Rebaudioside Production

Abstract

The present specification relates to a composition for producing a steviol glycoside containing rebaudioside M at a high purity using a biocatalyst including UDP-glycosyltransferase and UGT76G1, and a method for producing a steviol glycoside using the same.

Description

[Technical field]

[0001] The present invention relates to the production of rebaudioside using a biocatalytic reaction, for example, a microorganism for producing rebaudioside and a method for producing rebaudioside using the same, and more particularly to a recombinant microorganism for producing rebaudioside containing UDP-glycosyltransferase and a method for producing rebaudioside from a high-concentration stevia extract using the microorganism. The present invention also relates to an improved method for producing rebaudioside, and more particularly to a microorganism for producing rebaudioside that utilizes a freeze-dried microorganism that stably maintains enzyme activity for a long period of time, and a method for producing rebaudioside using the same. [Background technology]

[0002] Sweeteners are known to be the most commonly used ingredients in the food, beverage, and 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, 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 the perennial shrub, Stevia rebaudiana. Stevia extracts of various levels of purification are used as sensitivity flavors in foods and blends, and are commercially available alone as a table sweetener.

[0004] Extracts of the stevia plant contain rebaudiosides and other steviol glycosides that contribute sweetness, but conventional commercial products contain primarily rebaudioside A with small 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.

[0005] The composition of stevia extracts is also 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 has been reported to vary from 20 to 97% of the total steviol glycoside content during commercial production. Other steviol glycosides are also present in varying amounts in stevia extracts. Summary of the Invention [Problem to be solved by the invention]

[0006] However, stevia extracts produced from stevia plants contain many steviol glycosides and compounds that cause off-flavors, and their recovery and purification are labor-intensive and inefficient. Therefore, there is a need for a production system that can produce and accumulate steviol glycosides such as Reb D and Reb M in high yields. In particular, Reb M has a clean sweetness that is 200 to 350 times sweeter than sugar and has a slightly bitter aftertaste or a licorice aroma. Recently, the EFSA Panel on Food Additives and Flavorings announced that Reb M is safe as a food additive. Reb M is suitable for blending and applicable to various food and beverage products, so it can attract great interest and generate sustained demand as a natural sweetener in the global food industry. In addition, there is an urgent need to establish a manufacturing process and conditions for removing moisture contained in a biocatalyst, such as a microbial cell that produces an enzyme having steviol glycoside conversion activity, to provide a biocatalyst that maintains its conversion activity and has excellent storage and distribution properties, and to develop a mass production process for steviol glycoside, especially rebaudioside M, using the biocatalyst. [Means for solving the problem]

[0007] One example of the present invention relates to a composition for producing steviol glycoside, the composition comprising a biocatalyst including a first UDP-glucosyltransferase (uridine diphosphate-glucosyltransferase) that has 92% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 and that transfers glucose to a steviol glycoside substrate, and UGT76G1, and a reaction substrate, in which rebaudioside M is contained in an amount of 10% by weight or more of the steviol glycoside reaction product, or a method for producing the same.

[0008] One example of the present invention provides a recombinant microorganism for producing rebaudioside containing a novel UDP-glycosyltransferase derived from Triticum aestivum, preferably a biocatalyst containing UDP-glycosyltransferase and UDP-glycosyltransferase76G1, or more preferably a biocatalyst containing UDP-glycosyltransferase, UDP-glycosyltransferase76G1 and sucrose synthase, for producing high purity rebaudioside M.

[0009] One example of the present invention relates to a recombinant microorganism for producing rebaudioside containing a novel UDP-glycosyltransferase derived from Triticum aestivum, preferably a biocatalyst containing UDP-glycosyltransferase, UDP-glycosyltransferase76G1, or more preferably a biocatalyst containing UDP-glycosyltransferase, UDP-glycosyltransferase76G1 and sucrose synthase, and a composition for producing steviol glycoside containing rebaudioside M, in which rebaudioside M is contained in an amount of 50% by weight or more of a steviol glycoside reaction product.

[0010] Another example of the present invention relates to a method for producing steviol glycosides including rebaudioside M, comprising a step of reacting a reaction substrate including one or more selected from the group consisting of stevioside and rebaudioside A with a biocatalyst including a first UDP-glycosyltransferase derived from Triticum aestivum and UGT76G1.

[0011] Another embodiment of the present invention relates to a microorganism for producing rebaudioside using freeze-dried microbial cells that can stably maintain the enzyme activity of the microorganism in a dry state for a long period of time, and a method for producing rebaudioside using the same.

[0012] An additional embodiment of the present invention may be a microbial cell or a dried cell thereof having steviol glycoside, particularly rebaudioside M, conversion activity.

[0013] An additional embodiment of the present invention relates to a composition for producing steviol glycosides, particularly rebaudioside M, comprising a microbial cell having steviol glycoside, particularly rebaudioside M, conversion activity or a dried cell thereof, or a method for producing steviol glycosides, particularly steviol glycosides containing rebaudioside M. Effect of the Invention

[0014] The present invention relates to a transformed microorganism that expresses one or more enzymes selected from the group consisting of exogenous TaUGTm, SrUGT76G1, and sucrose synthase and has activity of converting Stevia to Rebaudioside M, and a method for producing Rebaudioside M using the same, wherein the transformed microorganism has high productivity of Rebaudioside M. [Brief description of the drawings]

[0015] [Figure 1] 1 is a cleavage map of a vector for producing a yeast strain producing a TaUGTm enzyme 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 a TaUGTm enzyme according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present specification relates to a composition for producing steviol glycosides containing rebaudioside M at high purity by utilizing a recombinant microorganism for producing rebaudioside containing a novel UDP-glycosyltransferase derived from Triticum aestivum, preferably a biocatalyst containing UDP-glycosyltransferase, UDP-glycosyltransferase76G1, or more preferably a biocatalyst containing UDP-glycosyltransferase, UDP-glycosyltransferase76G1 and sucrose synthase, and a method for producing steviol glycosides using the same.

[0017] One example of the present application relates to a composition for producing steviol glycoside containing rebaudioside M, which includes a biocatalyst containing UDP-glycosyltransferase derived from Triticum aestivum, specifically a first UDP-glycosyltransferase (uridine diphosphate-glucosyltransferase) having 92% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 and transferring glucose to a steviol glycoside substrate, and UGT76G1, and a reaction substrate, wherein rebaudioside M is contained in an amount of 50% by weight or more of the steviol glycoside reaction product.

[0018] Additional examples of the present application include a biocatalyst comprising a first UDP-glycosyltransferase (uridine diphosphate-glucosyltransferase) and UGT76G1 that have 92% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 and transfer glucose to a steviol glycoside substrate, and a reaction substrate, The biocatalyst is at least one selected from the group consisting of a microbial cell that produces the enzyme protein and a dried product of the microbial cell. The present invention relates to a composition for producing steviol glycosides containing rebaudioside M, wherein rebaudioside M is contained in an amount of 10% by weight or more of a steviol glycoside reaction product.

[0019] In addition, when a conversion reaction is carried out using the composition for producing a steviol glycoside containing rebaudioside M, for example, when a raw material substrate containing Reb A and stevioside is used, rebaudioside M may be 10% by weight or more, 15% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, 85% by weight or more, 86% by weight or more, 87% by weight or more, or 90% by weight or more of the steviol glycoside reaction product. The composition for producing a steviol glycoside containing rebaudioside M may have a total conversion rate of Reb M and Reb D of 30% by weight or more, 35% by weight or more, 40% by weight or more, 45% by weight or more, or 50% by weight or more.

[0020] The composition may further contain ethylenediaminetetraacetic acid (EDTA), preferably at a concentration of 1 to 20 mM, 3 to 20 mM, 4 to 20 mM, 1 to 15 mM, 3 to 15 mM, 4 to 15 mM, 1 to 10 mM, 3 to 10 mM, or 4 to 10 mM. When the composition for producing steviol glycosides containing rebaudioside M contains EDTA, when a conversion reaction is carried out using the composition for producing steviol glycosides containing rebaudioside M, for example, when a raw material substrate containing Reb A and stevioside is used, rebaudioside M may account for 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, 85% by weight or more, 86% by weight or more, 87% by weight or more, or 90% by weight or more of the steviol glycoside reaction product.

[0021] The composition may further include at least one reactant selected from the group consisting of stevia extract, uridine diphosphateglucose (UDP) and sucrose. In one embodiment, the conversion of reb A to reb D in the composition for producing steviol glycosides is carried out using MgCl 2 and / or MnCl 2 may additionally include:

[0022] Yet another example of the present application is a method for producing steviol glycosides including rebaudioside M, the method comprising the step of reacting a reaction substrate including one or more selected from the group consisting of stevioside and rebaudioside A with a biocatalyst including a first UDP-glycosyltransferase derived from Triticum aestivum and UGT76G1, The present invention relates to a method for producing steviol glycosides including rebaudioside M, wherein the first UDP-glycosyltransferase has an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO:1 and is an enzyme protein (uridine diphosphate-glucosyltransferase) having UDP-glycosyltransferase activity of transferring glucose to a steviol glycoside substrate.

[0023] One example of the present invention includes a method for producing one or more steviol glycosides selected from the group consisting of Reb D and Reb E by reacting one or more selected from the group consisting of a first 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 substrates selected from the group consisting of stevioside and Reb A in the presence of a glucose donor; 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, in the presence of a glucose donor.

