Glycosyltransferase mutants and methods for producing steviol glycosides using them

Abstract

This invention provides glycosyltransferase mutants, as well as compositions and methods for producing steviol glycosides utilizing these mutants. [Solution] The present invention relates to a rebaudioside glycosyltransferase and mutagenesis that transfer glucose to a steviol glycoside, a recombinant bacterial strain expressing the enzyme, and a method for producing rebaudioside, a steviol glycoside, using these.

Description

Technical Field

[0001] The present invention relates to a glycosyltransferase, a composition and a method for producing steviol glycosides using the same, and more particularly to a glycosyltransferase of rebaudioside that transfers glucose to steviol glycosides, a recombinant strain expressing the enzyme, and a method for producing rebaudioside of steviol glycosides using these.

Background Art

[0002] Sweeteners are known as the most commonly used ingredients in the food, beverage, or confectionery industries. Sweeteners can be incorporated into the final food products during production or used at home as a substitute for sugar in table sweeteners or baking when appropriately diluted for single use. Sweeteners include, for example, 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 Stevia rebaudiana, a perennial shrub. Stevia extracts purified at various levels are used as high-sensitivity seasonings in foods and blends or sold commercially as table sweeteners alone.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Stevia plant extracts contain rebaudioside and other steviol glycosides that contribute to sweetness, but conventional commercial products are mainly rebaudioside A, with other glycosides such as small amounts of rebaudioside C, D, and F. Stevia extracts from plants may contain contaminants such as induced compounds that cause off-flavors. Such off-flavors can be a problem depending on the selected food system or application. Furthermore, the composition of stevia extract varies considerably depending on the soil and climate in which the plant grows. Depending on the raw material plant, climatic conditions, and extraction process, the amount of rebaudioside A in commercial manufacturing processes is reported to vary from 20% to 97% of the total steviol glycoside content. Other steviol glycosides are present in varying amounts within the stevia extract. Stevia extracts produced from the stevia plant contain various steviol glycosides and off-odor compounds, and their recovery and purification are labor-intensive and inefficient. Therefore, there is still a need for recombinant manufacturing systems that can accumulate steviol glycosides such as Reb D and Reb M in high yield. For this reason, the development of efficient enzymes remains necessary. There is also still a demand for improved production of Reb D for the production of Reb M, a steviol glycoside, from recombinant hosts for commercial use. [Means for solving the problem]

[0005] An example of the present invention relates to a glycosyltransferase that transfers glucose to a steviol glycoside, a nucleic acid molecule that encodes the enzyme protein, a recombinant vector containing the nucleic acid molecule, and a transformed microorganism.

[0006] An example of the present invention relates to a composition for producing a 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 expressing the enzyme protein, the cells of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and extracts thereof.

[0007] One example of the present invention relates to a method for producing a steviol glycoside, comprising the 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, the cells of the microorganism, a cell lysate 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 a steviol glycoside by supplying UDP_Glucose, which is active glucose, as a cofactor necessary for transferring glucose to the steviol glycoside.

[0009] An additional example of the present invention relates to a method for producing a steviol glycoside via glucose transfer, wherein the cofactors required for transferring glucose to the steviol glycoside include UDP and sucrose to provide active glucose, and further include sucrose synthase. [Effects of the Invention]

[0010] The UDP-glucosyltransferase protein according to the present invention has substrate specificity that acts on multiple substrates of steviol glycosides, compared to conventionally known UGTs, and has the advantage of not producing reaction byproducts. Steviol glycosides can be produced by reacting one or more selected from the group consisting of the enzyme protein, recombinant microorganisms expressing the enzyme protein, the cells of the microorganisms, the cell lysates of the microorganisms, the cultures of the microorganisms, 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. Steviol glycosides can be used as high-intensity sweeteners by adding them to various foods and beverages. [Brief explanation of the drawing]

[0011] [Figure 1] This is a cleavage map of a vector for producing a yeast strain that produces an enzyme that converts to Reb D, according to an example of the present invention. [Figure 2] The graph shows the results obtained by analyzing the reaction product of an enzyme that converts to Reb D according to an example of the present invention using HPLC. [Modes for carrying out the invention]

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

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

[0014] Furthermore, the polynucleotide sequence that encodes the mutant enzyme includes the amino acid sequence of SEQ ID NO: 1 in which the 201st threonine from the N-terminus is replaced with serine and 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, it may include the polynucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 3. This is a mutant enzyme comprising the amino acid sequence of SEQ ID NO: 3 or the polynucleotide sequence that encodes the said amino acid sequence.

