Method for producing target stevioside

Abstract

The present invention discloses a method for producing a target stevioside. According to the method for producing the target stevioside, the distribution change amount of the target stevioside reaches 90% or above within 2 hours, so that a precipitate containing the target stevioside is formed. The target stevioside is rebaudioside D and / or rebaudioside M. The target stevioside is rebaudioside M.

Description

[0001] This application is a divisional application of Chinese patent application No. 2024111774654, entitled “Preparation method of rebaudioside M and its application therein”, with priority date of August 24, 2023 and filing date of August 26, 2024. Technical Field

[0002] This invention relates to the field of biotechnology, specifically to a method for preparing rebaudioside M and the application of β-1,2-glucosyltransferase therein. Background Technology

[0003] Steviol glycosides are sweeteners extracted from stevia (Steviarebaudiana Bertoni). They have advantages such as high sweetness (e.g., 250 to 450 times sweeter than sucrose), low calories (e.g., only 1 / 300 the calories of white sugar), economical use (only one-third the price of sucrose), stability (heat-resistant, acid-resistant, alkali-resistant, and not easily decomposed), safety (no toxic side effects), and non-participation in lipid metabolism in the human body. They also have antihypertensive, anti-inflammatory, and anti-tumor effects. Therefore, they have a wide range of uses and high commercial value.

[0004] Stevia leaves can accumulate up to 10-20% (based on dry weight) of steviol glycosides. The main glycosides found in stevia leaves are rebaudioside A (2-10%), steviol glycoside (2-10%), and steviol glycoside C (1-2%). Other glycosides, such as rebaudioside B, D, E, F, I, M, N, and O, as well as steviol diglycosides and steviol glycosides, are present in significantly lower amounts in the leaves (approximately 0-0.2%), and their content varies depending on the stevia variety and growing conditions.

[0005] The structural formulas of steviol glycosides are shown in Formula I below, and the specific compounds involved are shown in Table 1 below.

[0006]

[0007] Formula I

[0008] Table 1. List of Stevioside Compounds

[0009]

[0010] As shown in Table 1, different types of steviol glycosides share a common glycoside core: steviol. The differences lie in the number and type of sugar groups linked at positions C-13 and C-19. These mainly include steviol glycosides, rebaudioside A (Reb A), rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside I, dulcitin A, and steviol diglycosides.

[0011] Although steviol glycosides are high-intensity sweeteners, their lingering sweet-bitter aftertaste severely limits their application in food, beverage, and other fields with specific taste requirements. The root cause of this aftertaste lies in the intrinsic molecular structure of steviol glycosides. The more sugar groups linked to R1 and R2, the better the flavor. Generally, steviol glycosides are 110-270 times sweeter than sucrose, and rebaudioside A is 250-300 times sweeter. However, even in highly purified forms, steviol glycosides still possess undesirable taste properties such as bitterness, a lingering sweet aftertaste, a persistent sweetness, and a licorice-like flavor.

[0012] By using glucosyltransferases that transfer only glucose residues in an enzymatic reaction, more sugar residues can be added to the R1 and / or R2 positions of the steviol glycoside molecule, thus modifying the starting material containing steviol glycosides. The mechanism of action involves catalyzing the transfer of glucose residues from the sugar donor to the sugar acceptor molecule, thereby modulating the activity of the acceptor molecule.

[0013] Glycosyl donors include nucleoside diphosphate (NDP) sugars, such as UDP-glucose. UDP-glucose, an abbreviation for uridine diphosphate glucose, also known as UDP-Glc or UDPG, is a vitamin composed of uridine diphosphate and glucose. It can be considered "active glucose" and is widely distributed in the cells of plants, animals, and microorganisms. It serves as a glycosyl donor in the synthesis of sucrose, starch, glycogen, and other oligosaccharides and polysaccharides, making it the most common glycosyl donor. UDP-glucosyltransferase is a glucosyltransferase that uses UDP-glucose as a glycosyl donor.