[0024] Yet another example of the present invention is a method for producing steviol glycosides including rebaudioside M, comprising a step of reacting a reaction substrate including one or more selected from the group consisting of stevioside and rebaudioside A with a biocatalyst including a first UDP-glycosyltransferase and UGT76G1, The first UDP-glycosyltransferase is an enzyme protein (uridine diphosphate-glucosyltransferase) having an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO: 1 and having a UDP-glycosyltransferase activity of transferring glucose to a steviol glycoside substrate, The biocatalyst is at least one selected from the group consisting of a microbial cell that produces the enzyme protein and a dried product of the microbial cell. The present invention relates to a method for producing steviol glycosides, including rebaudioside M.

[0025] The second UDP-glucosyltransferase protein used in the production process of Reb M may be UGT76G1, etc.

[0026] The method for producing steviol glycosides according to the present invention can significantly shorten the production cycle, improve production capacity, and provide products with lower costs and higher purity, making it economically applicable to the food and beverage industries.

[0027] In the present invention, in the steviol glycoside containing rebaudioside M which is the reaction product, the purity of rebaudioside M may be 50% by weight or more.

[0028] The reaction substrate includes one or more steviol glycosides selected from the group consisting of stevioside and rebaudioside A, or is provided at 15 to 200 g / L. The reaction substrate may additionally include one or more selected from the group consisting of steviol monoside, rubusoside, rebaudioside E, rebaudioside C1, rebaudioside 1, stevioside, rebaudioside D, synthetic steviol glycosides, and combinations thereof.

[0029] The diversity of steviol glycosides in S. rebaudiana is due to glycosylation catalyzed by UDP-glycosyltransferases (UGTs), which transfer sugar residues from activated donors, mainly uridine diphosphate (UDP)-glucose, to recipient molecules. Rebaudioside M (Reb M) is a preferred natural, calorie-free, high-potency sweetener found in very low amounts in the leaves of Stevia rebaudiana. According to the composition for producing steviol glycosides or the method for producing steviol glycosides of the present invention, Reb M can be produced with high purity and high conversion rate.

[0030] The present invention will now be described in further detail. The composition for producing steviol glycoside or the method for producing steviol glycoside according to the present invention may use a biocatalyst containing a novel first UDP-glycosyltransferase and a second UDP-glycosyltransferase, UGT76G1. The biocatalyst may additionally contain sucrose synthase, and may contain a fusion protein with the first UDP-glycosyltransferase and / or the UDP-glycosyltransferase.

[0031] In another embodiment, as a third UDP-glucosyltransferase, an enzyme that can add at least one glucose unit to rebaudioside A to form rebaudioside D, e.g., a UDP-glucosyltransferase, can additionally include UGT91D2.

[0032] In the present specification, the biocatalyst containing the enzyme may comprise one or more selected from the group consisting of the enzyme protein, the microbial cells, a culture of the microbial cells, a disrupted product of the microbial cells, and an extract of the disrupted product or the culture.

[0033] In the present specification, the first UDP-glycosyltransferase may be an enzyme protein (uridine diphosphate-glucosyltransferase) having an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO:1 and having UDP-glycosyltransferase activity that transfers glucose to a steviol glycoside substrate.

[0034] The first UDP-glycosyltransferase can convert stevioside (STE) to Reb E and / or convert Reb A to Red D to produce Reb D and / or Reb E.

[0035] Specifically, the first 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 into 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 into Reb D and stevioside into Reb E, and has conversion activity for both Reb A and stevioside as single substrates or a mixture of substrates.

[0036] In addition, the conversion from Reb A to Reb D is a transfer of glucose via beta1-2 glycosidic bond in the steviol ring, with glycation occurring mainly at position 19 of the steviol ring. Steviol glycosides are formed by the binding of 1 to 3 glucose units via beta bonds.

[0037] The conversion reaction from Reb A to Reb D is a reaction in which one more glucose molecule is added, and is catalyzed by UGT enzymes. The glucose required for this reaction is in the active form of UDP-glucose, and UGT enzymes transfer glucose from UDP-glucose to produce steviol glycosides.

[0038] The first UDP-glucosyltransferase may be an enzyme derived from Triticum aestivum (wheat) (Triticum aestivum UDP-glycosyltransferase) (hereinafter referred to as TaUGT) or a mutant enzyme thereof (hereinafter referred to as TaUGTm). More specifically, the first UDP-glucosyltransferase is an enzyme protein having 92% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and having UDP-glycosyltransferase activity of transferring glucose to a steviol glycoside substrate, and the enzyme protein having the amino acid sequence of SEQ ID NO: 1 may be a wild-type enzyme.

[0039] The nucleic acid sequence of the wild-type TaUGT of the present invention may be a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:1, but is specifically, but not limited to, the nucleic acid sequence of SEQ ID NO:2.

[0040] The first UDP-glucosyltransferase may comprise an amino acid sequence that has a sequence identity of 92% or more, 95% or more, 97% or more, 99% or more, or 99.5% or more to the amino acid sequence of SEQ ID NO: 1, and also includes mutant enzyme proteins that contain an amino acid sequence in which part of the sequence has been deleted, modified, substituted or added, so long as the amino acid sequence exhibits efficacy equivalent to that of the enzyme protein.

[0041] It 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 the polynucleotide sequence encoding the first UDP-glucosyltransferase.

[0042] In the above, the term "homology" or "identity" refers to the degree of identity with a given amino acid sequence or nucleotide 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 nucleotide sequence is expressed as "% homology" or "% identity".

[0043] The mutant first UDP-glucosyltransferase may have an activity of 101% or more, for example, 101% to 200%, based on 100% activity of a wild-type enzyme comprising the amino acid sequence of SEQ ID NO: 1 in converting a rebaudioside A (Reb A) substrate into 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 be reacted 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 through the enzyme conversion reaction, and the highly functional sugar Reb M can be produced with high productivity by using this.

[0044] The mutant first UDP-glucosyltransferase has 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, can have the amino acid sequence of SEQ ID NO: 3.

[0045] Furthermore, the nucleic acid sequence encoding the mutant enzyme includes the amino acid sequence of SEQ ID NO: 1 in which threonine at position 201 from the N-terminus is replaced with serine, valine at position 202 is replaced with leucine, or threonine at position 201 is replaced with serine and valine at position 202 is replaced with leucine, and more specifically, may include a nucleic acid sequence encoded by the amino acid sequence of SEQ ID NO: 3. In the mutant enzyme including the amino acid sequence of SEQ ID NO: 3 or a nucleic acid sequence encoding the amino acid sequence, the nucleic acid sequence encoding the amino acid sequence may be the nucleic acid sequence of SEQ ID NO: 3.

[0046] The mutant enzyme TaUGTm(T201S) of the present invention is an enzyme 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, and TaUGTm(V202L) is an enzyme in which Val at position 201 is replaced with Leu, for example, GTA is replaced with TTA, or TaUGTm(T201SV202L) is an enzyme in which 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 is replaced with Ser-Leu, and more specifically, it may be an enzyme in which ACCGTA is replaced with TCATTA in the polynucleotide sequence of SEQ ID NO: 2.

[0047] The first mutant UDP-glucosyltransferase according to the present invention has an activity of converting 45% by weight or more of Reb A to Reb D in an enzyme reaction for 2 to 24 hours, specifically, has an activity of converting 45% by weight or more, 50% by weight or more, 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, or 95% by weight or more of Reb A to Reb D, preferably has an activity of converting 0% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, or 95% by weight or more to Reb D. More specifically, the conversion activity of the enzyme was measured under the conditions of 30°C, pH 7.2, and 150 rpm. The Reb D conversion rate (%) was calculated according to the following formula 1, and the details are described in Example 4. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / total HPLC peak area (Formula 1)

[0048] The first 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 single substrate to Red D is calculated according to Equation 1. Detailed measurement criteria are described in Example 4 below.

[0049] Furthermore, the first mutant UDP-glucosyltransferase has an activity (Reb D / E total conversion rate) 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 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.

[0050] The first mutant UDP-glucosyltransferase according to the present invention has an activity of converting 50% by weight or more of a mixed substrate containing stevioside and Reb A to Reb D and Reb E (total Reb D / E conversion rate) in an enzymatic 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% by weight or more, 105% by weight or more, 106% by weight or more, 108% by weight or more, 109% by weight or more, or 110% by weight or more, as a 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 that converts 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 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)

[0051] The wild-type first UDP-glucosyltransferase according to 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.

[0052] 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 enzyme known to be involved in this production process is UGT91D2 in stevia. An enzyme with similar activity is the EUGT11 enzyme present in rice. UGT91D2 in stevia 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.

[0053] Conventionally known UGT enzymes include UGT derived from stevia (Stevia rebaudiana) (hereinafter referred to as SrUGT), UGT derived from rice (Oryza sativa) (hereinafter referred to as EUGT11), and UGT derived from barley (Hordeum vulgare) (hereinafter referred to as HvUGT). Compared with conventionally known EUGT11 and 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. 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.

[0054] The amino acid sequence of the HvUGT enzyme is represented in SEQ ID NO:14, and the polynucleotide sequence is represented in SEQ ID NO:15.

[0055] Analysis of the amino acid sequence and sequence identity of the wild-type enzyme having the amino acid sequence of SEQ ID NO: 1 with HvUGT and EUGT11 revealed that it has 91% amino acid sequence identity with HvUGT and 66% with EUGT, and analysis of the polynucleotide sequence identity revealed that it has 36% polynucleotide sequence identity with HvUGT and 37% with EUGT11.