[0015] The mutant enzyme TaUGTm(T201S) according to the present invention is one in which the Thr at position 201 from the N-terminus in the wild-type enzyme amino acid sequence of SEQ ID NO: 1 is replaced with Ser, for example ACC is replaced with TCA, TaUGTm(V202L) is one in which the Val at position 201 is replaced with Leu, for example GTA is replaced with TTA, or TaUGTm(T201SV202L) is one in which the Thr-Val corresponding to the 201st and 202nd amino acids from the N-terminus in the wild-type enzyme amino acid sequence of SEQ ID NO: 1 is replaced with Ser-Leu, and more specifically, ACCGTA may be replaced with TCATTA in the polynucleotide sequence of SEQ ID NO: 2.

[0016] According to yet another example of the present invention, the mutant UDP-glucosyltransferase may contain an amino acid sequence with sequence identity of 92% or more, 95% or more, 97% or more, or 99% or more, and may also include mutant enzyme proteins containing amino acid sequences in which some sequences are deleted, modified, substituted, or added, as long as the amino acid sequence exhibits efficacy corresponding to that of the enzyme protein. However, the mutant UDP-glucosyltransferase does not include the wild-type enzyme consisting of the amino acid sequence of SEQ ID NO: 1.

[0017] The mutant UDP-glucosyltransferase may be encrypted by a polynucleotide sequence with a sequence identity of 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. However, the mutant UDP-glucosyltransferase does not contain the wild-type enzyme consisting of the polynucleotide sequence of SEQ ID NO: 2.

[0018] In the foregoing, the terms “homology” or “identity” mean the degree to which a given amino acid sequence or polynucleotide sequence matches, and are expressed as a percentage. In this specification, homologous sequences having the same or similar activity as a given amino acid sequence or polynucleotide sequence are 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% of the activity of converting the Rebaudioside A (Reb A) substrate of the wild-type enzyme containing the amino acid sequence of SEQ ID NO: 1 to Rebaudioside D (Reb D). That is, enzyme improvement by mutation is carried out to improve the activity of the enzyme, improve the production rates of Reb D and Reb E, and react with a second UDP-glucosyltransferase, for example, UGT76G1 derived from Stevia, to produce Rebaudioside M (Reb M). Thereby, the enzyme conversion reaction increases the productivity of Reb D and Reb E, and using this, a highly functional steviol glycoside called Reb M can be produced with high productivity.

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

[0021]

Chemical formula

[0022] As shown in reaction equation 1 above, the conversion from Reb A to Reb D involves the transfer of glucose via a beta1-2glycoside bond in the steviol ring, mainly producing steviol glycosides where glycation occurs at position 19 of the steviol ring. Steviol glycosides consist of 1 to 3 glucose molecules linked by β-bonds. Steviol glycosides are a group of compounds found in the leaves of Stevia rebaudiana Bertoni, and are structurally distinguished by the presence of carbohydrate residues at positions C13 and C19, resulting in different single bases called 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 carried out by the UGT enzyme in a reaction in which one more molecule of glucose is added. The glucose required for this reaction is the active form of UDP-glucose, and the UGT enzyme transfers glucose from UDP-glucose to produce a steviol glycoside. Therefore, the present invention relates to a fusion protein formed by fusing UDP-glucosyltransferase with a sucrose synthase (SUS) enzyme capable of producing UDP-glucose.

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

[0025] For example, if the conversion reaction is carried out using a fusion protein of UGT and SUS, the two reactions can form a single channel, allowing the enzymatic reactions to occur simultaneously, thus enabling an effective conversion reaction. Furthermore, when using the TaUGT_SUS fusion enzyme, conversion is possible using 1 / 10 the amount of UDP relative to the substrate, and in the case of UDP, the reaction can be carried out using an amount less than 1 / 100 of RA40. Economical substrate conversion is possible through the conversion reaction using the fusion enzyme, and steviol glycosides can be produced by high-concentration substrate reactions.

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

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

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

[0029] The mutant UDP-glucosyltransferase according to the present invention has the activity to convert 45% by weight or more of Reb A to Reb D in an enzymatic reaction over 2 to 24 hours. Specifically, it has the activity to convert 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 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 to Reb D. More specifically, the conversion activity of the enzyme was measured under conditions of 30°C, pH 7.2, and 150 rpm. The Reb D conversion rate (%) is calculated by the following formula 1, and further details are described in Example 4. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / Total HPLC peak area (Equation 1)

[0030] The mutant UDP-glucosyltransferase according to the present invention has, compared to the wild-type enzyme having the amino acid sequence of SEQ ID NO: 1, an activity that converts a Reb A substrate to Reb D in an enzymatic reaction of 2 to 24 hours, 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. The conversion activity of the enzyme was measured under conditions of 30°C, pH 7.2, and 150 rpm. The conversion rate of the enzyme from a Reb A substrate alone to Red D is calculated according to formula 1. Detailed measurement criteria are described in Example 4 below.