[0014] "Nucleoside" refers to a glycosamine containing a nucleobase (i.e., a nitrogenous base) and a 5-carbon sugar (such as ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine.

[0015] "Nucleoside diphosphate" refers to a glycosamine containing a nucleobase (i.e., a nitrogenous base), a 5-carbon sugar (such as ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) fragment. "Nucleoside diphosphate" can be abbreviated as "NDP". Non-restrictive examples of nucleoside diphosphates include cytidine diphosphate (CDP), uridine diphosphate (UDP), adenosine diphosphate (ADP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP).

[0016] Besides UDP-glucose, other NDP-glycosyl donors include ADP-glucose, TDP-glucose, dTDP (deoxythymidine diphosphate)-glucose, CDP-glucose, IDP-glucose, or GDP-glucose. These glycosyl donors are expensive, which is not conducive to industrial-scale production.

[0017] Glycosyl donors can be synthesized using a glycosyl donor generation system. Sucrose synthase (SUS, also abbreviated as SuSy / SS, EC2.4.1.13) reversibly catalyzes the synthesis of NDP and sucrose from nucleotide diphosphate (NDP) - glucose + D-fructose. It belongs to the glycosyltransferase-4 subfamily and was first discovered in wheat embryos. Each protein exists as a tetramer. The molecular weight of each subunit is approximately 90 kDa. The gene size is typically 5.9 kb, and the cDNA size is approximately 2.7 kb. The encoded amino acid sequence is approximately 800 residues long (Ross and Davies 1992). Sucrose synthases exhibit different affinities for different NDPs, with the affinity order being UDP > ADP > dTDP > CDP > GDP.

[0018] Rebaudioside A is the most abundant steviol glycoside in commercial stevia leaves. It is highly sweet, approximately 250-300 times sweeter than sucrose, low in calories, easily soluble, heat-resistant, and stable. The content or purity of rebaudioside A is a key indicator of the quality of stevia sweeteners. Furthermore, rebaudioside A is an important intermediate in the synthesis of other steviol glycosides, such as rebaudioside D (RebD, RD), rebaudioside E (RebE, RE), rebaudioside M (RebM, RM), and rebaudioside I (RebI, RI).

[0019] Rebaudioside D is another steviol glycoside with excellent taste and promising applications in the food and beverage industry. Compared to other steviol glycosides, it is significantly sweeter, approximately 300 to 350 times sweeter than sucrose. It has a pure sweetness close to that of sucrose, without any bitterness or licorice-like taste, and exhibits good stability, making it an ideal natural high-intensity sweetener.

[0020] However, the content of rebaudioside D in stevia leaves is very low (less than 5%), so the extraction method requires a large amount of stevia raw material. Furthermore, the purification process of rebaudioside D is quite complex, requiring multiple chromatographic column chromatography, desalting, decolorization, and recrystallization processes after extraction. This process generates a considerable amount of wastewater, resulting in high production costs and making it unsuitable for large-scale industrial production.

[0021] Rebaudioside D can be synthesized via a bioenzymatic process, starting with stevia leaf extract and using glucosyltransferase. This biosynthesis requires the addition of expensive UDP-glucose as one of the substrates. The process involves the action of UDP-glucosyltransferase (UGT), catalyzing the production of rebaudioside D from steviol glycosides or rebaudioside A as substrates. However, due to the extremely high price of UDP-glucose, the feasibility of industrial production of rebaudioside D is almost entirely limited, resulting in poor economic benefits and a lack of market competitiveness.