[0056] The composition for producing steviol glycoside or the method for producing steviol glycoside according to the present invention may use a biocatalyst containing a second UDP-glycosyltransferase, UGT76G1, together with a first UDP-glycosyltransferase. The SrUGT76G1 of the present invention is a type of SrUGT, ​​an enzyme having a family number of 76G1, and has an amino acid sequence of SEQ ID NO: 31, but is not limited thereto. The nucleic acid sequence of SrUGT76G1 is a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 31, specifically, the nucleic acid sequence of SEQ ID NO: 32, but is not limited thereto.

[0057] In the present invention, the second UDP-glycosyltransferase UGT76G1 is the UDP-glycosyltransferase UGT76G1 (Stevia glycosyltransferase UGT76G1), which is the main enzyme responsible for the production of Reb M in S. rebaudiana, and is the only enzyme reported for the biosynthesis of Reb M from Reb D. UGT76G1 is known to have a wide range of substrate specificity, and has been found to specifically catalyze eight different reactions in the biosynthesis of steviol glycosides, all of which include 1,3-glucosylation of steviol C13 and C19-linked glucose. For example, UGT76G1 can convert stevioside (STE) to Rebaudioside A (Reb A), Rebaudioside D (Red D) to Reb M, and Rebaudioside E (Reb E) to Reb M. The SrUGT76G1 has the activity of converting rebaudioside A to rebaudioside I. Since rebaudioside I is difficult to convert to rebaudioside M, in order to obtain rebaudioside M in high yield from stevia (e.g., stevia extract) and / or rebaudioside A, TaUGTm, which has the activity of converting rebaudioside E and / or rebaudioside A to rebaudioside D, can be used together to obtain rebaudioside M in high yield.

[0058] The composition for producing steviol glycoside or the method for producing steviol glycoside of the present invention can use a biocatalyst that additionally contains Sucrose Synthase (SUS) enzyme in addition to the first UDP-glycosyltransferase and the second UDP-glycosyltransferase UGT76G1.

[0059] Sucrose synthase (SUS) enzyme is an enzyme that breaks down sucrose to produce fructose and UDP-glucose. Therefore, by introducing SUS enzyme into the reaction of converting Reb A to Reb D or stevioside to Reb E and supplying UDP-glucose to UGT enzyme, the reaction can be carried out more economically by supplying UDP-glucose. When the SUS enzyme is contained in the biocatalyst, it is more preferable that the microorganism that produces the enzyme is a Saccharomyces microorganism lacking the SUC2 gene that encodes the invertase enzyme.

[0060] 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, and all sucrose synthases known in the art can be applied to the present invention. More specifically, the sucrose synthase is SUS1 enzyme derived from Arabidopsis thaliana, and may include, for example, the amino acid sequence of SEQ ID NO: 4, and may have a nucleic acid sequence encoded by the amino acid sequence of SEQ ID NO: 4, or a nucleic acid sequence of SEQ ID NO: 5. The sucrose synthase is derived from Glycine max, and may include, for example, the amino acid sequence of SEQ ID NO: 33, and may have a nucleic acid sequence encoded by the amino acid sequence of SEQ ID NO: 33, or a nucleic acid sequence of SEQ ID NO: 34.

[0061] In one specific example, when the conversion reaction is carried out by preparing a fusion protein of UGT and SUS, the two reactions form one channel and one enzymatic reaction occurs, so that an effective conversion reaction can be carried out.

[0062] For example, when the above conversion reaction is carried out by preparing a fusion protein of UGT and SUS, the two reactions form one channel and one enzymatic reaction occurs, so that an effective conversion reaction can be carried out.

[0063] 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. Economical enzymatic conversion is possible through the conversion reaction using the fusion enzyme, and steviol glycosides can be produced through a high-concentration substrate reaction.

[0064] More specifically, one or more enzymes selected from the group consisting of the first UDP-glucosyltransferase and the second UDP-glucosyltransferase (e.g., UGT76G1 enzyme) of the present invention can be fused to sucrose synthase directly or via a linker, and the linker is a peptide consisting of 4 to 15 amino acids, more specifically, one containing 4 to 15 amino acids of one or more types selected from the group consisting of G (Gly), S (Ser) and P (Pro), specifically 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).

[0065] In a specific example of the present invention, the first UDP-glucosyltransferase is a fusion protein linked to the Arabidopsis thaliana-derived SUS1 enzyme via a linker (GGGGSG), and specifically may include the amino acid sequence of SEQ ID NO: 7, or have a nucleic acid sequence encoded by the amino acid sequence of SEQ ID NO: 7 or a nucleic acid sequence of SEQ ID NO: 8.

[0066] Specific enzyme sequences described in the present specification are exemplified in Table 1 below, in which the mutant enzyme TaUGTm represents UGT(T201S / V202L).

[0067] [Table 1-1]

[0068] [Table 1-2]

[0069] [Table 1-3]

[0070] [Table 1-4]

[0071] [Table 1-5]

[0072] In the present specification, the biocatalyst containing the enzyme may comprise one or more selected from the group consisting of the enzyme protein, the microbial cells, a culture of the microbial cells, a disrupted product of the microbial cells, and an extract of the disrupted product or the culture.

[0073] The biocatalyst is one or more selected from the group consisting of a recombinant microorganism containing a first UDP-glycosyltransferase, further expressing one or more enzymes selected from the group consisting of UDP-glycosyltransferase76G1 (SrUGT76G1) and sucrose synthase, a cell of the microorganism, a culture of the microorganism, a disrupted product of the microorganism, and an extract of the disrupted product or culture. The biocatalyst is one or more selected from the group consisting of a transformed microorganism, a cell of the microorganism, a culture of the microorganism, a disrupted product of the microorganism, and an extract of the disrupted product or culture, which expresses all of TaUGT or TaUGTm, SrUGT76G1, and sucrose synthase and has an activity of converting Stevia to Rebaudioside M.

[0074] The disruptant refers to a disruptant obtained by disrupting the cells of a microorganism that produces UDP-glucosyltransferase, or a supernatant obtained by centrifuging the disruptant, and contains the enzyme produced by the microorganism that produces UDP-glucosyltransferase.

[0075] 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 contains or does 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.

[0076] The biocatalyst according to the present invention may be a microbial cell having an enzymatic activity or a dried microbial cell thereof. The dried microbial cell according to the present invention has a low water content, storage stability, and high conversion activity. The dried microbial cell has a reduced water content compared to the same weight of microbial cell, making it easy to store and distribute, and maintains a stable conversion activity for a long period of time, making it particularly suitable for mass production.

[0077] The yeast cell dry powder according to the present invention can stably maintain the transformation activity of the recombinant yeast in a dry state for a long period of time, i.e., it can be stored and maintained without excessively decreasing the enzyme activity when dried, even when stored at room temperature.

[0078] Furthermore, the production of steviol glycosides according to the present invention can be carried out by a whole cell reaction using microbial cells or the dried cells themselves having the conversion activity of steviol glycosides, particularly rebaudioside M. The biocatalyst not only has the conversion activity of steviol glycosides, particularly rebaudioside M, but also can produce steviol glycosides, particularly rebaudioside M, at a high conversion rate in a whole cell conversion reaction.

[0079] When the microorganism having the conversion activity of steviol glycosides, particularly rebaudioside M, according to the present invention is yeast, it shows low conversion activity in the whole cell reaction due to its thick cell wall, and therefore has a high conversion rate in the whole cell reaction using dried cells compared to the cells themselves isolated by microbial culture. Furthermore, the whole cell conversion reaction can be provided in a liquid raw material containing the substrate to be converted, and can be carried out by mixing the liquid raw material with the cells or dried cells. Therefore, the cells can be treated so that the cell walls of the cells have high permeability to the liquid substrate, or a substance that can increase the cell wall permeability of the recombinant yeast cells can be added. One example of a method for treating the cells so that the cell walls of the cells have high permeability to the liquid substrate is drying treatment. The method of the present invention does not have the purpose of increasing and maintaining the viability of such microorganisms, but rather, it is preferable that the microorganisms themselves are killed and only the enzyme activity possessed by the microorganisms is preserved.

[0080] In one embodiment of the present invention, the method of drying the bacterial cells is not particularly limited, and examples that can be used include, but are not limited to, freeze-drying, hot air drying using air or wind, spray drying, etc. As the bacterial cell drying process, a drying process having high permeability to a liquid substrate is more preferable, and may be, for example, freeze-drying. In the case of hot air drying or spray drying, the temperature at the inlet and outlet of the dryer can be set within a range that does not significantly reduce the activity of the enzyme.

[0081] The freeze-drying of the bacteria can be carried out by a conventional freeze-drying method, but since it is important to maintain the conversion activity of the enzyme, it is preferable to freeze the bacteria at a temperature range of -90 to 40°C or -90°C to -5°C, preferably at a temperature range of -60 to 40°C, -50 to 40°C, -60 to 20°C, or -50 to 20°C, and dry the bacteria at a temperature range of -60°C to 40°C under a pressure of less than 10 mtorr. In addition, it is not necessary to add a cryoprotectant or the like in the freeze-drying process of the bacteria. The moisture of the beads frozen in the freeze-drying stage was completely removed to produce dried beads.