[0031] Furthermore, the mutant UDP-glucosyltransferase has the activity to convert 50% or more, 60% or more, 70% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 85% or more, or 87% or more of the mixed substrate containing stevioside and Reb A to Reb D and Reb E in an enzymatic reaction for 2 to 24 hours. 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, compared to the wild-type enzyme having the amino acid sequence of SEQ ID NO: 1, an enzymatic reaction of 2 to 24 hours with a mixed substrate containing stevioside and Reb A in which 50% or more by weight of the mixed substrate containing Reb A and stevioside is converted to Reb D and Reb E (total Reb D / E conversion rate), which is the sum of the activity to convert the Reb A substrate to Reb D and the activity to convert stevioside to Reb E, and has 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. More specifically, the conversion activity of the enzyme was measured under conditions of 30°C, pH 7.2 and 150 rpm and using a mixed substrate having rebaudiosideA:STE(stevioside) = 40:60. The total relative Reb D / E conversion rate of the enzyme refers to the Reb D / E conversion rate of the wild-type enzyme, converted to a standard of 100% by weight of the wild-type enzyme. That is, the total Reb D / E conversion rate of the enzyme that converts to Reb D / E for a mixed substrate is calculated according to the following formula 2, and the relative total conversion rate of Reb D / E of the enzyme refers to the total Reb D / E conversion rate of the mutant TaUGT enzyme, converted to a standard of 100% by weight of the wild-type enzyme. Reb D / E conversion rate (%) = HPLC peak area of ​​Reb D / E / Total HPLC peak area (Equation 2)

[0033] The wild-type UDP-glucosyltransferase enzyme according to the present invention relates to an enzyme derived from Triticum aestivum, obtained by searching for plant-derived enzymes. The function of the said protein was elucidated and its UGT enzyme activity was confirmed, and the enzyme activity that converts 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 in stevia. An enzyme with similar activity is EUGT11, which is found in rice. In the case of stevia's UGT91D2, it has the activity to convert steviol glycoside Reb A to Reb D, but its conversion activity is low, making it difficult to produce high concentrations of Reb M in high yield.

[0035] In addition, 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 according to the present invention has substrate specificity that acts on multiple substrates of steviol glycosides, and has the advantage of not producing reaction byproducts, compared to the 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 Sequence ID No. 1 was analyzed for amino acid sequence identity with HvUGT and EUGT11. The results showed that it had 91% amino acid sequence identity with HvUGT and 66% with EUGT. Analysis of polynucleotide sequence identity showed that it had 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 to 50°C and a pH of 5.0 to 9.0. Preferably, the reaction can be carried out in an aqueous system at a temperature of 25 to 40°C and a pH of 6.0 to 8.0, and more preferably in an aqueous system at a temperature of 30°C and a pH of 7.2. According to a preferred example of the invention, the reaction can be carried out in a phosphate buffer at a pH of 7.2. An example of the present invention relates to a nucleic acid molecule that encodes a glycosyltransferase protein that transfers glucose to a 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 that encodes the fusion protein, so that it can be expressed in 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 glucose-transferring glycosyltransferase protein, the SUS enzyme, and their fusion protein are as described above.

[0039] According to the present invention, recombinant microorganisms or cells are microbial cells, and preferably include, but are not limited to, Escherichia coli, Saccharomyces strains, such as Saccharomyces cerevisiae, or Pichia strains.

[0040] In this specification, the term “transformation” means introducing a vector containing nucleic acids that encode a target protein into a host cell so that the protein encoded by the nucleic acids can be expressed in the host cell. Transformed nucleic acids may include all of the following, regardless of whether they are inserted into or extrachromosomal regions of the host cell, as long as they can be expressed in the host cell. The nucleic acids may also include DNA and RNA that encode the target protein. The nucleic acids may be introduced into the host cell in any form that allows them to be introduced and expressed. For example, the nucleic acids may be introduced into the host cell in the form of an expression cassette of a gene structure containing all the elements necessary for their expression. The expression cassette may typically include 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 also be in the form of a self-replicating expression vector. Furthermore, the nucleic acids may be introduced into the host cell in their own form and operably linked to the sequences necessary for expression in the host cell, but are not limited to such forms.

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

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

[0043] An example of a recombinant vector containing a nucleic acid molecule that encodes a glycosyltransferase protein that transfers glucose to a steviol glycoside according to an example of the present invention is shown in the cleavage map of Figure 1. For example, the nucleic acid molecule that encodes the enzyme according to the present invention may include a GAL10 promoter and a CYC1 terminator as transcriptional regulatory sequences that can be linked to and actuated by 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 for transforming cells with the vector of the present invention also includes a method for introducing nucleic acids into cells, and can be performed by selecting a standard technique suitable for the host cell, as is well known in the art. Examples include, but are 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] An example of the present invention relates to a composition for producing a 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 expressing the enzyme protein, the cells of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and extracts thereof.

