

[0075] 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:

[0076] No. 31 and No. 97;

[0077] No. 31 and No. 336;

[0078] No. 31 and No. 362;

[0079] No. 31 and No. 378;

[0080] No. 31 and No. 344;

[0081] No. 31 and No. 434;

[0082] No. 31 and No. 444;

[0083] No. 31, No. 362 and No. 434;

[0084] No. 31, No. 344, No. 362 and No. 434;

[0085] No. 31, No. 344, No. 362, No. 434 and No. 444;

[0086] No. 31, No. 336, No. 344, No. 362, No. 434 and No. 444;

[0087] 31, 97, 336, 344, 362, 434, and 444; or

[0088] Numbers 31, 97, 336, 344, 362, 378, 434, and 444.

[0089] For example, from the N-terminus of the amino acid sequence of sequence number 1

[0090] The amino acid corresponding to position 31 is isoleucine (I).

[0091] The amino acid corresponding to position 97 is arginine (R).

[0092] The amino acid corresponding to position 336 is threonine (T).

[0093] The amino acid corresponding to position 362 is threonine (T).

[0094] The amino acid corresponding to position 378 is serine (S).

[0095] The amino acid corresponding to position 344 is valine (V).

[0096] The amino acid corresponding to position 434 is threonine (T).

[0097] The amino acid corresponding to position 444 may be cysteine ​​(C).

[0098] In one embodiment, the UDP-glucosyltransferase variant may further comprise one or more substitutions of the amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of SEQ ID NO: 1, a substitution of serine (S) for the amino acid corresponding to position 202, and a substitution of leucine (L) for the amino acid corresponding to position 202.

[0099] For example, the amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of sequence number 1 may be threonine (T), and the amino acid corresponding to position 202 may be valine (V).

[0100]

[0101] Another aspect of the present invention provides a fusion protein comprising UDP-glucosyltransferase and sucrose synthase.

[0102] When UDP-glucosyltransferase and sucrose synthase are produced as a fusion protein and the conversion reaction of the steviol glycoside is performed, the two enzymatic reactions can occur simultaneously by forming a single channel, thereby causing an efficient conversion reaction. That is, when the fusion enzyme is used, economical substrate conversion is possible, and steviol glycosides can be produced through a high-concentration substrate reaction. Specifically, since the sucrose synthase of the fusion protein produces UDP-glucose from UDP, steviol glycosides can be produced even without supplying UDP-glucose when producing steviol glycosides using the fusion protein of the present invention. The supply may be, for example, separately adding or adding UDP-glucose during the production of steviol glycosides. In addition, the supply may include, but is not limited to, contacting a medium containing a starting material with the UDP-glucose to produce steviol glycosides. In addition, when UDP-glucose is supplied, glucose is transferred from UDP-glucose to steviol glycoside to produce UDP, and the sucrose synthase of the fusion protein produces UDP-glucose from the produced UDP. Therefore, when producing steviol glycoside using the fusion protein of the present invention, steviol glycoside can be produced even without supplying UDP. In other words, the fusion protein of the present invention can produce steviol glycoside without the addition of UDP-glucose or UDP.

[0103] In one embodiment, the fusion protein of the present invention may comprise a sucrose synthase variant according to the present invention and a UDP-glucosyltransferase or a variant thereof. The UDP-glucosyltransferase or a variant thereof is as described above. For example, the C-terminus or N-terminus of the sucrose synthase variant may be linked directly or via a linker to the N-terminus or C-terminus of the UDP-glucosyltransferase or a variant thereof.

[0104] In one embodiment, the fusion protein of the present invention may comprise a UDP-glucosyltransferase variant according to the present invention and a sucrose synthase or variant thereof. The sucrose synthase or variant thereof is as described above. For example, the C-terminus or N-terminus of the sucrose synthase or variant thereof may be linked directly or via a linker to the N-terminus or C-terminus of the UDP-glucosyltransferase variant.

