Glycosyltransferase mutants and method for producing steviol glycosides using the same

Abstract

The present invention relates to glycosyltransferase mutants, and compositions and methods for producing steviol glycosides using the same. More specifically, the present invention relates to rebaudioside glycosyltransferases and mutant enzymes that transfer glucose to steviol glycosides, recombinant strains that express the enzymes, and methods for producing steviol glycosides using the same.

Description

[Technical field]

[0001] The present invention relates to a glycosyltransferase, and a composition for producing steviol glycoside and a production method thereof using the same. More specifically, the present invention relates to a rebaudioside glycosyltransferase that transfers glucose to steviol glycoside, a recombinant strain expressing the enzyme, and a method for producing steviol glycoside rebaudioside using the same. [Background technology]

[0002] Sweeteners are known as the most commonly used ingredients in the food, beverage, or confectionery industries. They can be incorporated into the final food product during manufacturing, or used at home as a table sweetener or sugar substitute in baking when appropriately diluted for standalone use. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey, and artificial sweeteners such as aspartame, saccharin, and sucralose.

[0003] Stevia extract is a natural sweetener that can be extracted from the perennial shrub Stevia rebaudiana. Stevia extracts of various levels of purification are used as sensitivity flavorings in foods and blends, and are commercially available alone as table sweeteners. Summary of the Invention [Problem to be solved by the invention]

[0004] Extracts of the stevia plant contain rebaudiosides and other steviol glycosides that contribute sweetness, although conventional commercial products contain primarily rebaudioside A with smaller amounts of other glycosides such as rebaudioside C, D, and F. Stevia extracts extracted from the plant may contain contaminants such as derived compounds that cause off-flavors. Such off-flavors may be problematic depending on the food system or application selected. The composition of stevia extracts is highly variable depending on the soil and climate in which the plant is grown. Depending on the source plant, climatic conditions, and extraction process, the amount of rebaudioside A is reported to vary from 20 to 97% of the total steviol glycoside content in commercial production. Other steviol glycosides are present in varying amounts in stevia extracts. Since stevia extracts produced from stevia plants contain various steviol glycosides and compounds that cause off-flavors, and the recovery and purification are labor-intensive and inefficient, there remains a need for recombinant production systems that can accumulate steviol glycosides such as Reb D and Reb M in high yields. For this purpose, there remains a need for the development of efficient enzymes. There also remains a need for improved production of Reb D for the production of steviol glycoside Reb M from recombinant hosts for commercial use. [Means for solving the problem]

[0005] One embodiment of the present invention relates to a glycosyltransferase that transfers glucose to steviol glycosides, a nucleic acid molecule encoding the enzyme protein, a recombinant vector containing the nucleic acid molecule, and a transformed microorganism.

[0006] One example of the present invention relates to a composition for producing steviol glycoside, comprising one or more selected from the group consisting of a glycosyltransferase protein that transfers glucose to the steviol glycoside, a recombinant microorganism that expresses the enzyme protein, a bacterial cell of the microorganism, a disrupted bacterial cell of the microorganism, a culture of the microorganism, and extracts thereof.

[0007] One example of the present invention relates to a method for producing steviol glycoside, which includes a step of reacting one or more selected from the group consisting of a glycosyltransferase protein that transfers glucose to the steviol glycoside, a recombinant microorganism expressing the enzyme protein, a bacterial cell of the microorganism, a disrupted bacterial cell of the microorganism, a culture of the microorganism, and extracts thereof, with a substrate for the enzyme.

[0008] One example of the present invention relates to a method for producing steviol glycoside through glucose transfer by supplying UDP-Glucose, which is an activated form of glucose, as a cofactor required for the transfer of glucose to the steviol glycoside.

[0009] Another example of the present invention relates to a method for producing steviol glycoside through glucose transfer, in which cofactors required for transferring glucose to steviol glycoside additionally include sucrose synthase, which provides UDP and sucrose to provide activated glucose. Effect of the Invention

[0010] The UDP-glucosyltransferase protein of the present invention has substrate specificity acting on multiple substrates of steviol glycoside compared to conventionally known UGTs and has the advantage of not producing reaction by-products. Steviol glycoside can be produced by reacting one or more selected from the group consisting of the enzyme protein, a recombinant microorganism expressing the enzyme protein, a cell mass of the microorganism, a cell disruptant of the microorganism, a culture of the microorganism, and extracts thereof with one or more substrates selected from the group consisting of stevioside and Reb A in the presence of a glucose donor. The steviol glycoside can be added to various foods, beverages, etc. as a high-intensity sweetener. [Brief description of the drawings]

[0011] [Figure 1] 1 is a cleavage map of a vector for constructing a yeast strain that produces an enzyme that converts to Reb D according to an example of the present invention. [Diagram 2] 1 shows a graph of the results obtained by HPLC analysis of the reaction products of an enzyme that converts Reb D according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will now be described in further detail. One example of the present invention relates to a glycosyltransferase that transfers glucose to steviol glycoside, more specifically, a UDP-glucosyltransferase (i.e., uridine diphosphate glucosyltransferase, abbreviated as UGT).

[0013] Specifically, the UDP-glucosyltransferase of the present invention may be a mutant enzyme in which at least one or more amino acids selected from the group consisting of amino acids at positions 201 and 202 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more amino acids selected from the group consisting of serine and valine. More specifically, the mutant enzyme includes those in which the threonine at position 201 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with serine, the valine at position 202 is substituted with leucine, or the threonine at position 201 is substituted with serine and the valine at position 202 is substituted with leucine, and more specifically, the mutant enzyme may include the amino acid sequence of SEQ ID NO: 3.

[0014] Furthermore, the polynucleotide sequence encoding the mutant enzyme may include the amino acid sequence of SEQ ID NO: 1 in which the 201st threonine from the N-terminus is replaced with serine, the 202nd valine is replaced with leucine, or the 201st threonine is replaced with serine and the 202nd valine is replaced with leucine, and more specifically may include a polynucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 3. The mutant enzyme comprises the amino acid sequence of SEQ ID NO: 3 or a polynucleotide sequence encoding the amino acid sequence.