[0082] The microbial cell dry powder obtained by the present invention has an advantage that it can maintain the conversion activity of the microorganism immediately after drying or at least 90% of the conversion activity for more than one year even when stored at room temperature (25°C). In other words, based on the conversion activity of the microorganism immediately after drying being 100%, when stored at room temperature for 9 weeks, it can have steviol glycoside conversion activity of 90% or more, 95% or more, 97% or more, or 98% or more based on the steviol glycoside conversion activity of the microorganism at the start of storage being 100%. Thus, the freeze-dried microorganism according to the present invention can maintain high conversion activity for a long period of time. In contrast, the enzyme activity of the cell lysate or the coenzyme solution obtained therefrom is almost inactivated 24 hours after cell lysate, and the residual activity is reduced to 10% or less.

[0083] The recombinant microorganisms described herein include fungal or bacterial cells. In one embodiment, the bacterial cells include Escherichia, Lactobacillus, Lactococcus, Cornebacterium, Acetobacter, Acinetobacter, Aspergillus, or Pseudomonas microorganisms. The fungal cells are yeast cells, including, for example, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Ashbya, Cyberlindnera, Pichia, Kluyveromyces, and Hansenula microorganisms. Specifically, the microorganism includes, but is not limited to, Escherichia coli (E. coli), Saccharomyces sp. strains (eg, Saccharomyces cerevisiae), or Pichia sp. strains (eg, Pichia pastoris).

[0084] The transformed microorganism provided by the present invention may be transformed with an exogenous TaUGTm, SrUGT76G1 and / or sucrose synthase gene for expression of TaUGTm, SrUGT76G1 and / or sucrose synthase. The TaUGT or TaUGTm, SrUGT76G1 and / or sucrose synthase gene is cloned into a vector and transformed into a microorganism, but is not limited thereto.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The method of transforming the vector of the present invention includes a method for introducing a nucleic acid into a cell, and can be carried out by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO 4 ) precipitate, calcium chloride (CaCl 2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but are not limited to these.

[0090] In the present invention, the composition for producing steviol glycoside or the method for producing steviol glycoside performs a conversion reaction at a high substrate concentration, has a high conversion rate to rebaudioside M, and is suitable for industrial mass production.

[0091] In the present specification, the reaction substrate (reaction raw material) used in the conversion reaction for producing steviol glycoside contains one or more steviol glycoside substrates selected from the group consisting of stevioside and rebaudioside A, and may be, for example, a single substrate of stevioside or Reb A, or a mixed substrate thereof. The mixed substrate may be a mixture of stevioside and / or Reb A, or a stevia extract containing stevioside or Reb A.

[0092] In the present specification, "stevia" refers to a stevia extract, and specifically, rebaudioside A is present in an amount of 30(w / w)% or more, 35(w / w)% or more, 40(w / w)% or more, 50(w / w)% or more, 55(w / w)% or more, 58(w / w)% or more, 60(w / w)% or more, 70(w / w)% or more, or 80(w / w)% or more based on the total mass of steviol glycosides contained in the stevia extract, and specifically, 50 to 99 .5(w / w)%, 50~99(w / w)%, 50~95(w / w)%, 50~90(w / w)%, 50~85(w / w)%, 50~80(w / w)%, 50~70(w / w)%, 50~65(w / w)%, 50~62(w / w)%, 55~99.5(w / w%, 55~99(w / w)%, 55~95(w / w)%, 55~90(w / w)%, 55~85(w / w)%, 55~80(w / w)%, 55~70(w / w) ), 55-65(w / w)%, 55-62(w / w)%, 58-99.5(w / w)%, 58-99(w / w)%, 58-95(w / w)%, 58-90(w / w)%, 58-85(w / w)%, 58-80(w / w)%, 58-70(w / w)%, 58-65(w / w)%, 58-62(w / w)%, for example, 60(w / w)%, and the content of stevioside based on the total volume of the stevia extract may be The content of steviol glycoside may be other than the content of baudioside A, specifically, the content of steviol glycoside may be 30-50(w / w)%, 30-45(w / w)%, 30-42(w / w)%, 35-50(w / w)%, 35-45(w / w)%, 35-42(w / w)%, 38-50(w / w)%, 38-45(w / w)%, or 38-42(w / w)%, for example, 40(w / w)%, but is not limited thereto.

[0093] The present invention is capable of producing Reb M with high purity at a high conversion rate and productivity in an industrially applicable mass production process using a large amount of reaction substrate. In the present specification, the reaction substrate used in the conversion reaction for producing steviol glycoside can be provided in a liquid form, and has a concentration of 15 g / L or more, 20 g / L or more, 25 g / L or more, 50 g / L or more, 75 g / L or more, or 100 g / L or more, for example, 15 to 200 g / L, 20 to 200 g / L, 30 to 200 g / L, 40 to 200 g / L, 50 to 60 g / L, 70 to 80 g / L, 80 to 90 g / L, 90 to 100 g / L, 100 to 120 g / L, 120 to 140 g / L, 140 to 160 g / L, 160 to 180 g / L, 180 to 200 g / L, 180 to 220 g / L, 180 to 240 g / L, 180 to 260 g / L, 180 to 28 ... It can be provided in amounts of 200g / L, 60-200g / L, 70-200g / L, 80-200g / L, 90-200g / L, 15-150g / L, 20-150g / L, 30-150g / L, 40-150g / L, 50-150g / L, 60-150g / L, 70-150g / L, 80-150g / L, or 90-150g / L.

[0094] The composition may further include at least one reactant selected from the group consisting of stevia extract, uridine diphosphateglucose (UDP) and sucrose. In one embodiment, the conversion of reb A to Reb D in the composition for producing steviol glycosides is achieved by using MgCl 2 and / or MnCl 2 may additionally include:

[0095] The stevia extract of the present invention contains steviol glycosides at 80-99(w / w)%, 80-97(w / w)%, 80-96(w / w)%, 80-95(w / w)%, 85-99(w / w)%, 85-97(w / w)%, 85-96(w / w)%, 85-95(w / w)%, 90-99(w / w)%, 90-97(w / w)%, 90-96(w / w)%, 90-95(w / w)%, 93 The steviol glycoside may be, but is not limited to, 99(w / w)%, 93-97(w / w)%, 93-96(w / w)%, 93-95(w / w)%, 94-99(w / w)%, 94-97(w / w)%, 94-96(w / w)%, 94-95(w / w)%, 95-99(w / w)%, 95-97(w / w)%, or 95-96(w / v)%, for example, 95(w / v)%. The steviol glycoside may include rebaudioside A and stevioside.

[0096] The reactant materials for the composition for producing rebaudioside M provided by the present invention and / or the reactant materials used in the method for producing rebaudioside M provided by the present invention are reactant materials solutions, which contain sucrose at concentrations of 500-2000 mM, 500-1500 mM, 500-1200 mM, 500-1100 mM, 500-1050 mM, 500-1000 mM, 600-2000 mM, 600-1500 mM, 600-1200 mM, 600-1100 mM, 600-1050 mM, and 600-1000 mM based on the reactant materials, but are not limited thereto.

[0097] The composition may further include at least one reactant selected from the group consisting of stevia extract, uridine diphosphateglucose (UDP) and sucrose. In one embodiment, the conversion of reb A to Reb D in the composition for producing steviol glycosides is achieved by adding MgCl 2 and / or MnCl 2 may additionally include:

[0098] In the present invention, in the steviol glycoside containing rebaudioside M which is the reaction product, the purity of rebaudioside M may be 50% by weight or more, 55% by weight or more, 60% by weight or more, 65% by weight or more, 70% by weight or more, 75% by weight or more, 80% by weight or more, or 85% by weight or more, and specifically, may be 50% to 99% by weight, 55% to 99% by weight, 60% to 99% by weight, 65% to 99% by weight, 70% to 99% by weight, 75% to 99% by weight, 80% to 99% by weight, 85% to 99% by weight, or 87 to 99% by weight.

[0099] The conversion rate to rebaudioside M produced and / or prepared by the composition for producing rebaudioside M provided by the present invention and / or the method for producing rebaudioside M provided by the present invention relative to the weight of stevia extract contained in the reaction raw materials is 110% to 200%, 115% to 200%, 120% to 200%, 125% to 200%, 130% to 200%, or 135% to 200%, for example, 125%, 129%, 135%, or 136%, but is not limited thereto.

[0100] The content of rebaudioside M in a steviol glycoside produced by the composition for producing rebaudioside M provided by the present invention and / or the method for producing rebaudioside M provided by the present invention is 80 to 99% by weight, 85 to 99% by weight, 87 to 99% by weight, 90 to 99% by weight, 94 to 99% by weight, or 95 to 99% by weight, and may be, for example, 87.7% by weight, 90.2% by weight, 94.8% by weight, or 95.4% by weight.

[0101] The conversion reaction according to the present invention can be carried out in an aqueous system at a temperature of 20 to 60° C. and a pH of 4 to 9. 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, 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 solution at pH 7.2.

[0102] The pH of the stevia extract contained in the reaction raw material of the composition for producing rebaudioside M provided by the present invention or the pH of the conversion reaction is pH 4-9, pH 4-8.5, pH 4-8.2, pH 4-8, pH 4-7.8, pH 4-7.5, pH 4-7, pH 4-6.7, pH 4-6.5, pH 4-6.3, pH 4-6.2, pH 5-7, pH 5-6.7, pH 5-6.5, pH 5-6.3, pH 5-6.2, pH 5.5-7, pH 5.5- 6.7, pH5.5-6.5, pH5.5-6.3, pH5.5-6.2, pH5.7-7, pH5.7-67, pH5.7-6.5, pH5.7-6.3, pH5.7-6.2, pH5.9-7, pH5.9-6.7, pH5.9-6.5, pH5.9-6.3, pH5.9-6.2, pH6.0-7, pH6.0-6.7, pH6.0-6.5, pH6.0-6.3, pH6.0-6.2, but not limited thereto.