[0105] In one embodiment, the UDP-glucosyltransferase or variant thereof can be fused directly or via a linker to the sucrose synthase or variant thereof. The linker can be a peptide consisting of 4 to 15 amino acids, and specifically can include 4 to 15 amino acids selected from the group consisting of G (Gly), S (Ser), and P (Pro), but is not limited thereto. For example, it can be GGGS (SEQ ID NO: 8), GGGGS (SEQ ID NO: 9), GGGSGGGGS (SEQ ID NO: 10), GGGGPSPGGGGS (SEQ ID NO: 11), or GGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0106] In one embodiment, the fusion protein of the present invention comprises:

[0107] A variant having 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: 5 (a total of 1281 amino acids; UGT enzyme of SEQ ID NO: 1 at positions 1-461 + linker of SEQ ID NO: 7 at positions 462-476 + SUS of SEQ ID NO: 3 at positions 477-1281), and having UDP-glucosyltransferase and sucrose synthase activities,

[0108] (a) at least one substitution of 'an amino acid corresponding to position 487 from the N-terminus of the amino acid sequence of SEQ ID NO: 5 (i.e., position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3)' with glutamic acid (E) and 'an amino acid corresponding to position 932 from the N-terminus of the amino acid sequence of SEQ ID NO: 5 (i.e., position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3)' with lysine (K);

[0109] (b) a substitution of the amino acid corresponding to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) positions of the amino acid sequence of SEQ ID NO: 5 from the N-terminus: 31, 97, 336, 362, 378, 344, 434 and 444' as follows:

[0110] From the N-terminus of the amino acid sequence of sequence number 5,

[0111] Substitution of the amino acid corresponding to position 31 with leucine (L);

[0112] Substitution of the amino acid corresponding to position 97 with glutamine (Q);

[0113] Substitution of the amino acid corresponding to position 336 with leucine (L);

[0114] Substitution of the amino acid corresponding to position 362 with isoleucine (I);

[0115] Substitution of the amino acid corresponding to position 378 with methionine (M);

[0116] Substitution of the amino acid corresponding to position 344 with isoleucine (I);

[0117] Substitution of the amino acid corresponding to position 434 with lysine (K); and

[0118] Substitution of the amino acid corresponding to position 444 with leucine (L); or

[0119] (c) both (a) and (b)

[0120] may include.

[0121] In one embodiment, the fusion protein of the present invention may be a variant comprising a substitution of (a) to (c) 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.

[0122] For example, the substitution in (b) above may include substitution of amino acids corresponding to the following two or more positions of the amino acid sequence of SEQ ID NO: 5:

[0123] No. 31 and No. 97;

[0124] No. 31 and No. 336;

[0125] No. 31 and No. 362;

[0126] No. 31 and No. 378;

[0127] No. 31 and No. 344;

[0128] No. 31 and No. 434;

[0129] No. 31 and No. 444;

[0130] No. 31, No. 362 and No. 434;

[0131] No. 31, No. 344, No. 362 and No. 434;

[0132] No. 31, No. 344, No. 362, No. 434 and No. 444;

[0133] No. 31, No. 336, No. 344, No. 362, No. 434 and No. 444;

[0134] 31, 97, 336, 344, 362, 434, and 444; or

[0135] Numbers 31, 97, 336, 344, 362, 378, 434, and 444.

[0136] For example, from the N-terminus of the amino acid sequence of sequence number 5

[0137] The amino acid corresponding to position 487 is serine (S).

[0138] The amino acid corresponding to position 932 is arginine (R),

[0139] The amino acid corresponding to position 31 is isoleucine (I).

[0140] The amino acid corresponding to position 97 is arginine (R).

[0141] The amino acid corresponding to position 336 is threonine (T).

[0142] The amino acid corresponding to position 362 is threonine (T).

[0143] The amino acid corresponding to position 378 is serine (S).

[0144] The amino acid corresponding to position 344 is valine (V).

[0145] The amino acid corresponding to position 434 is threonine (T).

[0146] The amino acid corresponding to position 444 may be cysteine ​​(C).

[0147] In one embodiment, the fusion protein of the present invention may further comprise at least one substitution of the amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of SEQ ID NO: 5 with serine (S) and the amino acid corresponding to position 202 with leucine (L).

[0148] For example, the amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of SEQ ID NO: 5 may be threonine (T), and the amino acid corresponding to position 202 may be valine (V).

[0149]

[0150] Another aspect of the present invention provides a polynucleotide encoding a sucrose synthase variant according to the present invention; a UDP-glucosyltransferase variant according to the present invention; or a fusion protein according to the present invention.