[0015] The mutant enzyme TaUGTm(T201S) of the present invention has the Thr at position 201 from the N-terminus in the wild-type enzyme amino acid sequence of SEQ ID NO: 1 replaced with Ser, e.g., ACC replaced with TCA; TaUGTm(V202L) has the Val at position 201 replaced with Leu, e.g., GTA replaced with TTA; or TaUGTm(T201SV202L) has the Thr-Val corresponding to the amino acids at positions 201 and 202 from the N-terminus in the wild-type enzyme amino acid sequence of SEQ ID NO: 1 replaced with Ser-Leu, and more specifically, may have ACCGTA replaced with TCATTA in the polynucleotide sequence of SEQ ID NO: 2.

[0016] According to yet another embodiment of the present invention, the mutant UDP-glucosyltransferase may have an amino acid sequence with a sequence identity of 92% or more, 95% or more, 97% or more, or 99% or more, and may also include a mutant enzyme protein having an amino acid sequence with a partial deletion, modification, substitution, or addition, so long as the amino acid sequence shows an effect corresponding to that of the enzyme protein. However, the mutant UDP-glucosyltransferase does not include a wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1.

[0017] The mutant UDP-glucosyltransferase may be encoded by a polynucleotide sequence that has 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more sequence identity to a polynucleotide sequence encoding the mutant UDP-glucosyltransferase, provided that the mutant UDP-glucosyltransferase does not include a wild-type enzyme consisting of the polynucleotide sequence of SEQ ID NO:2.

[0018] In the above, the term "homology" or "identity" refers to the degree of identity with a given amino acid sequence or polynucleotide sequence, and is expressed as a percentage. In the present specification, a homologous sequence having the same or similar activity as a given amino acid sequence or polynucleotide sequence is expressed as "% homology" or "% identity".

[0019] The mutant enzyme may have an activity of 101% or more, for example, 101% to 200%, based on 100% activity of the wild-type enzyme containing the amino acid sequence of SEQ ID NO: 1 to convert rebaudioside A (Reb A) substrate to rebaudioside D (Reb D). That is, in order to improve the activity of the enzyme, the enzyme is improved by mutation to increase the production rate of Reb D and Reb E, which can react with a second UDP-glucosyltransferase, for example, UGT76G1 derived from stevia, to produce rebaudioside M (Reb M). This increases the productivity of Reb D and Reb E in the enzyme conversion reaction, and this can be used to produce a highly functional steviol glycoside called Reb M with high productivity.

[0020] The UDP-glucosyltransferase of the present invention has the activity of converting one or more substrates selected from the group consisting of stevioside and Reb A to steviol glycosides, such as one or more substrates selected from the group consisting of Reb D and Reb E, in the presence of a glucose donor, and more specifically, it can convert Reb A to Reb D and stevioside to Reb E (see Reaction Scheme 1 below), and has conversion activity for all of the single or mixed substrates of Reb A and stevioside.

[0021] [ka]

[0022] As shown in Reaction Scheme 1, the conversion from Reb A to Reb D is the transfer of glucose through the beta1-2 glycoside bond in the steviol ring, producing steviol glycosides that are mainly glycosylated at the 19th position of the steviol ring. Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, which are structurally differentiated by the presence of carbohydrate residues at the C13 and C19 positions, which are single bases, steviol. Steviol glycosides generally include, but are not limited to, stevioside, rebaudioside A (Reb A), rebaudioside B, C, D, E, F and M.

[0023] The conversion reaction from Reb A to Reb D is a reaction in which one glucose molecule is further added, and is catalyzed by UGT enzyme. The glucose required for this reaction is an active form of UDP-glucose, and the UGT enzyme transfers glucose to UDP-glucose to produce steviol glycoside. Thus, the present invention relates to a fusion protein in which UDP-glucosyltransferase is fused with Sucrose Synthase (SUS) enzyme capable of producing UDP-glucose.

[0024] Preferably, UDP-glucose is an expensive co-substrate and requires cheaper supply. Sucrose Synthase (SUS) enzyme is an enzyme that decomposes sucrose to produce fructose and UDP-glucose. Therefore, by adding SUS enzyme to the reaction of converting Reb A to Reb D or the reaction of converting stevioside to Reb E to supply UDP and sucrose, and finally supplying UDP-glucose to the UGT enzyme, the reaction can be more economically supplied with UDP-glucose to proceed. The SUS enzyme can be provided by itself in the composition for producing steviol glycoside containing the UDP-glucosyltransferase of the present invention, or in the form of a fusion protein with the UDP-glucosyltransferase of the present invention.

[0025] For example, when UGT and SUS are prepared as a fusion protein and the conversion reaction is carried out, the two reactions form one channel and the enzymatic reactions can occur simultaneously, resulting in an effective conversion reaction. In addition, when using TaUGT_SUS fusion enzyme, conversion is possible using 1 / 10 of UDP relative to the substrate, and in the case of UDP, a reaction using less than 1 / 100 of RA40 is possible. Through the conversion reaction using the fusion enzyme, economical substrate conversion is possible, and steviol glycosides can be produced through a high-concentration substrate reaction.

[0026] The sucrose synthase is derived from one or more microorganisms selected from the group consisting of Arabidopsis thaliana, Solanum lycopersicum, Glycine max, Nicotiana tabacum, and Thermosynechococcus elongatus, but is not limited thereto. More specifically, the sucrose synthase is an Arabidopsis thaliana-derived SUS1 enzyme, and may include, for example, the amino acid sequence of SEQ ID NO: 4, or may have a polynucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 4 or a polynucleotide sequence of SEQ ID NO: 5.

[0027] The UDP-glucosyltransferase according to the present invention can be fused to sucrose synthase directly or via a linker. The linker is a peptide consisting of 4 to 15 amino acids, more specifically, it contains 4 to 15 amino acids of one or more kinds selected from the group consisting of G (Gly), S (Ser) and P (Pro), and specifically may be GGGS (SEQ ID NO: 7), GGGGS (SEQ ID NO: 8), GGGSGGGGS (SEQ ID NO: 9), GGGSGGGGSGGGGS (SEQ ID NO: 10), or GGGGPSPGGGGS (SEQ ID NO: 11).

[0028] In a specific example of the present invention, the fusion protein is a fusion protein in which the UDP-glucosyltransferase of the present invention is linked to the Arabidopsis thaliana-derived SUS1 enzyme via a linker (GGGGSG), and specifically includes the amino acid sequence of SEQ ID NO: 7 or has a polynucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 7 or a polynucleotide sequence of SEQ ID NO: 8.