[0103] In the present specification, the composition for producing steviol glycoside or the method for producing steviol glycoside according to the present invention may be carried out at a reaction temperature of 20 to 60°C, 20 to 50°C, 20 to 45°C, 20 to 40°C, 20 to 37°C, or 20 to 35°C. EXAMPLES

[0104] The present invention will be described in more detail with reference to the following examples, but the scope of the invention is not limited to the following examples.

[0105] Example 1 Creation 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 prepared to overlap the GAL10 promoter (sequence number 16) and cyc1 terminator B (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 primer information used is shown in Table 2 below. The subcloned genes in the form of plasmids were transformed into E. coli (DH5a) and selected and amplified, and the sequence of the amplified plasmid was analyzed to confirm that the expression gene of the enzyme was correctly subcloned.

[0106] [Table 2]

[0107] 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. 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.

[0108] 1-2: Microbial culture 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 using the OD 600 Measure the absorbance to confirm, and measure the OD in a 250 mL flask containing 25 mL of SC-Ura liquid medium. 600 The mixture was inoculated so that the final absorbance became 0.1, and primary culture was carried out for 24 hours (30° C., 240 rpm). In order to induce enzyme expression in the primary cultured cells, the same amount of YPG medium was added and secondary cultured for 24 hours. The secondary 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), 0.7 g / L Drop-out mix, 50 g / L Glucose, and 50 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.

[0109] 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, a forward primer of EUGT11 linked to the GAL10 promoter (SEQ ID NO: 20), a reverse primer of EUGT11 linked to the CYC1 terminator (SEQ ID NO: 21), a forward primer of HvUGT linked to the GAL10 promoter (SEQ ID NO: 22), and a reverse primer of HvUGT linked to the CYC1 terminator (SEQ ID NO: 23) were used. The primer information used is shown in Table 2, and Sc stands for 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 cultivation were carried out.

[0110] 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 collected and washed twice with the same amount of water. The washed cells were resuspended in 100 mM phosphate buffer (pH 7.2) and the 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 mixture for enzyme activity evaluation contained the substrate Reb A (95%, Sinochem), MgCl 2 and UDP-Glucose, to which 0.1 mL of the coenzyme solution was added to prepare a total reaction volume of 0.5 mL. The final reaction solution contained 2 g / L of the substrate Reb A (95%, Sinochem), 100 mg / L of MgCl 2 The reaction mixture was subjected to an enzymatic reaction 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 depending on 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 3 below.

[0111] [Table 3]

[0112] The HPLC analysis graph of the conversion product is shown in Table 4. The Reb D conversion rate of the enzyme converting the single substrate Reb A 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 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)

[0113] [Table 4]

[0114] As shown in Table 4, 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 amount of Reb D produced in the 18-hour enzyme reaction solution was analyzed, and the order of the conversion rates of the three enzymes showed a similar tendency to that of the 3-hour reaction. The nucleic acid sequence of the TaUGT enzyme was analyzed by the base sequence analysis service of Macrogen Co., Ltd., and the amino acid sequence was analyzed based on the base sequence. The resulting amino acid sequence is shown in SEQ ID NO:1, and the nucleic acid sequence is shown in SEQ ID NO:2.

[0115] 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 explored based on the existing EUGT11 gene. The conserved region was confirmed through alignment, and the difference in amino acids between the N-term sugar acceptor and C-term sugar donor domains of UGT was compared. In the case of threonine at residue 201 and valine at residue 202 (serine and leucine amino acids in EUGT11, respectively), 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 (SEQ ID NOs: 24 and 25), perform mutation gene amplification (PCR) to change the base sequence of the corresponding site, and clone the amplified gene fragment into pRS426 vector through Gibson assembly. The resulting enzyme mutant cloned into the pRS426 vector was named TaUGTT201S / V202L. The sequences of the primers used in this example are shown in Table 5 below.

[0116] [Table 5]

[0117] 2-2: Preparation of enzyme mutant expression strains In order to confirm the expression of the enzyme, TaUGT was cloned into the pRS426 vector in a manner substantially similar to that described in Example 1-2. T201S / V202L The plasmids prepared in Example 2-1 were transformed into yeast strains and selected using the LiAc method. The transformed strains were cultured in substantially the same manner as in Example 1-2. Yeast transformed with the plasmids prepared in Example 2-1 was selected in SC (synthetic complete) medium using URA-drop out mix, and the selected yeasts were cultured in liquid medium to induce enzyme expression.

[0118] Example 3: Evaluation of conversion activity 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 mixture for enzyme activity evaluation contained the substrate Reb A (95%, Sinochem), MgCl 2 and UDP-Glucose, to which 0.1 mL of the coenzyme solution was added to prepare a total reaction volume of 0.5 mL. The final reaction solution contained 2 g / L of the substrate Reb A (95%, Sinochem), 100 mg / L of MgCl 2 The reaction mixture was subjected to an enzymatic reaction 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 depending on 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 in a manner substantially similar to that of Examples 1-4 to confirm the conversion ratio of the product. The coenzyme solution obtained from the cell lysate of the recombinant strain exhibits the activity of converting the Reb A substrate to Reb D, so the activity was measured by determining the production ratio of Reb D, and the HPLC analysis graph is shown in FIG. 2. The Reb D conversion rate of the enzyme converting the single substrate Reb A to Red D shown in Table 6 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 (100%). Reb D conversion rate (%) = HPLC peak area of ​​Reb D / total HPLC peak area (Formula 1) The enzyme reaction was carried out for 18 hours using a reaction solution containing the TaUGT mutant enzyme and the Reb A substrate, 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 6 below and Figure 2. Figure 2 is an HPLC graph analyzing the reaction product of the Reb D conversion reaction using the TaUGT mutant enzyme.

[0119] [Table 6]

[0120] Example 4: Conversion reaction using mixed substrates As in Example 3, when enzyme activity was evaluated using Reb A, only Reb A was converted to Reb D, and the enzyme activity was clearly confirmed. However, since a mixed substrate is used in actual industrial production, a conversion reaction was carried out on a mixed substrate containing stevioside and Reb A in order to evaluate the possibility of using the enzyme in industrial applications using a mixed substrate. That is, when a mixed substrate is used, a reaction in which stevioside is converted to Reb E and Reb A is converted to Reb D is carried out. In order to evaluate the enzyme activity against the mixed substrate, the recombinant strain obtained in Example 2-2 was 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 cell solution was centrifuged (13,000 rpm, 15 min) in a refrigerated state to obtain a supernatant, which was used as a coenzyme solution. The reaction mixture for enzyme activity evaluation contained the substrate RA40 (Reb A (rebaudioside A:STE (stevioside) = 40:60, Sinochem), MgCl 2 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. The final reaction solution contained 2 g / L of substrate RA40 (Reb A:STE=40:60, Sinochem), MgCl 2 The concentration of UDP-Glucose was 4 mM. The reaction solution was subjected to an enzymatic reaction at 30° C., pH 7.2, and 150 rpm, and the reaction was monitored for 18 hours after the reaction started. 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 7 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)

[0121] [Table 7]

[0122] As a result of the experiment, in the case of the 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 the 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.

[0123] Example 5: Preparation of TaUGT_SUS fusion enzyme and confirmation of activity 5-1: Construction of recombinant yeast producing TaUGT_SUS fusion enzyme A fusion protein was prepared with sucrose synthase (SUS1, derived from Arabidopsis thaliana) enzyme 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. The nucleic acid sequence encoding the TaUGT_T201SV202L (TaUGTm) mutant enzyme was the nucleic acid sequence of SEQ ID NO: 30 in Table 1, and the nucleic acid sequence of SEQ ID NO: 5 in Table 1 was used for sucrose synthase (SUS1, derived from Arabidopsis thaliana). 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.

[0124] [Table 8]

[0125] In the same manner as in Example 1-2, the constructed fusion enzyme expression vector was transformed into yeast to obtain a recombinant yeast expressing the fusion enzyme.

[0126] 5-2: Recombinant yeast culture and enzyme preparation The transformed strain was cultured in substantially the same manner as in Example 1-2. Yeast transformed with the plasmid prepared in Example 2-1 was selected in SC (synthetic complete) medium using URA-drop out mix, and the selected yeast was induced to express enzymes through liquid culture. The recombinant strain culture was centrifuged to recover the bacterial cells, which were then disrupted with a glass beader, in a manner substantially similar to that of Example 3. The disrupted bacterial cells were centrifuged (13,000 rpm, 15 min) in a refrigerated state to obtain the supernatant as a coenzyme solution.

[0127] 5-3: Evaluation of conversion activity of fusion enzyme The reaction solution for the conversion reaction consisted of RA40 reaction substrate (90% purity by weight), MgCl 2, UDP, and sucrose were mixed, and 0.1 mL of each of the coenzyme solutions of the TaUGTm_SUS fusion enzyme prepared in Example 5-2 was added thereto to prepare a total reaction volume of 0.5 mL. In the final reaction solution, the RA40 reaction substrate was 10 g / L, MgCl 2 The concentrations of 1 mM, UDP 1 mM, and sucrose 125 mM were adjusted. The 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 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. 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.