[0046] The composition for producing the steviol glycoside may additionally include a substrate selected from the group consisting of stevioside and Reb A, for example, a single substrate of stevioside or Reb A, or a mixture thereof. The mixture substrate may be a mixture of stevioside or Reb A, or a stevia extract (or stevia) containing stevioside or Reb A. Specifically, the substrate or reaction raw material may contain rebaudioside A in an amount of 50-70(v / v)%, 50-65(v / v)%, 50-62(v / v)%, 55-70(v / v)%, 55-65(v / v)%, 55-62(v / v)%, 58-70(v / v)%, 58-65(v / v)%, or 58-62(v / v)%, for example, 60(v / v)%, based on the total volume of the stevia extract. The stevia extract contains, but is not limited to, stevioside in amounts 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)%, based on the total volume.

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

[0048] The composition may further contain a glucose donor, for example, UDP-glucose or a sugar capable of producing UDP-glucose.

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

[0050] The culture contains the enzyme produced from a UDP-glucosyltransferase-producing microorganism, and may contain the strain or be in a cell-free form without the strain. The lysate refers to a lysate obtained by crushing the cells of a UDP-glucosyltransferase-producing microorganism, or the supernatant obtained by centrifugation of the lysate, and contains the enzyme produced from the UDP-glucosyltransferase-producing microorganism.

[0051] The culture of the strain contains the enzyme produced by the UDP-glucosyltransferase-producing microorganism and may be in a cell-free form, with or without the microbial cells. In this specification, unless otherwise specified, the UDP-glucosyltransferase-producing microorganism used means one or more selected from the group consisting of the microbial cells of the strain, the culture of the strain, the lysates of the microbial cells, the supernatant of the lysates, and extracts thereof.

[0052] When producing steviol glycosides using the aforementioned steviol glycoside production composition, the reaction temperature and reaction pH conditions using UDP-glucosyltransferase or enzyme-producing microorganisms 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 a steviol glycoside, comprising the 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, the cells of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and extracts thereof, with a substrate for the enzyme.

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

[0055] An example of the present invention is a case in which 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 the step of reacting one or more selected from the group consisting of UDP-glucosyltransferase protein, recombinant microorganism expressing the enzyme protein, microbial cells, microbial cell lysates, microbial cultures, 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] An additional example of the present invention is when the produced steviol glycoside is Reb M, and specifically involves a step of reacting one or more selected from the group consisting of UDP-glucosyltransferase protein, recombinant microorganism expressing the enzyme protein, microbial cells, microbial cell lysates, microbial cultures, 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 to produce one or more steviol glycosides selected from the group consisting of Reb D and Reb E, and The present invention relates to a method for producing Reb M of a steviol glycoside, comprising the step of reacting one or more selected from the group consisting of the produced steviol glycoside, a second UDP-glucosyltransferase protein, a recombinant microorganism expressing the enzyme protein, the cells of the microorganism, the cell lysate of the microorganism, the culture of the microorganism, and extracts thereof, in the presence of a glucose donor.

[0057] The second UDP-glucosyltransferase protein used in the manufacturing process of Reb M may be UGT76G1 or the like.

[0058] The glucose donor provided in the process of producing the steviol glycoside may be UDP-glucose, which may be provided as a compound or as sugar and the enzyme Sucrose Synthase (SUS). The SUS enzyme is an enzyme that breaks down sugar to produce fructose and UDP-glucose. Therefore, by introducing the SUS enzyme into the reaction that converts Reb A to Reb D, or the reaction that converts stevioside to Reb E, UDP-glucose can be supplied to the UGT enzyme, allowing the reaction to proceed more economically. Furthermore, if the conversion reaction is carried out using a fusion protein of UGT and SUS, the two reactions form a single channel, and the enzymatic reaction occurs in a single step, resulting in an effective conversion reaction. 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 significantly shortens the production cycle, improves production capacity, and provides high-purity products at low cost, making it economically applicable to the food and beverage industry.

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

[0061] The UDP-glucosyltransferase protein according to the present invention, and the steviol glycoside produced using recombinant microorganisms expressing the enzyme protein, are used as high-intensity sweeteners, added to various foods and beverages, or sold commercially as table sweeteners on their 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 exemplary examples.

[0063] Example 1: Production of microorganisms expressing wild-type enzymes 1-1: Vector creation to express wild-type enzyme A gene encoding a wild-type enzyme protein with the amino acid sequence of Sequence ID No. 1 from Triticum aestivum was synthesized (Gblock synthesis), and the polynucleotide sequence of the synthesized gene is shown in Sequence ID No. 2. The synthesized gene was subcloned into a plasmid expressed in S. cerevisiae to create a form that can be expressed. Specifically, to confirm protein activity, we used a pRS426 vector containing a uraauxotrophic marker (selection marker) that can be sorted using S. cerevisiae, a GAL10 promoter, and CYC1 as the terminator. The newly synthesized (IDT, gBlock) Sequence ID 2 gene was cloned into this plasmid. For the cloning of the TaUGT gene, a primer pair (Sequence ID 19 and Sequence ID 19) was constructed to overlap with the GAL10 promoter (Sequence ID 16) and cyc1 terminator B (Sequence ID 17). The enzyme gene was amplified by PCR, and the gene was subcloned by Gibson assembly (HIFi DNA master mix, NEW ENGLAND BIOLABS). The subcloned plasmid genes were transformed into E. coli (DH5a), sorted, and amplified. The sequence of the amplified plasmids was analyzed to confirm that the gene expressing the enzyme had been accurately subcloned.