[0029] The mutant UDP-glucosyltransferase according to the present invention has an activity of converting 45% or more by weight of Reb A to Reb D in an enzyme reaction for 2 to 24 hours, specifically, 45% or more by weight, 50% or more by weight, 70% or more by weight, 75% or more by weight, 80% or more by weight, 85% or more by weight, or 95% or more by weight of Reb A to Reb D, preferably 70% or more by weight, 75% or more by weight, 80% or more by weight, 85% or more by weight, or 95% or more by weight of Reb A to Reb D. More specifically, the conversion activity of the enzyme was measured under the conditions of a temperature of 30° C., pH 7.2, and 150 rpm. The Reb D conversion rate (%) was calculated according to the following formula 1, and details are described in Example 4. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / total HPLC peak area (Formula 1)

[0030] The mutant UDP-glucosyltransferase according to the present invention has a relative conversion rate of Reb D of 101% or more, 103% or more, 105% or more, 107% or more, 108% or more, 109% or more, or 110% or more, more preferably 105% or more, 107% or more, 108% or more, 109% or more, 110% or more, 115% or more, 120% or more, or 125% or more, as an activity of converting a Reb A substrate to Reb D in an enzyme reaction for 2 to 24 hours, compared to a wild-type enzyme having the amino acid sequence of SEQ ID NO: 1. The conversion activity of the enzyme is measured under the conditions of 30°C, pH 7.2, and 150 rpm. The Reb D conversion rate of the enzyme converting the Reb A substrate alone to Red D is calculated according to the above formula 1. Detailed measurement criteria are described in Example 4 below.

[0031] Furthermore, the mutant UDP-glucosyltransferase has an activity of converting 50% by weight or more, 60% by weight or more, 70% by weight or more, 79% by weight or more, 80% by weight or more, 81% by weight or more, 82% by weight or more, 83% by weight or more, 85% by weight or more, or 87% by weight or more of a mixed substrate containing stevioside and Reb A into Reb D and Reb E (total conversion rate of Reb D / E) in an enzymatic reaction for 2 to 24 hours with a mixed substrate containing stevioside and Reb A. More specifically, the conversion activity of the enzyme was measured under conditions of 30°C, pH 7.2, and 150 rpm.

[0032] The mutant UDP-glucosyltransferase according to the present invention has an activity of converting 50% or more by weight of a mixed substrate containing stevioside and Reb A to Reb D and Reb E (total Reb D / E conversion rate) in an enzyme reaction for 2 to 24 hours using a mixed substrate containing stevioside and Reb A, and a relative conversion rate of Reb D / E of 103% or more by weight, 105% or more by weight, 106% or more by weight, 108% or more by weight, 109% or more by weight, or 110% or more by weight, as the total activity of converting Reb A substrate to Reb D and converting stevioside to Reb E, as compared to a wild-type enzyme having the amino acid sequence of SEQ ID NO: 1. More specifically, the conversion activity of the enzyme was measured under conditions of 30°C, pH 7.2, and 150 rpm using a mixed substrate having rebaudioside A:STE (stevioside) = 40:60. The relative total Reb D / E conversion rate of the enzyme refers to the Reb D / E conversion rate of the wild-type enzyme converted to 100% by weight of the wild-type enzyme. That is, the total Reb D / E conversion rate of the enzyme converting the mixed substrate to Reb D / E is calculated according to the following Equation 2, and the relative total Reb D / E conversion rate of the enzyme refers to the total Reb D / E conversion rate of the mutant TaUGT enzyme converted to 100% by weight of the wild-type enzyme. Reb D / E conversion rate (%)=HPLC peak area of ​​Reb D / E / total HPLC peak area (Formula 2)

[0033] The wild-type UDP-glucosyltransferase of the present invention is an enzyme derived from Triticum aestivum, which was obtained by searching for enzymes derived from plants. The function of the protein was elucidated to confirm the UGT enzyme activity, and the enzyme activity of converting stevioside or Reb A to Reb E or Reb D, respectively, was confirmed.

[0034] Reb D, which has a high sweetness, is a sugar present in small amounts in the stevia plant and is produced from Reb A. The well-known enzyme involved in this production process is UGT91D2 of stevia. An enzyme with similar activity is EUGT11 enzyme present in rice. In the case of UGT91D2 of stevia, it has the activity of converting steviol glycoside Reb A to Reb D, but the conversion activity is low, making it difficult to produce high-concentration Reb M in high yields.

[0035] Conventionally known UGT enzymes include UGT derived from stevia (Stevia rebaudiana), UGT derived from rice (Oryza sativa) (hereinafter referred to as EUGT11), and UGT derived from barley (Hordeum vulgare) (hereinafter referred to as HvUGT). The UDP-glucosyltransferase of the present invention has substrate specificity acting on multiple substrates of steviol glycosides and has the advantage of not producing reaction by-products, compared to conventionally known EUGT11 and HvUGT. The amino acid sequence of the EUGT11 enzyme is shown in SEQ ID NO: 12, and the polynucleotide sequence is shown in SEQ ID NO: 13. The amino acid sequence of the HvUGT enzyme is shown in SEQ ID NO: 14, and the polynucleotide sequence is shown in SEQ ID NO: 15.

[0036] The wild-type enzyme having the amino acid sequence of SEQ ID NO: 1 was analyzed for amino acid sequence identity with HvUGT and EUGT11, and was found to have 91% amino acid sequence identity with HvUGT and 66% with EUGT. The wild-type enzyme having the amino acid sequence of SEQ ID NO: 1 was analyzed for sequence identity with HvUGT and EUGT11, and was found to have 36% polynucleotide sequence identity with HvUGT and 37% polynucleotide sequence identity with EUGT11.

[0037] The UDP-glucosyltransferase according to the present invention can be carried out in an aqueous system at a reaction temperature of 10-50°C and a pH of 5.0-9.0. Preferably, the reaction can be carried out in an aqueous system at a temperature of 25-40°C and a pH of 6.0-8.0, more preferably at a temperature of 30°C and a pH of 7.2. According to a preferred embodiment of the invention, the reaction can be carried out in a phosphate buffer at pH 7.2. One embodiment of the present invention relates to a nucleic acid molecule encoding a glycosyltransferase protein that transfers glucose to steviol glycoside, a recombinant vector containing the nucleic acid molecule, and a transformed microorganism.