[0128] Example 6 Production of microorganisms expressing UGT and SUS enzymes 6-1: Preparation of vector incorporating enzyme gene In order to prepare a recombinant yeast that produces all three of TaUGTm, SrUGT76G1, and sucrose synthase for the rebaudioside M conversion reaction, a vector into which the above genes were introduced was first prepared. Specifically, pRS423_UGT76G1_GmSUS was prepared as a vector for the fusion enzyme of UGT76G1 and GmSUS, and pRS426_TaUGTm_GmSUS was prepared as a vector for the fusion enzyme of TaUGTm and GmSUS, using substantially the same preparation method, primers and linkers as in Example 5-1. The TaUGTm was the TaUGT_T201SV202L (TaUGTm) mutant enzyme as in Example 5, except that the sucrose synthase was an enzyme derived from Glycine max (soybean), and the nucleic acid sequence of SEQ ID NO: 34 in Table 1 was used. In addition, the enzyme SrUGT76G1 used was a UDP-glycosyltransferase derived from Stevia rebaudiana (stevia plant), and the nucleic acid sequence encoding the fusion enzyme was introduced into the vector using the nucleic acid sequence shown in SEQ ID NO: 32 in Table 1.

[0129] 6-2: Preparation of host cells As a host cell for introducing the prepared vector, Saccharomyces cerevisiae CENPK2-1c(Euroscarf)△gal80△SUC2 was prepared by removing the SUC2 gene encoding the invertase enzyme in order to prevent sucrose decomposition by the invertase present in the yeast cell. Specifically, the SUC2 gene was removed using yeast homologous recombination, and 500-bp DNA sequences upstream and downstream of the SUC2 gene were amplified by PCR using Q5 DNA polymerase (NEB) under the following conditions: 98°C (1m)-[98°C (10s)-55°C (30s)-72°C (5m)] 30 cycles-72°C (5m)-16°C (storage). The information on the upstream and downstream sequences used is shown in Table 9 below.

[0130] [Table 9]

[0131] After amplification by PCR, the amplified DNA fragments were ligated using the PCR overlap extension method. The ligated DNA fragments were cloned into pRS26 vector and transformed with yeast using the LiAc method. The obtained transformants were subjected to colony PCR to select strains in which the SUC2 gene had been deleted.

[0132] 6-3: Production of transformed yeast In substantially the same manner as in Example 5, the pRS423_UGT76G1_GmSUS and pRS426_TaUGTm_GmSUS vectors prepared in 6-1 were transformed into Saccharomyces cerevisiae CENPK2-1c(Euroscarf)Δgal80ΔSUC2 prepared in 6-2 by the LiAc method. The transformed strain was cultured in substantially the same manner as in Example 1-2. Yeast transformed with the plasmid prepared in Example 2-1 was selected in SC (synthetic complete) medium using URA-drop out mix. The selected transformed yeast was fermented and cultured in 5 L to produce three kinds of enzymes in the cells. The volume of the production medium used in the culture was 2 L, and the composition of the production medium used is as shown in Table 10 below. A feeding solution having the composition in Table 11 below was additionally added during the fermentation culture, and the vitamin 1000x and trace metal 1000x information in the production medium is shown in Tables 12 and 13 below, respectively.

[0133] [Table 10]

[0134] [Table 11]

[0135] [Table 12]

[0136] [Table 13]

[0137] The yeast cells transformed with the genes for the three enzymes had a final OD 600 The amount of bacteria was adjusted so that the concentration was about 120 / mL. Then, the culture medium and the cells were separated by centrifugation (4,000 rpm, 4°C, 10 minutes).

[0138] 6-4: Conversion reaction using coenzyme solution The isolated cells were resuspended in 500 mL of 50 mM sodium phosphate buffer (pH 7.2) and disrupted using a high-pressure disperser (GEA Lab Homogenizer Panda PLUS 2000). The disrupted cells were used immediately as an enzyme source without a separate centrifugation process. The stevia extract, RA60_SG95 (Sinochem, China), was used as a control, with 50 g / L of RA60_SG95, 750 mM sucrose, 0.5 mM Muridine diphosphate glucose (UDP), and 3 mM MgCl 2 The above was dissolved in 500 mL of 50 mM sodium phosphate buffer (pH 7.2) to prepare a reaction solution for the conversion reaction. The RA60_SG95 refers to a stevia extract containing 95 (w / v)% steviol glycoside as an active ingredient, 60 (w / w)% Reb A based on 100% steviol glycoside by weight, and 40 (w / w)% stevioside. The enzyme-containing cell lysate and the prepared raw material (reaction solution) for the conversion reaction were mixed and stirred at 30° C. for 24 hours to convert rebaudioside M. To stop the conversion reaction, the reaction solution was boiled for 5 minutes and centrifuged (13,000 rpm, 10 minutes). The supernatant obtained by centrifugation was analyzed to confirm the product. The product was analyzed qualitatively and quantitatively using HPLC, and the content of steviol glycosides produced was confirmed. The column used in the HPLC 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 water containing 0.01% TFA (Trifluoroacetic acid, Sigma) and 100% acetonitrile (ACN), respectively, and the analysis was performed using a gradient. The mobile phase was run at 1 mL / min, and the analysis was performed for a total of 40 minutes. The HPLC analysis conditions are shown in Table 14 below.

[0139] [Table 14]

[0140] The analytical results showed that the retention times obtained by HPLC analysis were 15.3 minutes for STE and 15.1 minutes for Reb A in the substrate, and 10.6 minutes for Reb E, 14.7 minutes for Reb I, 10.9 minutes for Reb D, and 11.6 minutes for Reb M in the product.

[0141] Example 7 Confirmation of pH conditions for conversion reaction Using the cell lysate (coenzyme solution) of the transformed yeast cells producing the three enzymes obtained in Example 6-4, the reaction pH of the conversion reaction from stevia extract to rebaudioside M was optimized. Specifically, the raw material (reaction raw material solution) for the conversion reaction was prepared in the same manner as in Example 6, except that the pH of the reaction solution in Example 6 was adjusted to 5.8, 6.0, 6.2, or 6.5 using 5N NaOH. Using the pH-adjusted reaction solution, rebaudioside M conversion reaction was carried out in substantially the same manner as in Example 6, and the supernatant was obtained by centrifugation, and the steviol glycoside content of the product was analyzed by HPLC analysis. The analysis results are shown in Table 15 below. The values ​​in Table 15 below represent the composition of each steviol glycoside in weight % based on 100% by weight of the produced steviol glycoside.

[0142] [Table 15]

[0143] Analysis of the steviol glycoside content of the product revealed that the glycosyltransferase catalyzed glycosylation of steviol occurred at carbon atoms 13 and 19. In actual reactions, the glycosyltransferase rate was faster at C19 when the pH was 6.0 or higher, and faster at C13 when the pH was 6.0 or lower. It was confirmed that the optimal pH conditions in the enzymatic reactions using the three enzymes used, TaUGTm, SrUGT76G1, and sucrose synthase, acted complementary to each other, and the amount of rebaudioside M produced from stevia extract was greatest at pH 6.0-6.2.

[0144] Example 8 Check the temperature conditions of the conversion reaction The reaction temperature for conversion of stevia extract to rebaudioside M was optimized using the cell lysate (coenzyme solution) of the transformed yeast cells producing the three enzymes obtained in Example 6-4. Specifically, the raw material for the conversion reaction (reaction raw material solution) was prepared in the same manner as in Example 6, but the pH of the substrate raw material for the conversion reaction was adjusted to 6.0 using 5N NaOH, and the cell lysate of Example 6 was mixed and stirred at 30° C., 35° C., 40° C. or 45° C. The rebaudioside M conversion reaction was carried out under substantially the same conditions as in Example 6, and the supernatant was obtained by centrifugation, and the steviol glycoside content of the product was analyzed by HPLC analysis. The analysis results of the steviol glycoside content according to the reaction time are shown in Table 16 below. The content of Reb M was calculated as a conversion rate according to the following Equation 3 using the area of ​​the third peak corresponding to the peak of Reb M (g / L) in HPLC. Reb M content=([HPLC Area]-6324) / 938442 (Formula 3) The values ​​in Table 16 below indicate the Reb M content (g / L).

[0145] [Table 16]

[0146] Analysis of the conversion of rebaudioside M as a function of reaction temperature showed that the initial conversion reaction rate increased as the reaction temperature increased, but this resulted in rapid enzyme inactivation, and ultimately the highest conversion rate of rebaudioside M was confirmed at 35℃.