[0064] [Table 1]

[0065] The transformation method for introducing genes into S. cerevisiae CENPK2-1c (Euroscarf) involved treatment with 0.1M LiAc (Lithium Acetate) followed by thermal shock. The transformed strains were sorted on SC_Ura medium, and the enzyme activity of the sorted colonies was confirmed through culture.

[0066] The manufactured recombinant plasmid 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 SC-Ura solid medium consisted of 6.7 g / L of YNB, 0.7 g / L of Drop-out mix, 50 g / L of Glucose, and 20 g / L of agar, with the pH adjusted to 6.0.

[0067] 1-2: Culture of recombinant microorganisms The Plasmid-transformed yeast produced in Example 1-1 was sorted in a synthetic complete (SC) medium using a URA-drop-out mix, and enzyme expression was induced in the sorted yeast through liquid culture. Specifically, colonies selected on SC_Ura solid medium were inoculated into test tubes containing 3 mL of SC_Ura liquid medium and incubated overnight. The concentration of the cultured cells was measured using the OD system. 600 The absorbance was confirmed, and OD was added to a 250 mL flask containing 25 mL of SC-Ura liquid medium. 600 The cells were inoculated so that the absorbance was ultimately 0.1 (final), and primary culture was performed for 24 hours (30°C, 240 rpm). To induce enzyme expression in the primary cultured cells, the same amount of YPG medium was added, and secondary culture was performed for 24 hours. The secondary cultured cells were collected by centrifugation (4,000 rpm, 10 min). The SC_Ura liquid medium consisted of 6.7 g / L of YNB (yeast nitrogene 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 YPG medium consisted of 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.

[0068] 1-3: Microbial production expressing EUGT11 and HvUGT The EUGT11 gene from Oryza sativa (SEQ ID NO: 13) and the HvUGT gene from Hordeum vulgare (SEQ ID NO: 15), both known to have existing activity, were synthesized (Gblock synthesis). These genes were amplified in the same manner 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 CYC1 as the terminator. Specifically, the following primers were used: a forward primer for EUGT11 that ligates to the GAL10 promoter (SEQ ID NO: 20), a reverse primer for EUGT11 that ligates to the CYC1 terminator (SEQ ID NO: 21), a forward primer for HvUGT that ligates to the GAL10 promoter (SEQ ID NO: 22), and a reverse primer for HvUGT that ligates to the CYC1 terminator (SEQ ID NO: 23). In Table 1, the enzyme-producing gene carset produced is shown, where Sc stands for Saccharomyces cerevisiae. Subsequently, S. cerevisiae CENPK2-1c (Euroscarf) was transformed using substantially the same method as in Examples 1-1 and 1-2, followed by strain selection and microbial culture.

[0069] 1-4: Enzyme activity evaluation The cultures of the recombinant strains obtained in Examples 1-2 and 1-3 were collected by centrifugation (4,000 rpm, 10 min). 10 mL of the collected cells was collected and washed twice with the same amount of water. The washed cells were resuspended in 100 mM Phosphate buffer (pH 7.2) and OD (Oxygen-Dose). 600 After adjusting the value to 100, 100 mg of glass beads (Sigma) with a particle size of 0.4-0.6 mm was placed in a cap tube, and 1 ml of 100 mM Phosphate buffer (pH 7.2) was added. The bacterial cells were crushed using a bead beater for 30 seconds, repeated 5 times. The crushed bacterial cell solution was centrifuged under refrigeration (13,000 rpm, 15 min), and the supernatant was obtained as a coenzyme solution. The reaction solution for evaluating enzyme activity contained the substrate Reb A (95%, Sinochem), MgCl2, and UDP-Glucose. 0.1 mL of the coenzyme solution was added to this to prepare a total reaction volume of 0.5 mL, resulting in a final reaction solution with a concentration of 2 g / L of Reb A (95%, Sinochem), 3 mM of MgCl2, and 4 mM of UDP-Glucose. The prepared reaction solution was subjected to an enzymatic reaction at a temperature of 30°C, pH 7.2, and 150 rpm. Samples were taken at 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 reaction, and the supernatant obtained by centrifugation (13,000 rpm, 10 min) was analyzed to confirm the enzyme reaction products. Specifically, the enzyme reaction products were analyzed using HPLC-Chromatography to confirm the conversion ratio of the products. The coenzyme solution obtained from the cell lysate of the recombinant strain showed activity to convert Reb A substrate to Reb D, so the activity was measured by confirming the Reb D production ratio. Specifically, the column used for HPLC analysis of the conversion product was UG120 (C18 250mm × 46mm, 5um 110 A particle, Shideido), and the analysis was confirmed at 210nm. The mobile phase was water containing 0.01% TFA (Trifluoroacetic acid, Sigma) and acetonitrile, respectively, and the analysis was performed using a gradient. The mobile phase was flowed at 1 mL / min, and the analysis was confirmed after a total of 40 minutes. The HPLC analysis conditions are shown in Table 2 below.