[0038] The glycosyltransferase protein that transfers glucose to the steviol glycoside can be provided as a nucleic acid molecule encoding a fusion protein so that it can be expressed as a fusion protein fused with a Sucrose Synthase (SUS) enzyme capable of producing UDP-glucose, and is linked to the UDP-glycosyltransferase directly or via a linker. The glycosyltransferase protein that transfers glucose, the SUS enzyme, and the fusion protein thereof are as described above.

[0039] According to the present invention, the recombinant microorganism or cell is a microbial cell, preferably including, but not limited to, Escherichia coli, a Saccharomyces strain, such as Saccharomyces cerevisiae, or a Pichia strain.

[0040] As used herein, the term "transformation" refers to introducing a vector containing a nucleic acid encoding a target protein into a host cell so that the protein encoded by the nucleic acid can be expressed in the host cell. The transformed nucleic acid may include any nucleic acid, regardless of whether it is inserted into the chromosome of the host cell or extrachromosomally located, as long as it can be expressed in the host cell. The nucleic acid may also include DNA and RNA encoding the target protein. The nucleic acid may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the nucleic acid is introduced into the host cell in the form of an expression cassette of a gene structure containing all elements necessary for its own expression. The expression cassette usually includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the nucleic acid. The expression cassette may be in the form of an expression vector capable of self-replication. The nucleic acid may also be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0041] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. A polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosome insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the chromosomal insertion can be additionally included.

[0042] In addition, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a nucleic acid encoding a target protein of the present invention.

[0043] An example of a recombinant vector including a nucleic acid molecule encoding a glycosyltransferase protein that transfers glucose to steviol glycoside according to an embodiment of the present invention is shown in the cleavage map of Figure 1. For example, the vector may include a nucleic acid molecule encoding the enzyme according to the present invention and a GAL10 promoter and a CYC1 terminator as a transcriptional regulatory sequence that is operably linked to the nucleic acid molecule. The transcriptional promoter may include GAL10, GAL1, GAL2, TEF1, GPD1, and TDH3 promoters, and the transcriptional terminator may include CYC1, TEF1, and PGK1.

[0044] The method of transforming the vector of the present invention includes a method of introducing a nucleic acid into a cell, and can be carried out by selecting an appropriate standard technique as known in the art depending on the host cell, such as, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0045] One example of the present invention relates to a composition for producing steviol glycoside, comprising one or more selected from the group consisting of a glycosyltransferase protein that transfers glucose to the steviol glycoside, a recombinant microorganism that expresses the enzyme protein, a bacterial cell of the microorganism, a disrupted bacterial cell of the microorganism, a culture of the microorganism, and extracts thereof.

[0046] The composition for producing a steviol glycoside may further include a substrate containing one or more selected from the group consisting of stevioside and Reb A, and may be, for example, a substrate containing stevioside or Reb A alone, or a mixed substrate thereof. The mixed substrate may be a mixture of stevioside or Reb A, or a stevia extract (or stevia) containing stevioside or Reb A. The substrate or reaction raw material may specifically contain rebaudioside A in an amount of 50 to 70 (v / v)%, 50 to 65 (v / v)%, 50 to 62 (v / v)%, 55 to 70 (v / v)%, 55 to 65 (v / v)%, 55 to 62 (v / v)%, 58 to 70 (v / v)%, 58 to 65 (v / v)%, or 58 to 62 (v / v)%, for example, 60 (v / v)%, based on the total volume of the stevia extract. and stevioside is contained in a content of 30-50(v / v)%, 30-45(v / v)%, 30-42(v / v)%, 35-50(v / v)%, 35-45(v / v)%, 35-42(v / v)%, 38-50(v / v)%, 38-45(v / v)% or 38-42(v / v)%, for example, 40(v / v)%, but is not limited thereto, based on the total volume of the stevia extract.

[0047] The composition for producing steviol glycoside may additionally contain an enzyme that converts Reb D or Reb E to Reb M, for example, a second UDP-glycosyltransferase, specifically UGT76G1 derived from Stevia.

[0048] The composition can additionally include a glucose donor, such as UDP-glucose or a sugar capable of producing UDP-glucose.

[0049] The composition may further include a Sucrose Synthase (SUS) enzyme capable of producing UDP-glucose, which is an example of a glucose donor, and a sugar (sucrose) as its substrate. In one example of the present invention, a glycosyltransferase protein that transfers glucose to the steviol glycoside is provided as a fusion protein with a SUS enzyme protein, in which case steviol glycoside can be produced without the need for a SUS enzyme protein and sugar.

[0050] The culture contains the enzyme produced by a microorganism producing UDP-glucosyltransferase, and may be in a cell-free form containing or not containing the strain. The lysate refers to a lysate obtained by disrupting the cells of a microorganism producing UDP-glucosyltransferase, or a supernatant obtained by centrifuging the lysate, and contains the enzyme produced by a microorganism producing UDP-glucosyltransferase.

[0051] The culture of the strain contains the enzyme produced by the microorganism that produces the UDP-glucosyltransferase, and may be in a cell-free form that may or may not contain the microbial cells. Unless otherwise specified, the microorganism that produces the UDP-glucosyltransferase used herein means one or more selected from the group consisting of the cells of the strain, a culture of the strain, a disruption of the cells, a supernatant of the disruption, and extracts thereof.

[0052] When steviol glycoside is produced using the composition for producing steviol glycoside, the reaction temperature and reaction pH conditions using UDP-glucosyltransferase or a microorganism producing the enzyme are as described above for the reaction temperature and reaction pH conditions for the enzyme.

[0053] One example of the present invention relates to a method for producing steviol glycoside, which includes a step of reacting one or more selected from the group consisting of a glycosyltransferase protein that transfers glucose to the steviol glycoside, a recombinant microorganism expressing the enzyme protein, a bacterial cell of the microorganism, a disrupted bacterial cell of the microorganism, a culture of the microorganism, and extracts thereof, with a substrate for the enzyme.

[0054] The produced steviol glycoside may be one or more selected from the group consisting of Reb D and Reb E, or may be Reb M.

[0055] One example of the present invention is a case where the produced steviol glycoside is one or more selected from the group consisting of Reb D and Reb E, and specifically relates to a method for producing one or more steviol glycosides selected from the group consisting of Reb D and Reb E, comprising a step of reacting one or more selected from the group consisting of the UDP-glucosyltransferase protein of the present invention, a recombinant microorganism expressing the enzyme protein, a microbial cell, a microbial cell disruptant, a culture of the microbial cell, and extracts thereof with one or more substrates selected from the group consisting of stevioside and Reb A in the presence of a glucose donor.