[0022] Biosynthetic methods for converting rebaudioside molecules have been publicly disclosed. Ohta et al. (J. Appl. Glycosci., 57, 199~209 (2010)) first reported the isolation of rebaudioside I (Reb I, RI) from stevia. Indra Prakash et al. (Molecules 2014, 19, 17345-17355) first reported a biosynthetic method for preparing RI from RA (rebaudioside A) using the glucosyltransferase UGT76G1 mutant UGT76G1-r11-f12. This method uses UDP-glucose as a glycosyl donor. The reaction was carried out in a shake flask at 30°C and pH 7.5 using MgCl2 salt, yielding RI in 22.5% of the sample. Patent application CN106795523A, inventorled by Indra Prakash, discloses the GenBank accession number AAR06912.1 for the glucosyltransferase UGT76G1 and its DNA sequence. CN110914445A screened for a UGT76G1 mutant of glucosyltransferase catalyzing the synthesis of RI from RA. The screening conditions were pH 7.0, MgCl2 10.3 mM, and temperature 35°C. CN106795523A reported sweetness data for RI and the components containing RI. The aforementioned prior art only discloses glucosyltransferases or their mutants with catalytic activity for the synthesis of RI from RA, but these enzymes have low activity and are not suitable for industrial-scale production.

[0023] Rebaudioside M (Reb M, abbreviated as RM) has attracted considerable attention due to its superior taste and higher price. However, its content in leaf dry weight is even lower than that of rebaudioside D, typically less than 0.1%, leading to high separation costs and a high price. Biocatalytic methods for obtaining high concentrations of rebaudioside M have garnered significant attention. Currently, it has been reported that stevia-derived recombinant enzymes can catalyze the synthesis of rebaudioside M from rebaudioside D, but the yield is relatively low. This study proposes a method using rebaudioside D as a substrate, catalyzed by microbial enzyme production to obtain rebaudioside M. Compared to traditional extraction methods, this approach not only improves the production process but also reduces environmental pollution and increases the yield of rebaudioside M.

[0024] Currently, enzymatic catalysis is commonly used to prepare rebaudin M. For example, CN113186141B discloses an enzymatic method for preparing rebaudin M, using sucrose, steviol glycoside (ST), and uridine diphosphate (UDP) as raw materials. First, sucrose and UDP are catalyzed by an Arabidopsis-derived sucrose synthase to generate UDPG. UDPG and ST are then catalyzed by a stevia-derived glycosyltransferase to generate rebaudin A. Second, UDPG and rebaudin A are catalyzed by a rice-derived glycosyltransferase to generate rebaudin D. Finally, UDPG and rebaudin D are catalyzed by a stevia-derived glycosyltransferase to generate rebaudin M. The yield of rebaudin M is only about 37.9%, which is low and difficult to meet the yield requirements for industrial production. The current bio-enzymatic catalysis method for rebaudioside M has the following main problems: (1) The cost of producing rebaudioside M from rebaudioside D using bio-enzymatic catalysis is high, and the enzyme catalytic yield needs further optimization; (2) The glycosyltransferase used for catalysis is difficult to separate and recover from the product, is easily inactivated, and needs to be replenished, which increases the cost; (3) In natural plants, the content of rebaudioside A is very high, while the content of D is very low. Directly converting rebaudioside A to rebaudioside D at low cost is also a challenge.

[0025] Currently, the enzymes used in the bio-enzymatic preparation of steviol glycosides are mainly wild-type enzymes derived from plant cells. These wild-type enzymes typically suffer from low enzyme activity and poor stability, leading to high costs in the industrial-scale production of steviol glycosides. Therefore, it is necessary to improve glucosyltransferases to obtain modified enzymes with higher activity and better stability, thereby better serving industrial-scale production. Summary of the Invention

[0026] To overcome the shortcomings of slow conversion rates and low yields in the existing enzymatic methods for preparing desired rebaudioside products such as rebaudioside D and rebaudioside M, this invention provides a method for significantly improving the preparation of desired rebaudioside products such as rebaudioside D and rebaudioside M, and the application of β-1,2-glucosyltransferase in this process. This invention improves the yield of the intermediate rebaudioside D when β-1,2-glucosyltransferase catalyzes the conversion of rebaudioside A to rebaudioside M. Furthermore, the combined use of β-1,2-glucosyltransferase, β-1,3-glucosyltransferase, and sucrose synthase further enhances the yield of the final product, rebaudioside M.