[0147] Example 9 Confirmation of sucrose concentration in conversion reaction The inhibition level of the rebaudioside M conversion reaction depending on the concentration of sucrose used as a substrate for supplying UDP-glucose in the transglycosylation process of the rebaudioside M conversion reaction was confirmed using the cell lysate (coenzyme solution) of the transformed yeast cells producing the three enzymes obtained in Example 6-4. The theoretical amount of UDP-glucose required for the conversion of 50 g / L of stevia extract to rebaudioside M is expected to be about 320 mM. However, in the actual conversion reaction, about 200 mM of sucrose is decomposed by other enzymes in the cell lysate. Therefore, the concentration of sucrose used in the conversion reaction was set to at least 500 mM, and optimization of the sucrose concentration was started. Specifically, the raw materials for the conversion reaction were prepared in the same manner as in Example 6, except that instead of the 750 mM sucrose used in the conversion reaction of Example 6, reaction raw materials were prepared with sucrose concentrations of 600, 800, 1000, or 1200 mM, and the pH of the substrate raw material was adjusted to 6.0 using 5N NaOH. The prepared reaction raw materials were mixed with the cell lysate (coenzyme solution) of the recombinant yeast producing the three enzymes obtained in Example 6-4 and stirred at 35°C. Rebaudioside M conversion reaction was performed under substantially the same conditions as in Example 6, and the supernatant was obtained by centrifugation, and the steviol glycoside content of the product was analyzed by HPLC analysis. The analysis results of the steviol glycoside content according to the amount of sucrose used are shown in Table 17 below. The values ​​in Table 17 below indicate the conversion rate (%) of Reb M. Reb M conversion rate (%) = HPLC peak area of ​​Reb M / total HPLC peak area (Formula 4)

[0148] [Table 17]

[0149] From the experimental results, it was confirmed that rebaudioside M had the highest conversion rate when the sucrose concentration in the raw material for the conversion reaction was 1000 mM, and at concentrations higher than this, inhibition of the sucrose synthase enzyme occurred, resulting in a decrease in the overall conversion rate.

[0150] Example 10: Conversion of high-concentration stevia extract to rebaudioside M Based on the reaction conditions confirmed in Examples 6 to 9, the conversion rates of high-concentration stevia extract to rebaudioside M were compared. The overall conversion reaction scheme for producing rebaudioside M using the three enzymes of Example 6 is shown in Reaction Scheme 1 below. SUS: Sucrose+UDP+[H + ]→Fructose+UDP-glucose UGT: Steviol glycoside+UDP-glucose→Steviol glycoside-[glucose]+UDP+[H + ] (Reaction Scheme 1) In order to conduct experiments under the optimized reaction conditions confirmed in Examples 6 to 9, RA60_SG95 (stevia extract) substrate concentration was prepared at four concentrations of 25, 50, 75, and 100 g / L instead of 50 g / L used in the preparation of the conversion reaction raw material used in Example 6, and sucrose was adjusted to a concentration of 1000 mM and the pH was adjusted to 6.0 to prepare the raw material. The prepared reaction materials were mixed with the cell lysate (coenzyme solution) of the transformed yeast cells producing the three enzymes obtained in Example 6-4, and stirred at 35° C. Rebaudioside M conversion reaction was carried out under substantially the same conditions as in Example 6, and the supernatant was obtained by centrifugation. The steviol glycoside content of the product and the content ratio of the components in the steviol glycoside of the product were analyzed by HPLC. The amount of rebaudioside M produced (g / L) in the conversion reaction is shown in Table 18 below. The results of the analysis of the content ratio of the steviol glycoside components in the product are shown in Table 19 and FIG. 1. The values ​​shown in Table 19 below indicate the content ratio (wt%) of each steviol glycoside based on the total of STE, Reb A, Reb E, Reb I, Reb D, and Reb M being 100 wt%.

[0151] [Table 18]

[0152] [Table 19]

[0153] As a result of carrying out a conversion reaction using a high-concentration steviol extract under the reaction temperature, reaction pH, and sucrose concentration conditions confirmed in Examples 6 to 9, the absolute amount of rebaudioside M produced increased as the concentration of the substrate, steviol extract, increased. As shown in Table 19, the production rate of Rebaudioside M was the highest among the six steviol glycosides at all four substrate concentrations of 25, 50, 75, and 100g / L. Although the content of rebaudioside M in the total steviol glycosides decreased slightly as the substrate concentration increased, a high conversion rate of 87.7% was confirmed at a concentration of 100g / L, which is the minimum concentration for industrial mass production, confirming the high conversion rate in high-concentration stevia extracts. In addition, it was confirmed that the absolute production amount of rebaudioside M produced through the conversion process increases as the substrate concentration increases. Even if the content ratio of rebaudioside M in the product steviol glycoside is somewhat low, the absolute production amount increases, demonstrating that the recombinant microorganism according to the present invention has excellent properties under conditions for industrial mass production. It was confirmed that rebaudioside I was contained in the reaction products at four substrate concentrations of 25, 50, 75, and 100 g / L, and that the inclusion of rebaudioside I as a by-product was unavoidable. From the above results, it was confirmed that when stevia extract is converted into steviol glycoside, in terms of rebaudioside M conversion rate, it is possible to overcome the low conversion rate and low production amount seen in the conventional rebaudioside M conversion reaction using a high concentration stevia extract, thereby enabling mass production.

[0154] Example 11 Freeze-drying of microorganisms expressing UGT and SUS enzymes 11-1: Preparation of transformed yeast cells producing UGT and SUS enzymes The transformed microorganisms expressing UGT and SUS enzymes were prepared and secured by the methods described in Examples 6-1, 6-2 and 6-3.

[0155] 11-2: Production of freeze-dried microorganisms In order to increase the substrate permeability of the microbial cells and to increase storage stability at room temperature, the microbial cells prepared in Example 11-1 were freeze-dried by rapidly freezing at -40°C while maintaining a vacuum, and then freeze-drying for 32 hours using a freeze-drying device.

[0156] Example 12: Evaluation of transformation activity of freeze-dried cells In a whole cell reaction using the freeze-dried cells produced in Example 11-2, a conversion reaction of Reb M was carried out with a reaction substrate to evaluate the conversion activity of the freeze-dried cells. As a control group, the transformed microbial cells harvested after culture in Example 11-1 without freeze-drying were prepared. To evaluate the enzyme activity of the bacteria, 1 g / L of substrate RA60 (95%, Sinochem), 1 mM UDP, and 250 mM sucrose were added to double distilled water (DDW) to prepare 1 mL of reaction solution. The reaction solution was added with the pre-freeze-dried cells prepared in Example 11-2 to prepare freeze-dried cells (experimental group) or the non-freeze-dried cells prepared in Example 11-1 (control group) to prepare a concentration of 9.2 g / L. The prepared reaction solution was subjected to a conversion reaction at a temperature of 30°C, pH 7.0 and 150 rpm. One hour after the start of the reaction, the whole cell reaction solution was boiled at 100°C for 5 minutes to terminate the conversion reaction. The whole cell reaction product was analyzed using the supernatant obtained by centrifugation (13,000 rpm, 10 minutes). The whole cell reaction product was analyzed by HPLC-Chromatography to confirm the conversion ratio of steviol glycosides contained in the product. Specifically, the HPLC analysis method of the conversion product was substantially the same as that in Example 6-4. The recombinant microorganism converted the Reb A substrate to Reb D, and converted Reb D to Reb M, and the production ratio of Reb D and Reb M was confirmed. The conversion rates of RA60 substrate to Red D and Reb M are shown in the table below. The conversion rates of Reb D and Reb M were calculated according to the following Equations 1 and 4, respectively. Reb D conversion rate (%) = HPLC peak area of ​​Reb D / total HPLC peak area (Formula 1) Reb M conversion rate (%) = HPLC peak area of ​​Reb M / total HPLC peak area (Formula 4)

[0157] [Table 20]

[0158] As a result of calculating the Reb D conversion rate and the Reb M conversion rate, it was confirmed that Reb D and Reb M were not produced in the conversion reaction using non-lyophilized cells, but Reb D and Reb M were produced in the conversion reaction using freeze-dried cells. The Reb M conversion rate was 14.7%, and the Reb D conversion rate was 47.5%. It was confirmed that freeze-dried whole cells can produce Reb D and M. When a cell lysate (coenzyme solution) containing three enzymes was used in Example 10, the content ratio of Reb M component in the product steviol glycoside was 80% or more, but when a precellular cell reaction was used in this example, the cell wall permeability of the substrate was low, and the Reb M production ratio tended to be relatively low.

[0159] Example 13: Stability of freeze-dried cells In order to evaluate the stability of the freeze-dried microorganism obtained in Example 11-2, it was confirmed whether the enzyme activity was maintained after storage at room temperature (25° C.) for 9 weeks. To confirm the maintenance of the enzyme activity, the enzyme activity was measured once a week in substantially the same manner as in Example 12. The enzyme activity of the freeze-dried microbial cells during the storage period was confirmed to be maintained for the entire test period of 9 weeks, demonstrating that the cells can be stored at room temperature. Specifically, when freeze-dried cells were stored for 9 weeks, the enzyme activity of the cells was confirmed to be maintained at 97% or more based on 100% activity at the start of storage. In contrast, the enzyme activity of the cell lysate used in the conversion reaction in Example 10 or the coenzyme solution obtained therefrom was almost inactivated 24 hours after cell lysation, with the residual activity decreasing to 10% or less.

[0160] Example 14 Optimization of pre-cell transformation reaction using freeze-dried cells 14-1: pH optimization of whole-cell reactions In order to confirm the optimal pH of the whole cell reaction using freeze-dried cells, a pre-cell conversion reaction was carried out in substantially the same manner as in Example 12 using the freeze-dried microbial cells obtained in Example 11-2, except that 100 mM potassium phosphate buffer was added to the reaction mixture to adjust the pH to 6.0-7.4. The whole cell reaction solution was then boiled at 100°C for 5 minutes to terminate the whole cell reaction, and the supernatant obtained after centrifugation (13,000 rpm, 10 minutes) was analyzed by HPLC under the HPLC conditions of Example 12 to confirm the whole cell reaction product. The relative conversion rate was determined as 100 for the highest amount of Reb M produced within the entire pH range, and the rest were expressed as a relative percentage (%). The results are shown in the table below.