[0070] [Table 2]

[0071] Table 3 shows the HPLC analysis graph of the conversion product. The Reb D conversion rate of the enzyme that converts Reb A alone to Red D, as shown in Table 3 below, is calculated according to Formula 1 below, and the relative Reb D conversion rate of the enzyme refers to the Reb D conversion rate of the TaUGT or HvUGT enzyme converted to a baseline of 100% Reb D conversion rate for EUGT11. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / Total HPLC peak area (Equation 1)

[0072] [Table 3]

[0073] As shown in Table 3, the conversion rate by the enzymatic reaction, after the initial 3-hour reaction, confirmed that TaUGT converted 40-45% of Reb A to Reb D, similar to EUGT11, and exhibited approximately 30% higher activity than HvUGT (30-35% conversion). Furthermore, analysis of the Reb D content of the enzyme reaction solution after 18 hours showed that the order of conversion rates of the three enzymes followed a similar trend to that of the 3-hour reaction. The polynucleotide sequence of the TaUGT enzyme was analyzed using Macrogen Co., Ltd.'s polynucleotide sequence analysis service. Based on the polynucleotide sequence, the amino acid sequence was analyzed, and the result is shown in SEQ ID NO: 1, and the polynucleotide sequence is shown in SEQ ID NO: 2.

[0074] Example 2: Creation of microorganisms expressing enzyme mutants 2-1: Vector preparation for enzyme mutant expression To develop the mutations in the TaUGT enzyme of Example 1, mutations were searched for based on the existing EUGT11 gene. The preserved region was identified through alignment, and amino acid differences were compared at the positions between the N-term sugar acceptor domain and the C-term sugar donor domain of UGT. In the case of threonine at residue 201 and valine at residue 202 (where serine and leucine amino acids are located in EUGT11), threonine was changed to serine, and valine was changed to alanine or leucine. The mutation method involved synthesizing a primer for the corresponding position in TaUGT (SEQ ID NOs. 24 and 25) and performing mutant gene amplification (PCR) to alter the polynucleotide sequence at the corresponding site. The amplified gene fragment was then cloned into the pRS426 vector via Gibson assembly. The resulting pRS426 vector was cloned and named TaUGTT201S / V202L. The primer sequences used in this example are listed in Table 4 below.

[0075] [Table 4]

[0076] 2-2: Creation of enzyme mutant-expressing bacterial strains In substantially the same manner as in Examples 1-2, TaUGTT201S / V202L cloned into the pRS426 vector was transformed into yeast strains and selected using the LiAc method to confirm enzyme expression. The transformed strains were cultured in substantially the same manner as in Examples 1-2. The yeast transformed with Plasmid produced in Example 2-1 was selected in SC (synthetic complete) medium using URA-drop-out mix, and enzyme expression was induced in the selected yeast through liquid culture.