[0151] The polynucleotide of the present invention may 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 invention is to be expressed.

[0152] The polynucleotide of the present invention may be DNA or RNA.

[0153]

[0154] Another aspect of the present invention provides a vector comprising a polynucleotide according to the present invention.

[0155] 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.

[0156] 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 a self-replicating expression vector. 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.

[0157] 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.

[0158] 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.

[0159] The vector of the present invention may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0160] 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.

[0161]

[0162] Another aspect of the present invention provides a microorganism comprising a polynucleotide according to the present invention or a vector comprising the same.

[0163] 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.

[0164] 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).

[0165]

[0166] Another aspect of the present invention provides a composition for producing steviol glycosides, comprising at least one selected from the group consisting of a fusion protein according to the present invention; a UDP-glucosyltransferase variant according to the present invention; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0167] When the composition or microorganism comprises the fusion protein, since the sucrose synthase of the fusion protein produces UDP-glucose from UDP, steviol glycosides can be produced even without externally supplying UDP-glucose when producing steviol glycosides using the composition of the present invention. The supply may be, for example, separately adding or adding UDP-glucose when producing steviol glycosides. In addition, the supply may include, but is not limited to, contacting a medium containing a starting material with the UDP-glucose to produce steviol glycosides. In addition, when UDP-glucose is supplied, glucose is transferred from UDP-glucose to steviol glycosides to produce UDP, and since the sucrose synthase of the fusion protein produces UDP-glucose from the produced UDP, steviol glycosides can be produced even without supplying UDP when producing steviol glycosides using the composition of the present invention. That is, when the composition for producing steviol glycosides of the present invention or the microorganism included therein comprises the fusion protein of the present invention, the composition for producing steviol glycosides of the present invention is capable of producing steviol glycosides without the addition of UDP-glucose or UDP.

[0168] 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.

[0169] 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.

[0170] 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.

[0171]

[0172] The composition of the present invention may further comprise, as a substrate, (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, but is not limited thereto. The steviol glycoside of (i) and the steviol glycoside produced using the same are as described below.

[0173] The composition or microorganism of the present invention may further comprise sucrose synthase or a variant thereof. The sucrose synthase variant is as described above, and specifically, may be one in which the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3 is substituted with glutamic acid and / or the amino acid corresponding to position 456 is substituted with lysine. When the microorganism comprises sucrose synthase or a variant thereof, the microorganism comprises a polynucleotide encoding the sucrose synthase or a variant thereof, and thus may express the enzyme, but is not limited thereto.

[0174] The composition or microorganism of the present invention may further comprise an 'enzyme that converts Reb D or Reb E into Reb M' to produce Reb M. For example, the composition may comprise a second UDP-glucosyltransferase, such as UGT76G1. The UGT76G1 is a stevia-derived UDP-glycosyltransferase known to have a wide substrate specificity, and has, for example, an activity of converting stevioside into Reb A, an activity of converting Reb A into Reb I, an activity of converting Reb E into Reb D, and / or an activity of converting Reb D into Reb M. When the microorganism further comprises an 'enzyme that converts Reb D or Reb E into Reb M', the microorganism may comprise a polynucleotide encoding the 'enzyme that converts Reb D or Reb E into Reb M', and may thus express the enzyme, but is not limited thereto.

[0175]

[0176] 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.

[0177] 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.

[0178] When the composition or microorganism comprises the fusion protein, since the sucrose synthase of the fusion protein produces UDP-glucose, steviol glycosides can be produced even without externally supplying UDP-glucose when producing steviol glycosides according to the method of the present invention. The supply may be, for example, separately adding or adding UDP-glucose when producing steviol glycosides. In addition, the supply may include, but is not limited to, contacting a medium containing a starting material with the UDP-glucose to produce steviol glycosides. In addition, when UDP-glucose is supplied, glucose is transferred from UDP-glucose to steviol glycosides to produce UDP, and since the sucrose synthase of the fusion protein produces UDP-glucose from the produced UDP, steviol glycosides can be produced even without supplying UDP when producing steviol glycosides according to the method of the present invention. That is, when the composition for producing steviol glycosides of the present invention or the microorganism included therein comprises the fusion protein of the present invention, the method for producing steviol glycosides of the present invention enables production of steviol glycosides without the addition of UDP-glucose or UDP.