[0027] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0028] The first aspect of the present invention provides a β-1,2-glucosyltransferase having an amino acid sequence as shown in SEQ ID NO: 4, or having a mutant amino acid sequence having at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4.

[0029] In some embodiments, compared to the amino acid sequence shown in SEQ ID NO: 4, the mutant amino acid sequence comprises any four or more of the following amino acid residue differences: D323N; H324K; M181L; F185L; E188F; A196V; A198F; G201P; R373K; L375M; L325F; V326A. As another example, compared to the amino acid sequence shown in SEQ ID NO: 4, the mutant amino acid sequence comprises four or five of the following: D323N; H324K; M181L; F185L; E188F; A196V; A198F; G201P; R373K; L375M; L325F; V326A.

[0030] In some embodiments, compared to the amino acid sequence shown in SEQ ID NO: 4, the mutant amino acid sequence comprises: D323N, H324K, and any two or more of the following amino acid residue differences: M181L; F185L; E188F; A196V; A198F; G201P; R373K; L375M; L325F; V326A. As another example, compared to the amino acid sequence shown in SEQ ID NO: 4, the mutant amino acid sequence comprises: D323N, H324K, and two or three of the following amino acid residue differences: M181L; F185L; E188F; A196V; A198F; G201P; R373K; L375M; L325F; V326A.

[0031] In some embodiments, the mutant amino acid sequence contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, M181L, F185L, and E188F.

[0032] In other embodiments, the mutant amino acid sequence contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, A196V, A198F, and G201P.

[0033] In other embodiments, the mutant amino acid sequence contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, R373K, and L375M.

[0034] In other embodiments, the mutant amino acid sequence contains the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, L325F, and V326A.

[0035] In some specific embodiments, the mutant amino acid sequence is as shown in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22.

[0036] A second aspect of the present invention provides an isolated nucleic acid molecule that encodes the β-1,2-glucosyltransferase as described in the first aspect.

[0037] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21.

[0038] A third aspect of the present invention provides a recombinant expression vector comprising a backbone vector and a separated nucleic acid molecule as described in the second aspect integrated into the backbone vector.

[0039] In some specific embodiments, the backbone vector comprises a phage vector, a plasmid vector, or a viral vector. The plasmid includes pET28a or pET21a.

[0040] A fourth aspect of the present invention provides a transformant, which is a gene-recombinant cell obtained by introducing a separated nucleic acid molecule as described in the second aspect or a recombinant expression vector as described in the third aspect into a host cell.

[0041] In some implementations, the host cell is a eukaryotic cell or a prokaryotic cell.

[0042] In some embodiments, the prokaryotic cell is Escherichia coli, such as BL21(DE3).

[0043] In some embodiments, the transformant is selected from any one or more of the following: RMIS708, RMIS828, RMIS829, RMIS830 and RMIS831 from Yikel Biotechnology (Shanghai) Co., Ltd.

[0044] The fifth aspect of the present invention provides a method for preparing β-1,2-glucosyltransferase as described in the first aspect, comprising culturing the transformant as described in the fourth aspect in a culture medium to obtain the β-1,2-glucosyltransferase.

[0045] In some embodiments, the culture medium is selected from LB liquid medium and / or TB liquid medium.

[0046] In some embodiments, the culture conditions are: shaking culture at a temperature of 37±1℃.

[0047] The sixth aspect of the present invention provides a method for preparing rebaudioside D, comprising the steps of forming a reaction system with at least rebaudioside A, a glycosyl donor and β-1,2-glucosyltransferase as described in the first aspect and reacting therewith.