[0077] Example 3: Evaluation of enzyme activity of enzyme mutants The culture of the recombinant strain obtained in Example 2-2 was centrifuged (4,000 rpm, 10 min) to collect the bacterial cells. 10 mL of the collected bacterial cells was collected and washed twice with the same amount of water. The washed bacterial cells were resuspended in 100 mM Phosphate buffer (pH 7.2) and OD (Oxygen-Dose). 600 After adjusting the value to 100, 100 mg of glass beads (Sigma) with a particle size of 0.4-0.6 mm was placed in a cap tube, and 1 ml of 100 mM Phosphate buffer (pH 7.2) was added. The bacterial cells were crushed using a bead beater for 30 seconds, repeated 5 times. The crushed bacterial cell solution was centrifuged under refrigeration (13,000 rpm, 15 min), and the supernatant was obtained as a coenzyme solution. The reaction solution for evaluating enzyme activity contained the substrate Reb A (95%, Sinochem), MgCl2, and UDP-Glucose. 0.1 mL of the coenzyme solution was added to this to prepare a total reaction volume of 0.5 mL, resulting in a final reaction solution with a concentration of 2 g / L of Reb A (95%, Sinochem), 3 mM of MgCl2, and 4 mM of UDP-Glucose. The prepared reaction solution was subjected to an enzymatic reaction at a temperature of 30°C, pH 7.2, and 150 rpm. Samples were taken at 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 reaction, and the supernatant obtained by centrifugation (13,000 rpm, 10 min) was analyzed to confirm the enzyme reaction products. Specifically, the enzyme reaction products were analyzed using HPLC-Chromatography to confirm the conversion ratio of the products. The coenzyme solution obtained from the cell lysate of the recombinant strain showed activity to convert Reb A substrate to Reb D, so the activity was measured by confirming the Reb D production ratio. Specifically, the column used for HPLC analysis of the conversion product was UG120 (C18 250mm × 46mm, 5um 110 A particle, Shiseido), and the analysis was confirmed at 210nm. The mobile phase was water containing 0.01% TFA (Trifluoroacetic acid, Sigma) and acetonitrile, respectively, and the analysis was performed using a gradient. The mobile phase was flowed at 1 mL / min, and the analysis was confirmed over 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 Figure 2. The Reb D conversion rate of the enzyme that converts from Reb A alone to Red D, as shown in Table 4 below, was calculated according to Equation 1 below, and the relative Reb D conversion rate of the enzyme represents the Reb D conversion rate of the TaUGT mutant enzyme converted to a standard of 100% Reb D conversion rate of the wild-type enzyme. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / Total HPLC peak area (Equation 1)

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

[0079] [Table 5]

[0080] Example 4 Enzyme conversion using mixed substrates The bacterial cells were resuspended in 100 mM Phosphate buffer (pH 7.2), and OD (Oxygen Dissociation) was performed. 600After adjusting the value to 100, 100 mg of glass beads (Sigma) with a particle size of 0.4-0.6 mm was placed in a cap tube, and 1 ml of 100 mM Phosphate buffer (pH 7.2) was added. The bacterial cells were crushed using a bead beater for 30 seconds, repeated 5 times. The crushed bacterial cell solution was centrifuged under refrigeration (13,000 rpm, 15 min), and the supernatant was obtained as a coenzyme solution. The reaction solution for evaluating enzyme activity contained the substrate RA40 (Reb A (rebaudioside A:STE (stevioside) = 40:60, Sinochem)), MgCl2, and UDP-Glucose. 0.2 mL of the coenzyme solution from Example 3 was added to this to prepare a total reaction volume of 0.5 mL, resulting in a final reaction solution with a concentration of 2 g / L of substrate RA40 (Reb A:STE = 40:60, Sinochem), 3 mM of MgCl2, and 4 mM of UDP-Glucose. The prepared reaction solution was subjected to an enzymatic reaction at a temperature of 30°C, pH 7.2, and 150 rpm, and the degree of reaction was confirmed 18 hours after the start of the reaction. The conversion product was analyzed by HPLC using substantially the same method as in Example 3. The total Reb D / E conversion rate of the enzyme that converts to the RA40 mixed substrate shown in Table 6 below is calculated according to formula 2 below, and the relative total Reb D / E conversion rate of the enzyme represents the total Reb D / E conversion rate of the TaUGT enzyme converted based on the total Reb D / E conversion rate of EUGT11, which is 100%. Reb D / E conversion rate (%) = HPLC peak area of ​​Reb D / E / Total HPLC peak area (Equation 2)

[0081] [Table 6]

[0082] The results of the aforementioned experiment showed that in the case of the mutant enzyme in which Threonine 201 was replaced with Serine, the relative conversion rate was 106% relative to the wild-type enzyme. In the case of the double mutant enzyme, which included both the change of Threonine 201 to Serine and the change of Valine 202 to Leucine, the relative conversion rate increased to 114% relative to the wild-type enzyme. This allowed us to identify the residue whose activity was improved by the mutation and to secure the mutant enzyme with increased activity.

[0083] Example 5: Preparation and activity confirmation of TaUGT_SUS fusion enzyme To supply the necessary glucose donor (UDP-Glucose) for the glycosylation reaction of steviol glycosides using the TaUGT_T201SV202L(TaUGTm) mutant enzyme, which showed improved activity in Example 3, a fusion protein was produced with the succrose synthase (SUS1, derived from Arabidopsis thaliana) enzyme. Nucleic acid distribution that encodes the aforementioned TaUGT_T201SV202L(TaUGTm) mutant enzyme The column contains the nucleic acid sequence of sequence number 30 in Table 7 below, and the sucrose synthase (SUS1, derived from Arabidopsis thaliana) uses the nucleic acid sequence of sequence number 5 in Table 7 below.