[0179] 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.

[0180] In one embodiment, the method of the present invention can produce Reb E from stevioside and / or Reb D from Reb A.

[0181] 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.

[0182] The method of the present invention may further include a step [step (b)] of reacting the steviol glycoside produced in step (a) with an 'enzyme that converts Reb D or Reb E into Reb M' to produce Reb M. The 'enzyme that converts Reb D or Reb E into Reb M' is as described above. In the present invention, step (b) may be performed simultaneously with step (a) or after step (a). In this case, the method of the present invention can produce Reb M from steviol glycoside.

[0183] 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, or 45 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.

[0184] The method of the present invention may further include a step of recovering the steviol glycoside produced from the reactant.

[0185] 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.

[0186] Another aspect of the present invention is a use for producing steviol glycosides, comprising a composition comprising at least one selected from the group consisting of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

[0187] Another aspect of the present invention is the use of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; or an extract thereof for producing a composition for producing steviol glycosides.

[0188] The production of the above fusion protein, UDP-glucosyltransferase mutant, microorganism, culture, lysate, extract, and steviol glycosides is as described above.

[0189] The sucrose synthase variant, UDP-glucosyltransferase variant and fusion enzyme using the same according to the present invention 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.

[0190] Figure 1 shows the results comparing the production of Reb E and D using TaUGT-GmSUS (template) and mutants.

[0191] 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.

[0192]

[0193] Example 1. Production of microorganisms expressing primary mutant TaUGT.

[0194]

[0195] 1-1: Construction of a vector for the expression of the first mutant TaUGT

[0196] A gene encoding a wild-type UDP-glucosyltransferase (TaUGT) enzyme protein having an amino acid sequence of sequence number 1 derived from Triticum aestivum was synthesized (Gblock synthesis), and the polynucleotide sequence of the synthesized gene is shown in sequence number 2.

[0197] A primary mutant TaUGT was prepared in which threonine at position 201 was substituted with serine and valine at position 202 was substituted with leucine in sequence number 1. Specifically, primers (SEQ ID NOs: 48 and 49) at the corresponding positions of TaUGT were synthesized, and amplification of the mutant gene (PCR) that changed the base sequence of the corresponding region was performed, and the amplified gene fragment was cloned into the pRS426 vector through Gibson assembly. The resulting mutant cloned into the pRS426 vector was called TaUGT. T201S / V202L It was named as such, and its amino acid sequence and base sequence are shown in sequence numbers 50 and 51, respectively.

[0198]

[0199]

[0200]

[0201]

[0202] 1-2: Production and cultivation of primary mutant TaUGT-expressing microorganisms

[0203]

[0204] The pRS426 vector prepared in Example 1-1 was transformed into the S. cerevisiae CENPK2-1c (Euroscarf) strain. Recombinant strains with inserted gene cassettes were selected using a selection marker on SC-Ura solid medium (SC_Ura medium + 2% agar). The composition of SC-Ura solid medium included YNB 6.7 g / L, Drop out mix 0.7 g / L, Glucose 50 g / L, and agar 20 g / L, and the pH was adjusted to 6.0.

[0205] Colonies selected from the SC_Ura solid medium were inoculated into test tubes containing 3 mL of SC_Ura liquid medium and cultured overnight. The concentration of cultured cells was determined by OD 600 Measure the absorbance to confirm, and OD into a 250 mL flask containing 25 mL of SC-Ura liquid medium. 600 The cells were inoculated until the absorbance reached 0.1 (final) and cultured for 24 hours (30°C, 240 rpm). To induce enzyme expression of the primary cultured cells, an equal amount of YPG medium was added and cultured for a second time for 24 hours. The secondary cultured cells were collected by centrifugation (4,000 rpm, 10 min). The composition of the SC_Ura liquid medium included 6.7 g / L of YNB (yeast nitrogen base), g / L of Drop-out mix, g / L of Glucose, and mM of MES, and the pH of the medium was adjusted to 6.0 using NaOH. The composition of the YPG medium included 20 g / L of Bacto peptone, 10 g / L of Yeast Extract, 20 g / L of Galactose, and 100 mM of Phosphate buffer (sodium salt), and the pH of the medium was adjusted to 6.0.