[0056] In a further example of the present invention, the produced steviol glycoside is Reb M, and specifically, the method comprises reacting one or more selected from the group consisting of a UDP-glucosyltransferase protein of the present invention, a recombinant microorganism expressing the enzyme protein, a cell mass of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof with one or more substrates selected from the group consisting of stevioside and Reb A in the presence of a glucose donor to produce one or more steviol glycosides selected from the group consisting of Reb D and Reb E; The present invention relates to a method for producing steviol glycoside Reb M, which includes a step of reacting the produced steviol glycoside, a second UDP-glucosyltransferase protein, a recombinant microorganism expressing the enzyme protein, a cell mass of the microorganism, a cell disruptant of the microorganism, a culture of the microorganism, and an extract thereof, with one or more members selected from the group consisting of the steviol glycoside, a second UDP-glucosyltransferase protein, a recombinant microorganism expressing the enzyme protein, a cell mass of the microorganism, a cell disruptant of the microorganism, and an extract thereof, in the presence of a glucose donor.

[0057] The second UDP-glucosyltransferase protein used in the production process of Reb M may be UGT76G1, etc.

[0058] The glucose donor provided in the process of producing the steviol glycoside may be UDP-glucose, which is provided as a compound or as a sugar and Sucrose Synthase (SUS) enzyme. The SUS enzyme is an enzyme that breaks down sugar to produce fructose and UDP-glucose. Therefore, if the SUS enzyme is introduced into the reaction of converting Reb A to Reb D or the reaction of converting stevioside to Reb E, and UDP-glucose is supplied to the UGT enzyme, the reaction can be proceeded with more economical supply of UDP-glucose. In addition, if the conversion reaction is performed by producing SUS as a fusion protein with UGT, the two reactions form one channel and the enzyme reaction occurs in one, so that an effective conversion reaction can be performed. The SUS enzyme and the fusion protein of UGT and SUS are as described above.

[0059] The method for producing steviol glycosides according to the present invention can significantly shorten the production cycle, improve production capacity, and provide high-purity products at low cost, and therefore can be economically utilized in the food and beverage industries.

[0060] The substrate for the UDP-glucosyltransferase protein of the present invention is a stevia extract containing 50% Reb A and 90% steviol polysaccharides in the extract. For example, enzymatic conversion can be performed using a stevia extract from SINOCHEM (China) containing 50% Reb A and 90% steviol polysaccharides in the extract.

[0061] The UDP-glucosyltransferase protein of the present invention and steviol glycoside produced using a recombinant microorganism expressing the enzyme protein can be used as a high-intensity sweetener by adding it to various foods and beverages, or can be sold commercially as a table sweetener on its own. EXAMPLES

[0062] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the scope of the following illustrative examples.

[0063] Example 1: Construction of microorganisms expressing wild-type enzymes 1-1: Preparation of vector expressing wild-type enzyme A gene encoding a wild-type enzyme protein having the amino acid sequence of SEQ ID NO: 1 derived from Triticum aestivum was synthesized (Gblock synthesis), and the polynucleotide sequence of the synthesized gene is shown in SEQ ID NO: 2. The synthesized gene was subcloned into a plasmid expressed in S. cerevisiae to create an expressible form. Specifically, the vector prepared to confirm the activity of the protein was the pRS426 vector, which contains a uraauxotrophic marker that can be selected in S. cerevisiae, and the GAL10 promoter and CYC1 terminator were used. The newly synthesized (IDT, gBlock) gene of sequence number 2 was cloned into this plasmid. To clone the TaUGT gene, a primer pair (sequence numbers 19 and 19) was created to overlap with the GAL10 promoter (sequence number 16) and cyc1 terminatorB (sequence number 17), the enzyme gene was amplified through PCR amplification, and the gene was subcloned through Gibson assembly (HIFi DNA master mix, NEW ENGLAND BIOLABS). The subcloned gene in the form of a 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 correctly subcloned.

[0064] [Table 1]

[0065] The gene was introduced into S. cerevisiae CENPK2-1c (Euroscarf) by transformation using heat shock with LiAc (Lithium Acetate) at 0.1 M. The transformed strain was selected on SC_Ura medium, and the selected colonies were cultured to confirm the enzyme activity.

[0066] The recombinant plasmid was transformed into S. cerevisiae CENPK2-1c (Euroscarf) strain. The recombinant strain with the gene cassette inserted via the selection marker was selected on SC-Ura solid medium (SC_Ura medium + 2% agar). The composition of SC-Ura solid medium was YNB 6.7g / L, Drop out mix 0.7g / L, Glucose 50g / L, and agar 20g / L, and the pH was adjusted to 6.0.

[0067] 1-2: Cultivation of recombinant microorganisms The yeast transformed with the plasmid prepared in Example 1-1 was selected in a synthetic complete (SC) medium using URA-drop out mix, and the selected yeast was cultured in liquid medium to induce enzyme expression. Specifically, the colonies selected from the SC_Ura solid medium were inoculated into a test tube containing 3 mL of SC_Ura liquid medium and cultured overnight. The concentration of the cultured bacteria was measured by OD 600 The OD was confirmed by measuring the absorbance in a 250 mL flask containing 25 mL of SC-Ura liquid medium. 600 The medium was inoculated so that the absorbance was 0.1 (final) and then cultured for 24 hours (30°C, 240 rpm). In order to induce enzyme expression in the cultured cells, the same amount of YPG medium was added and cultured for 24 hours. The cultured cells were collected by centrifugation (4,000 rpm, 10 min). The composition of SC_Ura liquid medium included 6.7 g / L YNB (yeast nitrogene base), 10 g / L Drop-out mix, 10 g / L Glucose, and 1 mM MES, and the pH of the medium was adjusted to 6.0 using NaOH. The composition of the YPG medium included 20 g / L Bacto peptone, 10 g / L Yeast Extract, 20 g / L Galactose, and 100 mM Phosphate buffer (sodium salt), and the pH of the medium was adjusted to 6.0.