[0048] In this invention, the reaction system refers to the solution system in which the various reactants participating in the reaction are located when the reaction occurs. In an embodiment of the sixth aspect of this invention, the reaction system includes components such as a crude enzyme solution or reaction enzyme solution containing β-1,2-glucosyltransferase, a substrate, a product, and a solvent. Other embodiments are similar.

[0049] In some embodiments, the β-1,2-glucosyltransferase is used in the form of wet cells, bacterial powder, liquid enzyme, solid enzyme powder, or immobilized enzyme.

[0050] In this invention, wet bacterial cells refer to the precipitate obtained after centrifuging and discarding the supernatant from the culture medium of bacterial cells expressing glycosyltransferases. Bacterial powder refers to the powder obtained by drying the aforementioned wet bacterial cells to remove the solvent water and then pulverizing them. Liquid enzyme (such as crude enzyme solution) refers to an enzyme solution obtained by resuspending the aforementioned wet bacterial cells in a solvent (e.g., water) at a certain ratio; preferably, it is the supernatant obtained by homogenizing the resuspended enzyme solution under a certain pressure and centrifuging it at a certain speed. Reactive enzyme solution refers to a liquid enzyme that has been kept at a constant temperature of 80±10℃ for a period of time to remove impurities. Solid enzyme powder refers to the powder obtained by drying and pulverizing the aforementioned liquid enzyme. Immobilized enzyme refers to the enzyme component of the aforementioned liquid enzyme immobilized on an immobilization carrier. The immobilization carrier includes resins, such as epoxy resin, amino resin, and adsorption resin. More specifically, the epoxy resin may be SEPABEADS® ECHFA, ReliZymeTM HFA403, ReliZymeTM EP113, ReliZymeTM EP403 and / or SEPABEADS® ECEP.

[0051] In some embodiments, the amount of lebodiin A added to the reaction system is 20-120 g / L, preferably 40-110 g / L, and more preferably 50-100 g / L.

[0052] In some embodiments, the glycosyl donor is NDP-glucose; NDP is preferably selected from any one or more of adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP).

[0053] In some embodiments, the pH of the reaction is 5.0-8.0, preferably 6.0.

[0054] In some embodiments, the reaction temperature is 20-90°C, preferably 60°C.

[0055] In some implementations, the reaction time is 10 minutes to 24 hours, preferably 30 minutes.

[0056] In some embodiments, the β-1,2-glucosyltransferase is used in the form of wet bacterial cells, and the mass ratio of the wet bacterial cells to the added rebaudioside A in the reaction system can be (0.1-0.8):1, (0.12-0.6):1, or (0.24-0.3):1.

[0057] In this invention, the added mass refers to the initial mass of components such as wet bacterial cells or substrates when added to the reaction system.

[0058] In this invention, one example of preparing the crude enzyme solution is to resuspend the wet bacterial cells expressing the β-1,2-glucosyltransferase in a buffer solution at a ratio of 1:5 (g / mL), then homogenize the resuspended solution at 550 bar for 1.5 min (to rupture the bacterial cells), centrifuge at 12000 rpm for 2 min, and then collect the supernatant to obtain the crude enzyme solution.

[0059] In this invention, the buffer solution can be a phosphate buffer with a pH of 6.0.

[0060] In some embodiments, the amount of wet bacterial cells added to the reaction system can be 6-30 g / L or 12-24 g / L.

[0061] In some embodiments, the β-1,2-glucosyltransferase is used as a liquid enzyme; the liquid enzyme is preferably a crude enzyme solution or a reaction enzyme solution; the crude enzyme solution is prepared from wet bacterial cells expressing the β-1,2-glucosyltransferase; the reaction enzyme solution is obtained by incubating the crude enzyme solution at a constant temperature of 80±10℃ for a period of time.