[0206]

[0207] Example 2. Production and cultivation of microorganisms expressing the fusion enzyme, TaUGT-GmSUS.

[0208]

[0209] 2-1: Construction of a vector expressing the fusion enzyme, TaUGT-GmSUS

[0210] The first mutant TaUGT (TaUGT) prepared in Example 1 T201S / V202L: The gene encoding the wild-type sucrose synthase (GmSUS) enzyme protein having the amino acid sequence of SEQ ID NO: 3 derived from Glycine max and the gene encoding TaUGT-GmSUS (SEQ ID NO: 50) were synthesized by codon optimization for optimal expression in yeast (IDT, Gblock synthesis), and the base sequences of the synthesized genes are shown in SEQ ID NOs: 51 and 4, respectively. To fuse the synthesized genes, GGGGSGGGGSGGGGS (SEQ ID NO: 7) was used as a linker. The enzyme gene was amplified through PCR amplification, and the genes were fused and subcloned using Gibson assembly (2X HiFi DNA master mix, NEW ENGLAND BIOLABS). The amino acid sequence of the fused TaUGT-GmSUS is shown in SEQ ID NO: 52, and the base sequence is shown in SEQ ID NO: 53. In addition, the amino acid sequence and base sequence of the fusion enzyme TaUGT-GmSUS, in which wild-type TaUGT (SEQ ID NO: 1) and wild-type GmSUS (SEQ ID NO: 3) are connected by the same linker, are shown in SEQ ID NOs: 5 and 6, respectively. Specifically, the vector manufactured to confirm the activity of the protein was the pRS426 vector containing the Ura auxotrophic marker (selectable marker) that can be selected in S. cerevisiae, and the pRS426_TaUGT-GmSUS recombinant vector was constructed. The pRS426 vector was cut using restriction enzymes XhoI and EcoRI, and the amplified genes and the restriction enzyme-treated pRS426 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 enzyme was accurately subcloned.

[0211] 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.

[0212] The prepared recombinant plasmid was transformed into the S. cerevisiae CENPK2-1c△gal80△SUC2 strain. Recombinant strains with inserted gene cassettes were selected through selection markers on SC-Ura solid medium (SC-Ura medium + 2% agar). The composition of SC-Ura solid medium was YNB (Difco TM ) 6.7 g / L, CSM-Ura 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.

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] - In sequence numbers 5, 6, 52 and 53, bold indicates the sequence of TaUGT, underline indicates the sequence of the linker, and the remaining portion indicates the sequence of GmSUS.

[0223]

[0224] 2-2: Mutation of the fusion enzyme, TaUGT-GmSUS gene

[0225] Based on the amino acid sequence analysis of homologous genes and the analysis of the 3D structure model of the active site and metal binding site, 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 TaUGT-GmSUS enzyme were analyzed. The amino acid candidates identified through the analysis are I31L, T434K, T362I, V344I, S487E, R932K, C444L, T336L, R97Q, and S378M. In order to introduce additional substitution mutations into the primary mutant TaUGT-GmSUS, pRS426_TaUGT-GmSUS of Example 2-1 was used as a template DNA. In order to substitute each amino acid residue (I31L, T434K, T362I, S487E, V344I, R932K, C444L, T336L, R97Q, S378M), two PCR fragments that overlapped with the substituted amino acids were constructed. Specifically, to construct mutant fragment 1, a polymerase chain reaction (PCR) reaction solution containing 10 ng of pRS426_TaUGT-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 was prepared in a final volume of 50 μl and amplified by PCR. Then, to construct mutant fragment 2, PCR was amplified by changing the sequence of the primers. The primer information used is shown in Table 3 below. The pRS426 vector was cut using restriction enzymes XhoI and EcoRI, and then the amplified Fragment 1 and Fragment 2 were inserted into the 2X HiFi DNA master mix (NEB) to create the pRS426 / TaUGT-GmSUS mutant vector. In the case of a combination of two or more mutants, the template DNA was changed and created using the same method as above.For sequence verification and transformation, it is the same as Example 2-1.

[0226]

[0227]

[0228] 2-3: Microbial cultivation

[0229] The yeast selected through transformation in Examples 2-1 and 2-2 were induced to express enzymes through liquid culture. Specifically, the colonies selected from the SC-URA 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.