[0161] [Table 21]

[0162] The conversion rate of the whole cell reaction according to the pH conditions of the conversion reaction was confirmed. As the pH increased, the relative conversion rate increased, starting from pH 6.0, and the conversion rate was highest at pH 7.2. It was confirmed that the optimal pH for the freeze-dried whole cell reaction is 7.2.

[0163] 14-2: Optimization of reaction temperature for whole-cell reactions In order to confirm the optimal temperature for the whole-cell reaction using freeze-dried cells, the freeze-dried microbial cells obtained in Example 11-2 were used to carry out a whole-cell conversion reaction in substantially the same manner as in Example 12, except that the whole-cell reaction was carried out at temperatures of 30, 33, 36, 39, 42, 45, 48, 52, or 55°C, with the addition of 100 mM potassium phosphate buffer, at pH 7.2. The whole cell reaction solution was then boiled at 100°C for 5 minutes to terminate the whole cell reaction, and centrifuged (13,000 rpm, 10 minutes) to obtain a supernatant, which was analyzed by HPLC under the HPLC conditions of Example 12-2 to confirm the whole cell reaction product, with the highest amount of Reb M produced within the entire temperature range being set as 100, and the rest being expressed as a relative percentage (%). The results are shown in the table below.

[0164] [Table 22]

[0165] As a result of checking the conversion rate of the whole cell reaction according to the temperature conditions of the conversion reaction, it was found that the conversion rate increased as the temperature increased from 30°C to approximately 35°C, and then the conversion reaction was stable up to approximately 52°C, with the highest conversion rate being confirmed at 45°C. It was therefore confirmed that the optimal temperature for the freeze-dried whole cell reaction is 45°C.

[0166] Example 15 Mass production of highly purified Reb M using freeze-dried cells In order to test whether high-concentration Reb M can be produced using the lyophilized cells obtained in Example 11-2, a conversion reaction was carried out using whole cells. Specifically, to carry out a mass production process for producing the high-concentration Reb M, 2 L of a whole cell reaction solution was prepared containing 75 g / L of substrate RA60 (95%, Sinochem), 0.3 mM UDP, 750 mM sucrose, 45 g / L lyophilized cells, and 5 mM EDTA in double distilled water (DDW). The prepared reaction solution was adjusted to pH 7.2 by adding 5 mM potassium phosphate buffer, and the whole cell reaction was carried out at 45° C. and 250 rpm. The whole cell reaction solution was boiled at 100° C. for 5 minutes to terminate the whole cell reaction, and centrifuged (4000 rpm, 20 min) to obtain a supernatant. The supernatant was used for HPLC analysis under the HPLC conditions of Example 12 to confirm the amount of Reb M produced. As a result of the analysis, the Reb M conversion rate was 90.4%, and the production amount was 97 g / L. As a result of checking the amount of Reb M produced, it was confirmed that a high concentration Reb M with a purity of 90% by weight could be obtained using the freeze-dried cells, confirming that mass production of high purity Reb M is possible. Although a small amount of substrate solution was used for evaluating the production activity of Reb M from the freeze-dried cells and the reaction conditions were somewhat different, it was confirmed that the conversion rate in this example was significantly increased compared to Reb M in Example 12.

Claims

1. The present invention relates to a method for the production of a steviol glycoside substrate comprising: (a) a first UDP-glycosyltransferase (uridine diphosphate-glucosyltransferase) that has an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO: 1; and (b) a biocatalyst that includes UGT76G1 and a first UDP-glycosyltransferase that transfers glucose to a steviol glycoside substrate; and (c) a reaction substrate; A composition for producing steviol glycoside containing rebaudioside M, wherein rebaudioside M is contained in an amount of 10% by weight or more of a steviol glycoside reaction product.

2. The composition of claim 1, wherein the first UDP-glycosyltransferase comprises an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of SEQ ID NO: 1 in which at least one amino acid selected from the group consisting of the 201st and 202nd amino acids from the N-terminus is substituted with one or more amino acids selected from the group consisting of serine and valine.

3. 10. The composition of claim 1, wherein the biocatalyst additionally comprises a sucrose synthase protein.

4. The composition according to claim 3, wherein the sucrose synthase is provided as a fusion protein linked directly or via a linker to one or more enzymes selected from the group consisting of a first UDP-glucosyltransferase and a UGT76G1 enzyme.

5. 2. The composition of claim 1, wherein rebaudioside M is present in an amount of 50% or more by weight of the steviol glycoside reaction product.

6. The composition according to claim 1, wherein the biocatalyst comprises one or more selected from the group consisting of the enzyme protein, a microbial cell, a culture of the microbial cell, a disrupted product of the microbial cell, and an extract of the disrupted product or culture.

7. The present invention relates to a method for the production of a steviol glycoside substrate comprising: (a) a first UDP-glycosyltransferase (uridine diphosphate-glucosyltransferase) that has an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO: 1; and (b) a biocatalyst that includes UGT76G1 and a first UDP-glycosyltransferase that transfers glucose to a steviol glycoside substrate; and (c) a reaction substrate; The biocatalyst is at least one selected from the group consisting of a microbial cell that produces the enzyme protein and a dried product of the microbial cell.

2. The composition for producing steviol glycoside containing rebaudioside M according to claim 1, wherein rebaudioside M is contained in an amount of 10% by weight or more of the steviol glycoside reaction product.

8. The composition according to claim 7, wherein the dried bacterial cell has a total conversion rate of Reb M and Reb D of 30% or more.

9. 8. The composition according to claim 7, wherein the enzyme activity of the dried bacterial cells after storage at room temperature for 9 weeks is 90% or more of the rebaudioside M converting activity of the bacterial cells immediately after drying.

10. The composition according to claim 7, wherein the freeze-dried bacterial cells are freeze-dried at a temperature of -60 to 40°C under reduced pressure.

11. 8. The composition of claim 7, wherein the composition additionally comprises ethylenediaminetetraacetic acid.

12. The composition according to claim 11, wherein the concentration of the ethylenediaminetetraacetic acid is 1 to 20 mM.

13. 2. The composition of claim 1, wherein the reactive substrate comprises one or more steviol glycosides selected from the group consisting of stevioside and rebaudioside A.

14. 2. The composition of claim 1, wherein the reaction substrate is provided at 15 to 150 g / L.

15. 2. The composition of claim 1, wherein the biocatalyst is Escherichia coli, a Saccharomyces strain, or a Pichia strain.

16. The composition of claim 1 , wherein the microorganism is a Saccharomyces microorganism in which the SUC2 gene encoding the invertase enzyme has been deleted.

17. The composition comprises MgCl 2 or MnCl 2 The composition of claim 1 additionally comprising:

18. 2. The composition of claim 1, further comprising one or more selected from the group consisting of uridine diphosphate (UDP) and sucrose.

19. 19. The composition of claim 18, comprising the sucrose at a concentration of 500 to 2000 mM.

20. The composition of claim 1, wherein the reaction substrate has a pH of 4 to 9.

21. A method for producing steviol glycosides including rebaudioside M, comprising a step of reacting a reaction substrate including one or more selected from the group consisting of stevioside and rebaudioside A with a biocatalyst including a first UDP-glycosyltransferase and UGT76G1, The first UDP-glycosyltransferase has an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO: 1 and is an enzyme protein (uridine diphosphate-glucosyltransferase) having a UDP-glycosyltransferase activity of transferring glucose to a steviol glycoside substrate, The method, wherein rebaudioside M comprises 10% or more by weight of the steviol glycoside reaction products.

22. The method according to claim 21, wherein the biocatalyst comprises one or more selected from the group consisting of the enzyme protein, a microbial cell, a culture of the microbial cell, a disrupted product of the microbial cell, and an extract of the disrupted product or culture.

23. A method for producing steviol glycosides including rebaudioside M, comprising a step of reacting a reaction substrate including one or more selected from the group consisting of stevioside and rebaudioside A with a biocatalyst including a first UDP-glycosyltransferase and UGT76G1, The first UDP-glycosyltransferase has an amino acid sequence identity of 92% or more with the amino acid sequence of SEQ ID NO: 1 and is an enzyme protein (uridine diphosphate-glucosyltransferase) having a UDP-glycosyltransferase activity of transferring glucose to a steviol glycoside substrate, The biocatalyst is at least one selected from the group consisting of a microbial cell that produces the enzyme protein and a dried product of the microbial cell. A method for producing steviol glycosides comprising rebaudioside M according to claim 21.

24. The method according to any one of claims 21 to 23, wherein the reaction temperature is 20 to 60°C and the reaction pH is pH 4 to 9.

25. 24. The method of any one of claims 21 to 23, wherein the reaction substrate is provided at 15 to 150 g / L.

26. The method according to claim 23, wherein the dried matter of the cells has a total conversion rate of Reb M and Reb D of 30% or more.

27. 24. The method according to claim 23, wherein after storing the dried product of the cells at room temperature for 9 weeks, the enzyme activity of the cells is 90% or more of the rebaudioside M converting activity of the cells at the start of the storage.

28. The method according to claim 23, wherein the dried product of the bacterial cells is freeze-dried at a temperature of -60 to 40°C under reduced pressure conditions.

29. The method according to claim 23, wherein the reacting step is carried out by adding ethylenediaminetetraacetic acid at a concentration of 1 to 20 mM.

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