[0062] In some embodiments, the crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacterial cells with a solvent, wherein the ratio of the added mass of the wet bacterial cells to the added volume of the solvent is 1 g: (5-10) mL. For example, the crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacterial cells with a solvent, wherein the ratio of the added mass of the wet bacterial cells to the added volume of the solvent is 1 g: 5 mL. The solvent is water or a buffer solution (e.g., phosphate buffer). As another example, the ratio of the added mass of the wet bacterial cells to the added volume of the solvent can also be 1 g: 10 mL.

[0063] In some embodiments, the ratio of the volume of crude enzyme solution added to the mass of rebaudioside A in the reaction system can be (0.5-5):1, (0.6-3):1, or (1.2-1.5):1, in mL / g.

[0064] A seventh aspect of the present invention provides an enzyme combination system comprising: a β-1,2-glucosyltransferase as described in the first aspect, wherein the β-1,2-glucosyltransferase is capable of catalyzing the reaction of rebaudioside A with a glycosyl donor to generate rebaudioside D; and a β-1,3-glucosyltransferase, wherein the β-1,3-glucosyltransferase is capable of catalyzing the reaction of rebaudioside D with a glycosyl donor to generate rebaudioside M.

[0065] In some embodiments, the β-1,2-glucosyltransferase is used in the form of wet cells, for example, referred to as the second wet cell; the β-1,3-glucosyltransferase is used in the form of wet cells, for example, referred to as the third wet cell. The relevant definition of the wet cell is as described above.

[0066] In some embodiments, the enzyme assembly further includes a glycosyl donor synthase capable of catalyzing the reaction of sugars with NDP to generate the glycosyl donor. In some embodiments, the glycosyl donor synthase is used in the form of wet cells, for example, referred to as a first wet cell.

[0067] In some embodiments, the sugar is sucrose, the glycosyl donor is NDP-glucose, and the glycosyl donor synthase is sucrose synthase.

[0068] In some embodiments, the glycosyl donor synthase is obtained by fermentation culture of a first transformant, which is a recombinant cell obtained by introducing a nucleotide sequence as shown in SEQ ID NO: 1 into a host cell, or a recombinant cell capable of expressing an amino acid sequence as shown in SEQ ID NO: 2.

[0069] In some embodiments, the β-1,2-glucosyltransferase is obtained by fermentation culture of a second transformant; the second transformant is a gene-recombinant cell obtained by introducing a nucleic acid molecule isolated as described in the second aspect or a recombinant expression vector as described in the third aspect into a host cell.

[0070] In some embodiments, the β-1,3-glucosyltransferase is obtained by fermentation culture of a third transformant; the third transformant is a recombinant cell obtained by introducing a nucleotide sequence as shown in SEQ ID NO: 23 into a host cell, or a recombinant cell capable of expressing an amino acid sequence as shown in SEQ ID NO: 24.

[0071] In some implementations, the first transformant is strain RMIS609 from Yikelei Biotechnology (Shanghai) Co., Ltd.

[0072] In some embodiments, the second transformant is selected from any one or more of the strains numbered RMIS708, RMIS828, RMIS829, RMIS830 and RMIS831 of Yikelai Biotechnology (Shanghai) Co., Ltd.

[0073] In some implementations, the third transformant is strain RMIS109 from Yikelei Biotechnology (Shanghai) Co., Ltd.

[0074] The eighth aspect of the present invention provides a method for preparing rebaudine M, comprising: a step of forming an enzyme catalytic system by rebaudine A, a glycosyl donor and an enzyme combination system as described in the seventh aspect to carry out a reaction (i.e., a one-pot reaction); or a step of forming an enzyme catalytic system by rebaudine A, a glycosyl donor and β-1,2-glucosyltransferase and reacting, and then adding β-1,3-glucosyltransferase and continuing the reaction (i.e., a stepwise reaction).

[0075] In some embodiments, the β-1,2-glucosyltransferase is the β-1,2-glucosyltransferase described in the first aspect of the present invention.