[0230]

[0231] Example 3. Measurement of enzyme activity of TaUGT-GmSUS and mutants against RA60 raw material.

[0232]

[0233] 3-1: Freeze-drying process of microorganisms expressing TaUGT-GmSUS and mutant enzymes

[0234] To perform whole-cell reactions using yeast expressing the primary mutant TaUGT-GmSUS manufactured in Example 2-1 and the mutant enzyme further mutated in Example 2-2, cells recovered after culture in Example 2-3 were freeze-dried. The freeze-drying method was performed under vacuum, and the temperature change is shown below.

[0235] ① Change the temperature from 20℃ to -40℃ for 0 to 30 minutes

[0236] ② Maintain at -40℃ for 30 to 270 minutes

[0237] ③ Change the temperature to -30℃ for 270 to 510 minutes

[0238] ④ Change the temperature to -20℃ for 510 to 750 minutes

[0239] ⑤ Change the temperature to -10℃ for 750 to 990 minutes

[0240] ⑥ Change the temperature to 0℃ for 990 to 1230 minutes

[0241] ⑦ Change the temperature to 10℃ for 1230 to 1470 minutes

[0242] ⑧ Temperature change of 25℃ for 1470 to 1710 minutes

[0243] ⑨ Maintain 25℃ for 1710 to 1950 minutes

[0244]

[0245] 3-2: Measurement of enzyme activity of lyophilized strains expressing TaUGT-GmSUS and mutant enzymes

[0246] In order to measure the enzyme activity of the freeze-dried strain expressing the TaUGT-GmSUS and mutant enzyme obtained in Example 3-1, the productivity of Rebaudioside E and Rebaudioside D was compared and confirmed through whole-cell reaction.

[0247] To prepare a reaction solution for enzyme activity evaluation, substrate RA60 (95%, Sinochem) containing 60% Rebaudioside A and 40% Stevioside, UDP, Sucrose, EDTA, and lyophilized cells were dissolved in 50 mM phosphate buffer. A total reaction volume of 1 mL was prepared. The final reaction solution contained 10 g / L of substrate RA60 (95%, Sinochem), 0.3 mM UDP, 750 mM Sucrose, 5 mM EDTA, and 9.3 g / L of lyophilized 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.

[0248] 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 E and Reb A into Reb D, the enzyme activity was measured using the ratio of Reb E and Reb D production.

[0249] 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 4 below.

[0250] Time (min)0.01% TFA (%)Acetonitrile (%)0802012643617643622010027010027.18020348020

[0251]

[0252] The results of the above analysis are shown in Table 5 below. In Table 5 below, the substrate conversion activity is expressed as relative conversion activity based on 100% substrate conversion activity of the strain producing the template enzyme.

[0253] Genetic relative conversion activity (%) Primary mutation TaUGT-GmSUS (template) 100 TaUGT-GmSUS (I31L) 112 TaUGT-GmSUS (V344I) 102 TaUGT-GmSUS (T362I) 116 TaUGT-GmSUS (T434K) 112 TaUGT-GmSUS (S487E) 111 TaUGT-GmSUS (R932K) 121 TaUGT-GmSUS (I 31L-R932K)131TaUGT-GmSUS(I31L-S487E-R932K)138TaUGT-GmSUS(I31L-S487E-R932 K-T434K)144TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I)155TaUGT-GmSUS(I31L-S 487E-R932K-T434K-T362I-V344I)157TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I -V344I-C444L)172TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336 L)188TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q)426 TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q-S378M)551

[0254]

[0255] As a result of confirming the substrate conversion activity of the enzyme using a strain producing the above enzyme, a recombinant strain expressing the I31L, T434K, T362I, V344I, and R932K mutant enzymes showed an increased activity of up to 121% or more compared to a recombinant strain expressing the template enzyme, and when the mutations were combined (I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q-S378M), an increased conversion activity of up to 551% compared to a recombinant strain expressing the template enzyme was confirmed.

[0256]

[0257] 3-3: Confirmation of the thermal stability of the lyophilized strain expressing TaUGT-GmSUS and the mutant enzyme.