[0068] 1-3: Production of microorganisms expressing EUGT11 and HvUGT The EUGT11 gene (SEQ ID NO: 13) from Oryza sativa and the HvUGT gene (SEQ ID NO: 15) from Hordeum vulgare, whose activities are known, were synthesized (Gblock synthesis), amplified in the same way as the TaUGT gene, and cloned into the pRS426 vector. To induce enzyme expression under the same conditions, the GAL10 promoter was used as the promoter and the CYC1 terminator was used. Specifically, the forward primer of EUGT11 linked to the GAL10 promoter (SEQ ID NO: 20), the reverse primer of EUGT11 linked to the CYC1 terminator (SEQ ID NO: 21), the forward primer of HvUGT linked to the GAL10 promoter (SEQ ID NO: 22), and the reverse primer of HvUGT linked to the CYC1 terminator (SEQ ID NO: 23) were used. In Table 1, the enzyme-producing gene cassette thus constructed is shown as Sc, which means Saccharomyces cerevisiae. Thereafter, the vector was transformed into S. cerevisiae CENPK2-1c (Euroscarf) in substantially the same manner as in Examples 1-1 and 1-2, and strain selection and microbial culture were carried out.

[0069] 1-4: Enzyme activity evaluation The cultures of the recombinant strains obtained in Examples 1-2 and 1-3 were centrifuged (4,000 rpm, 10 min) to recover the cells. 10 mL of the recovered cells were pooled and washed twice with the same amount of water. The washed cells were resuspended in 100 mM phosphate buffer (pH 7.2) and incubated at OD 600 After adjusting the value to 100, 100 mg of glass beads (Sigma) with a particle size of 0.4 to 0.6 mm were placed in a cap tube, and 1 ml of 100 mM phosphate buffer (pH 7.2) was added. The cells were disrupted five times for 30 seconds using a bead beater. The disrupted cell solution was centrifuged (13,000 rpm, 15 min) in a refrigerated state, and the supernatant was obtained as a coenzyme solution. The reaction solution for enzyme activity evaluation contained the substrate Reb A (95%, Sinochem), MgCl2, and UDP-Glucose, to which 0.1 mL of the coenzyme solution was added to prepare a total reaction volume of 0.5 mL, so that the final reaction solution contained 2 g / L of the substrate Reb A (95%, Sinochem), 3 mM MgCl2, and 4 mM UDP-Glucose. The enzyme reaction was carried out on the prepared reaction solution at a temperature of 30°C, pH 7.2, and 150 rpm, and samples were taken 3 and 18 hours after the start of the reaction to confirm the degree of reaction according to the reaction time. The enzyme reaction solution was boiled for 5 minutes to terminate the enzyme reaction, and centrifuged (13,000 rpm, 10 min) to obtain a supernatant, which was then analyzed to confirm the enzyme reaction product. Specifically, the enzyme reaction product was analyzed using HPLC-Chromatography to confirm the conversion ratio of the product. The coenzyme solution obtained from the cell lysate of the recombinant strain exhibited activity in converting the Reb A substrate to Reb D, so the activity was measured by confirming the production ratio of Reb D. Specifically, the column used for the HPLC analysis of the conversion products was UG120 (C18 250 mm x 46 mm, 5 um 110 A particle, Shideido), and the analysis was performed at 210 nm. The mobile phase was analyzed using a gradient of water and acetonitrile containing 0.01% TFA (Trifluoroacetic acid, Sigma), respectively, and the mobile phase was run at 1 mL / min for a total of 40 minutes. The HPLC analysis conditions are shown in Table 2 below.

[0070] [Table 2]

[0071] The HPLC analysis graph of the conversion product is shown in Table 3. The Reb D conversion rate of the enzyme converting the single substrate Reb A to Red D shown in Table 3 below was calculated according to the following Equation 1, and the relative Reb D conversion rate of the enzyme means the Reb D conversion rate of the TaUGT or HvUGT enzyme converted based on the Reb D conversion rate of EUGT11 (100%). Reb D conversion rate (%) = HPLC peak area of ​​Reb D / total HPLC peak area (Formula 1)

[0072] [Table 3]

[0073] As shown in Table 3, the conversion rate of the enzymatic reaction was measured after the first 3 hours of reaction. TaUGT converted 40-45% of Reb A to Reb D, similar to EUGT11, and was confirmed to be an enzyme with approximately 30% improved activity compared to HvUGT (30-35% conversion). In addition, the analysis of the amount of Reb D produced in the 18-hour enzyme reaction solution showed that the order of magnitude of the conversion rates of the three enzymes showed a similar tendency to that of the 3-hour reaction. The polynucleotide sequence of the TaUGT enzyme was analyzed through the polynucleotide sequence analysis service of Macrogen Co., Ltd., and the amino acid sequence was analyzed based on the polynucleotide sequence. As a result, the amino acid sequence is shown in SEQ ID NO:1, and the polynucleotide sequence is shown in SEQ ID NO:2.

[0074] Example 2: Preparation of microorganisms expressing mutant enzymes 2-1: Construction of vectors for expressing mutant enzymes In order to develop the mutation of TaUGT enzyme in Example 1, mutations were searched for based on the existing EUGT11 gene. Conserved regions were identified through alignment, and the amino acid differences were compared at the positions between the domains that function as the sugar acceptor at the N-terminus and the sugar donor at the C-terminus of UGT. In the case of threonine at residue 201 and valine at residue 202 (serine and leucine amino acids in EUGT11), threonine was changed to serine, and valine was changed to alanine or leucine. The mutation method was to synthesize primers at the corresponding positions of TaUGT (sequence numbers 24 and 25) and perform mutation gene amplification (PCR) to change the polynucleotide sequence at the corresponding site, and the amplified gene fragment was cloned into the pRS426 vector via Gibson assembly. The resulting fragment was cloned into pRS426 vector and named TaUGTT201S / V202L. The sequences of the primers used in this example are shown in Table 4 below.

[0075] [Table 4]

[0076] 2-2: Preparation of enzyme mutant expression strains In order to confirm the expression of the enzyme, TaUGTT201S / V202L cloned into the pRS426 vector was transformed into a yeast strain by the LiAc method and selected in the same manner as in Example 1-2. The transformed strain was cultured in the same manner as in Example 1-2. The yeast transformed with the plasmid prepared in Example 2-1 was selected in a synthetic complete (SC) medium using a URA-drop out mix, and the selected yeast was cultured in liquid medium to induce the expression of the enzyme.