[0076] In some embodiments, the β-1,2-glucosyltransferase and / or the β-1,3-glucosyltransferase are used in the form of wet cells, bacterial powder, liquid enzyme, solid enzyme powder or immobilized enzyme.

[0077] In some embodiments, the amount of lebodiin A added to the enzyme catalytic system can be 20-120 g / L, 40-110 g / L, or 50-100 g / L.

[0078] In some embodiments, the glycosyl donor in the reaction system is NDP-glucose; NDP is selected from any one or more of adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP).

[0079] In some embodiments, the pH of the reaction is 5.0-8.0, preferably 6.0.

[0080] In some embodiments, the reaction temperature is 20-90°C, preferably 60°C.

[0081] In some implementations, the reaction time is 10 minutes to 24 hours, preferably 30 minutes to 6 hours.

[0082] In some embodiments, the β-1,2-glucosyltransferase is used in the form of wet bacterial cells, and the mass ratio of the wet bacterial cells to the added rebaudioside A in the enzyme catalytic system can be (0.1-0.8):1, (0.12-0.6):1, or (0.24-0.3):1.

[0083] In some embodiments, the amount of wet bacterial cells added in the enzyme catalysis system can be 6-30 g / L or 12-24 g / L.

[0084] In some embodiments, the β-1,2-glucosyltransferase is used as a liquid enzyme; the liquid enzyme is preferably a crude enzyme solution or a reaction enzyme solution; the crude enzyme solution is prepared from a second wet bacterial cell expressing the β-1,2-glucosyltransferase; the reaction enzyme solution is obtained by incubating the crude enzyme solution at a constant temperature of 80±10℃ for a period of time.

[0085] In some embodiments, the crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacterial cells with a solvent, and the ratio of the added mass of the wet bacterial cells to the added volume of the solvent is 1 g: (5-10) mL.

[0086] In some embodiments, the ratio of the volume of crude enzyme solution added to the mass of rebaudioside A in the enzyme catalytic system can be (0.5-5):1, (0.6-3):1, or (1.2-1.5):1, in mL / g.

[0087] In some embodiments, the wet bacterial cells expressing the β-1,2-glucosyltransferase or the liquid enzyme containing the β-1,2-glucosyltransferase are prepared from a second transformant capable of expressing the β-1,2-glucosyltransferase; further, the second transformant is selected from any one or more of the strains numbered RMIS708, RMIS828, RMIS829, RMIS830, and RMIS831 of Yikelai Biotechnology (Shanghai) Co., Ltd.

[0088] In some embodiments, the β-1,3-glucosyltransferase is used in the form of wet bacterial cells, and the mass ratio of the wet bacterial cells to the rebaudioside A in the enzyme catalytic system can be (0.1-0.8):1, (0.12-0.6):1, or (0.24-0.3):1.

[0089] In some embodiments, the amount of wet bacterial cells added to the enzyme catalysis system can be 6-30 g / L or 12-30 g / L.

[0090] In some embodiments, the β-1,3-glucosyltransferase is used as a liquid enzyme; the liquid enzyme is preferably a crude enzyme solution or a reaction enzyme solution; the crude enzyme solution is prepared from a third wet cell expressing the β-1,3-glucosyltransferase; the reaction enzyme solution is obtained by incubating the crude enzyme solution at a constant temperature of 80±10℃ for a period of time.

[0091] In some embodiments, the crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacterial cells with a solvent, and the ratio of the added mass of the wet bacterial cells to the added volume of the solvent is 1 g: (5-10) mL.

[0092] In some embodiments, the ratio of the volume of crude enzyme solution added to the mass of rebaudioside A in the enzyme catalytic system can be (0.5-5):1, (0.6-3):1, or (1.2-1.5):1, in mL / g.

[0093] In some embodiments, the wet bacterial cells expressing the β-1,3-glucosyltransferase or the liquid enzyme containing the β-1,3-glucosyltransferase are prepared from a third transformant capable of expressing the β-1,3-glucosyltransferase; further, the third transformant is strain RMIS109 of Yikelai Biotechnology (Shanghai) Co., Ltd.