[0258] To test the increased thermostability of strains producing selected mutant enzymes, whole-cell reactions were performed at temperatures higher than 45°C using strains in which enzyme mutations were confirmed in Example 3-2. Specifically, the reaction temperature was increased from 45°C to 50°C, 55°C, and 60°C to identify the optimal temperature for enzyme activity.

[0259] A reaction solution substantially identical to that in Example 3-2 was prepared, and an enzyme reaction was performed on the prepared reaction solution under the conditions of 45°C, 50°C, 55°C, and 60°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 identify the whole-cell reaction product. The whole-cell reaction product was analyzed in the same manner as in Example 3-2. The analysis results are shown in Table 6 below.

[0260] 유전자상대적 전환 활성 (%)45℃50℃55℃60℃TaUGT-GmSUS (주형)100900TaUGT-GmSUS(I31L)1004220.30TaUGT-GmSUS(V344I)1003314.40TaUGT-GmSUS(T362I)10052280TaUGT-GmSUS(T434K)1003413.80TaUGT-GmSUS(S487E)1009.100TaUGT-GmSUS(R932K)100900TaUGT-GmSUS(I31L-R932K)10042.220.50TaUGT-GmSUS(I31L-S487E-R932K)10043.121.20TaUGT-GmSUS(I31L-S487E-R932K-T434K)10071.636.24.2TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I)81100587.4TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I)8010071.313.8TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L)66.385.710036TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L)66.284.510035TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q)65.983.210059.8TaUGT-GmSUS(I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q-S378M)65.281.910079.9

[0261]

[0262] As a result of confirming the substrate conversion activity of the enzyme according to temperature using the strain producing the above enzyme, in the case of the recombinant strain expressing the template enzyme, it was confirmed that 9% of the activity was shown at 50°C compared to 45°C, and no activity was shown at 55°C and 60°C, but when the mutations were combined (I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q-S378M), the highest activity was shown at 55°C.

[0263]

[0264] Example 4. Comparison of Reb E and D production using TaUGT-GmSUS and mutants.

[0265]

[0266] To test the productivity of Reb E and D of the strain producing the selected mutant enzyme, whole-cell reactions were performed at 45°C and 55°C using the strain in which the enzyme mutation was confirmed in Example 3-2 and the strain containing the template enzyme. The reaction solution to confirm the productivity of Reb E and D contained substrate RA60 (95%, Sinochem) containing 60% Rebaudioside A and 40% Stevioside, UDP, Sucrose, EDTA, and lyophilized cells. A total reaction volume of 100 mL was prepared. The final reaction solution contained 50 g / L of substrate RA60 (95%, Sinochem), 0.3 mM UDP, and 750 mM Sucrose, 5 mM EDTA, and 15.3 g / L of lyophilized cells. Enzyme reaction was performed on the above-prepared reaction solution at a temperature of 45°C or 55°C, pH 7.2, and 150 rpm, and the degree of reaction was checked from 30 minutes to 180 minutes after the start of the reaction.

[0267] 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 product. The analysis results are shown in Figure 1.

[0268] As a result of confirming the productivity of Reb E and D using the strain producing the above enzyme, in the case of the recombinant strain expressing the template enzyme, 10.3 g / L of Reb E and D were produced at 45°C in 180 minutes, and 0 g / L of Reb E and D were produced at 55°C in 180 minutes. In the case of the recombinant strain expressing the I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q-S378M mutant enzyme, 39.2 g / L of Reb E and D were produced at 45°C in 180 minutes, and 59 g / L of Reb E and D were produced at 55°C in 180 minutes.

[0269]

[0270] 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 sucrose synthase variant comprising an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 3, wherein the sucrose synthase 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 a substitution of an amino acid corresponding to position 456 to lysine (K).

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

3. In the second paragraph, the fusion protein is a fusion protein capable of producing steviol glycosides without addition of UDP-glucose or UDP.

4. In the second paragraph, the UDP-glucosyltransferase is a fusion protein derived from a plant of the genus Triticum, a plant of the genus Stevia, a plant of the genus Oryza, or a plant of the genus Hordeum.