[0077] Example 3: Enzyme activity evaluation of enzyme mutants The culture of the recombinant strain obtained in Example 2-2 was centrifuged (4,000 rpm, 10 min) to recover the cells. 10 mL of the recovered cells were collected and washed twice with the same amount of water. The washed cells were resuspended in 100 mM phosphate buffer (pH 7.2) and incubated at OD 600 After adjusting the value to 100, 100 mg of glass beads (Sigma) with a particle size of 0.4 to 0.6 mm were placed in a cap tube, and 1 ml of 100 mM phosphate buffer (pH 7.2) was added. The cells were disrupted five times for 30 seconds using a bead beater. The disrupted cell solution was centrifuged (13,000 rpm, 15 min) in a refrigerated state, and the supernatant was obtained as a coenzyme solution. The reaction solution for enzyme activity evaluation contained the substrate Reb A (95%, Sinochem), MgCl2, and UDP-Glucose, to which 0.1 mL of the coenzyme solution was added to prepare a total reaction volume of 0.5 mL, so that the final reaction solution contained 2 g / L of the substrate Reb A (95%, Sinochem), 3 mM MgCl2, and 4 mM UDP-Glucose. The enzyme reaction was carried out on the prepared reaction solution at a temperature of 30°C, pH 7.2, and 150 rpm, and samples were taken 3 and 18 hours after the start of the reaction to confirm the degree of reaction according to the reaction time. The enzyme reaction solution was boiled for 5 minutes to terminate the enzyme reaction, and centrifuged (13,000 rpm, 10 min) to obtain a supernatant, which was then analyzed to confirm the enzyme reaction product. Specifically, the enzyme reaction product was analyzed using HPLC-Chromatography to confirm the conversion ratio of the product. The coenzyme solution obtained from the cell lysate of the recombinant strain exhibited activity in converting the Reb A substrate to Reb D, so the activity was measured by confirming the production ratio of Reb D. Specifically, the column used for the HPLC analysis of the conversion product was UG120 (C18 250 mm x 46 mm, 5 um 110 A particle, Shiseido), and the analysis was performed at 210 nm. The mobile phase was analyzed using a gradient of water and acetonitrile containing 0.01% TFA (Trifluoroacetic acid, Sigma), respectively, and the mobile phase was run at 1 mL / min for a total of 40 minutes. The HPLC analysis conditions are shown in Table 4 below. The HPLC analysis graph of the conversion product is shown in FIG. 2. The Reb D conversion rate of the enzyme converting Reb A single substrate to Red D shown in Table 4 below was calculated according to the following Equation 1, and the relative Reb D conversion rate of the enzyme means the Reb D conversion rate of the TaUGT mutant enzyme converted based on the Reb D conversion rate of the wild-type enzyme being 100%. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / total HPLC peak area (Formula 1)

[0078] The reaction mixture containing the TaUGT mutant enzyme and the Reb A substrate was used to carry out the enzyme reaction for 18 hours, and the resulting conversion product was analyzed by HPLC analysis. The activity analysis results of the TaUGT mutant enzyme used in the HPLC analysis are shown in Table 5 below and Figure 2. Figure 2 is an HPLC graph of the reaction product of the Reb D conversion reaction using the TaUGT mutant enzyme.

[0079] [Table 5]

[0080] Example 4 Enzymatic conversion using mixed substrates The cells were resuspended in 100 mM phosphate buffer (pH 7.2) and the OD 600After adjusting the value to 100, 100 mg of glass beads (Sigma) with a particle size of 0.4 to 0.6 mm were placed in a cap tube, and 1 ml of 100 mM phosphate buffer (pH 7.2) was added. The cells were disrupted five times for 30 seconds using a bead beater. The disrupted cells were centrifuged (13,000 rpm, 15 min) under refrigerated conditions, and the supernatant was obtained as a coenzyme solution. The reaction solution for evaluating the enzyme activity contained the substrate RA40 (Reb A (rebaudioside A: STE (stevioside) = 40: 60, Sinochem), MgCl2, and UDP-Glucose, to which 0.2 mL of the coenzyme solution according to Example 3 was added to prepare a total reaction volume of 0.5 mL, so that the final reaction solution had a substrate RA40 (Reb A: STE = 40: 60, Sinochem) of 2 g / L, MgCl2 of 3 mM, and UDP-Glucose of 4 mM. The enzyme reaction was carried out on the prepared reaction solution at a temperature of 30°C, pH 7.2, and 150 rpm, and the reaction degree was confirmed 18 hours after the start of the reaction. The conversion product was analyzed by HPLC analysis in a manner substantially similar to that of Example 3. The total Reb D / E conversion rate of the enzymes converting the RA40 mixed substrate shown in Table 6 below to Reb D / E was calculated according to the following Equation 2, and the relative total Reb D / E conversion rate of the enzymes means the total Reb D / E conversion rate of the TaUGT enzyme converted based on the total Reb D / E conversion rate of EUGT11 (100%). Reb D / E conversion rate (%)=HPLC peak area of ​​Reb D / E / total HPLC peak area (Formula 2)

[0081] [Table 6]

[0082] As a result of the above experiment, in the case of a mutant enzyme in which threonine 201 was changed to serine, the relative conversion rate was 106% compared to the wild-type enzyme, and in the case of a double mutant enzyme in which threonine 201 was changed to serine and valine 202 was changed to leucine, the relative conversion rate increased to 114% compared to the wild-type enzyme. Thus, the residues whose activity was improved by mutation were identified, and a mutant enzyme with increased activity was obtained.

[0083] Example 5: Preparation of TaUGT_SUS fusion enzyme and confirmation of activity In order to supply the necessary glucose donor (UDP-glucose) for the transglycosylation reaction of steviol glycoside using the TaUGT_T201SV202L (TaUGTm) mutant enzyme, whose activity was confirmed to be improved in Example 3, a fusion protein was prepared with the sucrose synthase (SUS1, derived from Arabidopsis thaliana) enzyme. The nucleic acid sequence encoding the TaUGT_T201SV202L (TaUGTm) mutant enzyme is the nucleic acid sequence of SEQ ID NO: 30 in Table 7 below, and the nucleic acid sequence of SEQ ID NO: 5 in Table 7 below was used as sucrose synthase (SUS1, derived from Arabidopsis thaliana).