[0084] [Table 7-1]

[0085] [Table 7-2]

[0086] To create the fusion enzyme, a primer was synthesized and a linker (GGGGSG, SEQ ID NO: 8) was used to link the TaUGTm and SUS amino acids. For each gene, a primer was synthesized at the corresponding position (SEQ ID NO: 26, SEQ ID NO: 27), and the base sequence of that region was amplified (PCR). Gene amplification was performed using PCR, and the amplified gene fragment was cloned into the pRS426 vector via Gibson assembly to produce 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 in the production of the fusion protein has the sequence 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 fusion enzyme expression vectors prepared were transformed into yeast and then selected and secured using the same method as in Examples 1-2. A coenzyme solution was prepared using the lysate of the cultured bacterial cells in substantially the same manner as in Examples 1-3. Specifically, in substantially the same manner as in Example 3, bacterial cells were recovered from the culture of the recombinant strain by centrifugation, and the cells were crushed with a glass bead and 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. To this, 0.1 mL each of coenzyme solution of TaUGTm_SUS fusion enzyme was added to prepare a total reaction volume of 0.5 mL. The final reaction solution was then adjusted to contain 10 g / L of RA40 reaction substrate, 1 mM of MgCl2, 1 mM of UDP, and 125 mM of sucrose. The prepared reaction solution was subjected to an enzymatic reaction at a temperature of 30°C, pH 7.2, and 150 rpm for 20 hours to confirm the enzymatic conversion. The conversion products of the reaction solution were analyzed and confirmed by HPLC analysis using substantially the same method as in Examples 1-4 above. In the case of the fusion enzyme, it was confirmed that RA40 (Reb A:STE=40:60) was converted to Reb D / E by UDP-glucose supply using UDP. STE represents stevioside.

[0089] With 100% by weight of the substrate before the reaction, approximately 33% by weight of the substrate was converted to Reb D / E by the initial fusion enzyme conversion reaction. Conversion of the mixed substrate (RA40 (Reb A:STE=40:60)) resulted in conversion of Reb D (including D derivatives) and Reb E to 21.7% and 12.1%, respectively. The conversion of steviol glycosides was confirmed by UDP_Glucose production using sugar by the fusion enzyme and subsequent substrate saccharification using this glucose. 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 the conversion of RA40 (Reb A:STE=40:60), if a 10 g / L substrate is used, 10-20 mM or more of UDP-glucose is required to convert to a substrate equivalent to approximately 10 mM. Therefore, when performing conversions with high concentrations and large volumes, an excess amount of expensive UDP-glucose is required. When performing enzymatic reactions using high-concentration substrates, it is advisable to use an excess of UDP-glucose, but this is undesirable because the excess UDP-glucose inhibits enzyme activity. According to this experiment, when using the TaUGT_SUS fusion enzyme, conversion is possible using 1 / 10 the amount of UDP relative to the substrate, and in the case of UDP, the reaction can be carried out using less than 1 / 100 the amount relative to RA40. We confirmed that economical enzymatic conversion is possible through the conversion reaction using the fusion enzyme, and that the production of steviol glycosides is induced by a high-concentration substrate reaction.

Claims

1. i) Substitution of the 201st threonine from the N-terminus to serine in the amino acid sequence of Sequence ID No. 1; or, ii) In the amino acid sequence of Sequence ID No. 1, the substitution of threonine at position 201 from the N-terminus to serine, and the substitution of valine at position 202 from the N-terminus to leucine, It has, An enzyme protein possessing uridine diphosphonate-glucosyltransferase (UDP-glucosyltransferase) activity, which transfers glucose to a steviol glycoside substrate.

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

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

5. A fusion protein comprising a UDP-glucosyl transfer enzyme protein and a sugar synthase enzyme protein linked together, as described in claim 1.

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

7. A recombinant microorganism comprising a gene that encodes an enzyme protein according to any one of claims 1 to 4 or a fusion protein according to claim 5 or 6.

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

9. The present invention comprises one or more selected from the group consisting of an enzyme protein according to any one of claims 1 to 4, a recombinant microorganism expressing the enzyme protein, the cells of the microorganism, a lysate of the cells of the microorganism, a culture of the microorganism, and extracts thereof. A composition for producing steviol glycosides comprising the recombinant microorganism, the bacterial cells, the bacterial cell lysate, the culture, and the extract, the enzyme protein described in any one of claims 1 to 4.

10. The composition according to claim 9, wherein the composition further comprises a substrate selected from the group consisting of stevioside and rebaudioside A.

11. The composition according to claim 10, wherein the substrate is a mixed substrate containing stevioside and rebaudioside A.

12. The composition according to claim 9, further comprising an enzyme that converts rebaudioside D or rebaudioside E to rebaudioside M.

13. The composition according to claim 12, wherein the enzyme that converts to rebaudioside M is UGT76G1 derived from stevia.

14. The composition according to claim 9, wherein the enzyme protein is a fusion protein in which a enzyme synthase enzyme protein is linked.

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

16. The composition according to claim 15, wherein the glucose donor is UDP-glucose.

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

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