5. In the second paragraph, the UDP-glucosyltransferase is a fusion protein comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1, and a variant comprising at least one of the following substitutions: From the N-terminus of the amino acid sequence of sequence number 1, Substitution of the amino acid corresponding to position 31 with leucine (L); Substitution of the amino acid corresponding to position 97 with glutamine (Q); Substitution of the amino acid corresponding to position 336 with leucine (L); Substitution of the amino acid corresponding to position 362 with isoleucine (I); Substitution of the amino acid corresponding to position 378 with methionine (M); Substitution of the amino acid corresponding to position 344 with isoleucine (I); Substitution of the amino acid corresponding to position 434 with lysine (K); and Substitution of the amino acid corresponding to position 444 with leucine (L).

6. A fusion protein in the second paragraph, wherein the sucrose synthase mutant and UDP-glucosyltransferase are connected via a linker.

7. In the second paragraph, the fusion protein comprises an amino acid sequence having 85% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, and comprises at least one substitution of an amino acid corresponding to position 487 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 932 with lysine (K).

8. In the 7th paragraph, the fusion protein further comprises one or more 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 31 with leucine (L); Substitution of the amino acid corresponding to position 97 with glutamine (Q); Substitution of the amino acid corresponding to position 336 with leucine (L); Substitution of the amino acid corresponding to position 362 with isoleucine (I); Substitution of the amino acid corresponding to position 378 with methionine (M); Substitution of the amino acid corresponding to position 344 with isoleucine (I); Substitution of the amino acid corresponding to position 434 with lysine (K); and Substitution of the amino acid corresponding to position 444 with leucine (L).

9. A UDP-glucosyltransferase variant comprising an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the UDP-glucosyltransferase variant comprises at least one of the following substitutions: From the N-terminus of the amino acid sequence of sequence number 1, Substitution of the amino acid corresponding to position 31 with leucine (L); Substitution of the amino acid corresponding to position 97 with glutamine (Q); Substitution of the amino acid corresponding to position 336 with leucine (L); Substitution of the amino acid corresponding to position 362 with isoleucine (I); Substitution of the amino acid corresponding to position 378 with methionine (M); Substitution of the amino acid corresponding to position 344 with isoleucine (I); Substitution of the amino acid corresponding to position 434 with lysine (K); and Substitution of the amino acid corresponding to position 444 with leucine (L).

10. A fusion protein comprising a UDP-glucosyltransferase variant according to claim 9 and sucrose synthase.

11. In claim 10, the fusion protein is a fusion protein capable of producing steviol glycosides without addition of UDP-glucose or UDP.

12. A polynucleotide encoding a sucrose synthase variant according to claim 1; a fusion protein according to any one of claims 2 to 8, 10 and 11; or a UDP-glucosyltransferase variant according to claim 9.

13. A vector comprising a polynucleotide according to Article 12.

14. A microorganism comprising a polynucleotide according to Article 12 or a vector comprising the same.

15. In claim 14, the microorganism comprises at least one selected from the group consisting of microorganisms of the genus Escherichia, microorganisms of the genus Saccharomyces, microorganisms of the genus Bacillus, microorganisms of the genus Pichia, and microorganisms of the genus Corynebacterium.

16. A composition for producing steviol glycosides, comprising at least one selected from the group consisting of a fusion protein according to any one of claims 2 to 8, 10 and 11; a UDP-glucosyltransferase variant according to claim 9; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.

17. A composition in accordance with claim 16, wherein the composition or microorganism comprises the fusion protein, and is capable of producing steviol glycosides without addition of UDP-glucose or UDP.

18. In paragraph 16, the composition or microorganism further comprises sucrose synthase or a variant thereof, A composition wherein the mutant comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 3, and 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 and a substitution of an amino acid corresponding to position 456 to lysine.

19. In paragraph 16, 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.

20. A composition in claim 16, wherein the composition or microorganism further comprises an enzyme that converts rebaudioside D or rebaudioside E into rebaudioside M. 21.(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 16.

22. In the 21st paragraph, a method for producing steviol glycosides without addition of UDP-glucose or UDP, when the composition or microorganism comprises the fusion protein.

23. In paragraph 21, the composition or microorganism further comprises sucrose synthase or a variant thereof, A method wherein the variant comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 3, and 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 and a substitution of an amino acid corresponding to position 456 to lysine.

24. A method according to claim 21, wherein the composition or microorganism further comprises an enzyme that converts rebaudioside D or rebaudioside E into rebaudioside M.

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