[0084] [Table 7-1]

[0085] [Table 7-2]

[0086] To prepare the fusion enzyme, a primer was synthesized and a linker (GGGGSG, sequence number 8) was used to link the amino acid sequence between TaUGTm and SUS. Primers were synthesized for the corresponding positions of each gene (sequence numbers 26 and 27), and the base sequence of the corresponding sites was amplified (PCR). A gene fragment amplified by PCR was cloned into the pRS426 vector through Gibson assembly to prepare pRS426TaUGTm_SUS. The amino acid sequence of the SUS enzyme protein used is shown in SEQ ID NO: 4, the nucleotide sequence encoding the enzyme protein is shown in SEQ ID NO: 5, and the amino acid sequence of the fusion enzyme protein is shown in SEQ ID NO: 6. The linker sequence used to prepare the fusion protein has GGGGSG (SEQ ID NO: 8), and the amino acid and nucleotide sequences of the SUS1 enzyme protein and the primer information used are shown in Table 8 below.

[0087] [Table 8]

[0088] The constructed fusion enzyme expression vector was transformed into yeast in the same manner as in Example 1-2, and then selected and isolated. A coenzyme solution was prepared using the cultured bacterial cell disruption solution in substantially the same manner as in Examples 1 to 3. Specifically, in substantially the same manner as in Example 3, bacterial cells were collected from the culture of the recombinant strain by centrifugation, and disrupted with glass beads to be used as a coenzyme solution. Specifically, the reaction solution was prepared by mixing the RA40 reaction substrate (90% by weight purity), MgCl2, UDP, and sucrose, and adding 0.1 mL of each of the TaUGTm_SUS fusion enzyme coenzyme solutions to prepare a total reaction volume of 0.5 mL, so that the final reaction solution had 10 g / L of the RA40 reaction substrate, 1 mM MgCl2, 1 mM UDP, and 125 mM sucrose. The enzyme reaction was carried out on the prepared reaction solution at a temperature of 30°C, pH 7.2, and 150 rpm for 20 hours to confirm the enzyme conversion. The conversion products of the reaction solution were analyzed and confirmed by HPLC analysis in a manner substantially similar to that of Examples 1-4. It was confirmed that the fusion enzyme converted RA40 (Reb A:STE=40:60) to Reb D / E by supplying UDP-Glucose using UDP. STE stands for stevioside.

[0089] In the conversion reaction of the initial fusion enzyme, about 33% by weight of the substrate before the reaction was converted to Reb D / E. In the conversion reaction of the mixed substrate (RA40 (Reb A:STE = 40:60)), Reb D (including D derivatives) and Reb E were converted to 21.7% and 12.1%, respectively. The conversion of steviol glycosides was confirmed by the production of UDP-Glucose using the sugar of the fusion enzyme and the saccharification of the substrate using this, and it was confirmed that the enzyme conversion reaction can be increased by optimizing the amount of substrate and reaction conditions. In the case of conversion using fusion enzymes, for conversion of RA40 (Reb A:STE = 40:60), 10-20 mM or more of UDP-Glucose is required for conversion reaction with a substrate equivalent to about 10 mM for a 10 g / L substrate. Therefore, when converting a large amount at a high concentration, an excess of expensive UDP-Glucose is required. When performing an enzymatic reaction using a high concentration substrate, an excess of UDP-Glucose should be used, as an excess of UDP-Glucose inhibits enzyme activity, which is not preferable. According to this experiment, when using TaUGT_SUS fusion enzyme, conversion is possible using 1 / 10 of UDP relative to the substrate, and in the case of UDP, a reaction using less than 1 / 100 of RA40 is possible. It was confirmed that economical enzyme conversion is possible through the conversion reaction using the fusion enzyme, and that the production of steviol glycosides is induced through a high-concentration substrate reaction.

Claims

1. An enzyme protein having uridine diphosphate-glucosyltransferase (UDP-glucosyltransferase) activity that transfers glucose to a steviol glycoside substrate, in which at least one or more amino acids selected from the group consisting of the 201st and 202nd amino acids from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more amino acids selected from the group consisting of serine and valine.

2. The enzyme protein according to claim 1, wherein the steviol glycoside substrate is one or more selected from the group consisting of stevioside and rebaudioside A.

3. 2. The enzyme protein according to claim 1, wherein the enzyme protein has an activity of converting 45% by weight or more of rebaudioside A into rebaudioside D in an enzymatic reaction for 2 to 24 hours.

4. 2. The enzyme protein according to claim 1, wherein the enzyme protein has an activity of converting a rebaudioside A substrate to rebaudioside D in an enzyme reaction for 2 to 24 hours of 101% or more based on 100% activity of a wild-type enzyme comprising the amino acid sequence of SEQ ID NO:

1.

5. A fusion protein in which the UDP-glucosyl transferase enzyme protein according to claim 1 is linked to a sucrose synthase enzyme protein.

6. The fusion protein according to claim 5, wherein the sucrose synthase is derived from one or more microorganisms selected from the group consisting of Arabidopsis thaliana, Solanum lycopersicum, Glycine max, Nicotiana tabacum, and Thermosynechococcus elongatus.

7. A recombinant microorganism comprising a gene encoding the enzyme protein according to any one of claims 1 to 6.

8. 8. The recombinant microorganism of claim 7, wherein the microorganism is selected from the group consisting of Escherichia coli, Saccharomyces microorganisms, and Pichia microorganisms.

9. A composition for producing steviol glycosides, comprising at least one selected from the group consisting of the UDP-glucosyl transferase enzyme protein according to any one of claims 1 to 4, a recombinant microorganism expressing the enzyme protein, a bacterial cell of the microorganism, a disrupted bacterial cell of the microorganism, a culture of the microorganism, and extracts thereof.

10. 10. The composition of claim 9, wherein the composition additionally comprises a substrate comprising one or more selected from the group consisting of stevioside and rebaudioside A.

11. 11. The composition of claim 10, wherein the composition is a mixed substrate comprising stevioside and rebaudioside A.

12. 10. The composition of claim 9, wherein the composition additionally comprises an enzyme that converts rebaudioside D or rebaudioside E to rebaudioside M (Reb).

13. 13. The composition of claim 12, wherein the enzyme that converts Reb M is UGT76G1 from Stevia.

14. The composition according to claim 9, wherein the UDP-glucosyl transferase enzyme protein is a fusion protein linked to a sucrose synthase enzyme protein.

15. The composition of claim 9 , wherein the composition comprises a glucose donor.

16. The composition of claim 9, wherein the glucose donor is UDP-glucose.

17. The composition according to claim 9, further comprising a sucrose synthase enzyme protein, or when the sucrose synthase enzyme protein is provided as a fusion protein with a UDP-glucosyl transferase enzyme protein, the composition comprises sucrose and UDP.

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