[0094] In some embodiments, the glycosyl donor synthase is used in the form of wet bacterial cells, and the mass ratio of the wet bacterial cells to the rebaudioside A in the enzyme catalytic system can be (0.1-0.8):1, (0.12-0.6):1, or (0.24-0.3):1.

[0095] In some embodiments, the amount of wet bacterial cells added to the enzyme catalysis system can be 6-30 g / L or 12-30 g / L.

[0096] In some embodiments, the glycosyl donor synthase is used as a liquid enzyme; the liquid enzyme is preferably a crude enzyme solution or a reaction enzyme solution; the crude enzyme solution is prepared from a first transformant expressing the glycosyl donor synthase; the reaction enzyme solution is obtained by incubating the crude enzyme solution at a constant temperature of 80±10℃ for a period of time. In some embodiments, the glycosyl donor synthase is a sucrose synthase.

[0097] In some embodiments, the crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacterial cells with a solvent, and the ratio of the added mass of the wet bacterial cells to the added volume of the solvent is 1 g: (5-10) mL.

[0098] In some embodiments, the ratio of the volume of crude enzyme solution added to the mass of rebaudioside A in the enzyme catalytic system can be (0.5-5):1, (0.6-3):1, or (1.2-1.5):1, in mL / g.

[0099] In some embodiments, the wet bacterial cells expressing the glycosyl donor synthase in the enzyme combination system or the liquid enzyme containing the glycosyl donor synthase are prepared from a first transformant capable of expressing the glycosyl donor synthase; further, the first transformant is strain RMIS609 of Yikelai Biotechnology (Shanghai) Co., Ltd.

[0100] The ninth aspect of the present invention provides the use of the β-1,2-glucosyltransferase as described in the first aspect or the enzyme combination system as described in the seventh aspect in the preparation of rebaudioside D or rebaudioside M using rebaudioside A and a glycosyl donor as substrates.

[0101] The tenth aspect of the present invention provides an enzyme-catalyzed reaction solution comprising: rebaudine A, a glycosyl donor, rebaudine D, and β-1,2-glucosyltransferase as described in the first aspect.

[0102] In some embodiments, the enzyme-catalyzed reaction solution further includes β-1,3-glucosyltransferase and lebodiin M.

[0103] In some embodiments, the enzyme-catalyzed reaction solution further includes: sucrose synthase, NDP, sucrose, NDP-glucose, and D-fructose.

[0104] The eleventh aspect of this invention provides an enzyme-catalyzed end-product system comprising: rebaudine A; rebaudine D; rebaudine M; a β-1,2-glucosyltransferase capable of catalyzing the formation of rebaudine D from rebaudine A and a glycosyl donor, wherein the β-1,2-glucosyltransferase is the β-1,2-glucosyltransferase described in this invention; and a β-1,3-glucosyltransferase capable of catalyzing the formation of rebaudine M from rebaudine D and the glycosyl donor.

[0105] In some embodiments, in the enzyme-catalyzed end product system, the content of rebaudioside A accounts for less than 1% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M; the content of rebaudioside D accounts for less than 9.5% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M; and the content of rebaudioside M accounts for more than 84% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0106] In some embodiments, the content of rebaudioside A in the enzyme-catalyzed end product system is less than or equal to 0.88% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, the content of rebaudioside A in the enzyme-catalyzed end product system is less than or equal to 0.36% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0107] In some embodiments, the content of rebaudioside D in the enzyme-catalyzed end product system is less than or equal to 7.42% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, the content of rebaudioside D in the enzyme-catalyzed end product system is less than or equal to 3.57% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0108] In some embodiments, the content of rebaudioside M in the enzyme-catalyzed end product system is greater than or equal to 92.22% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, the content of rebaudioside M in the enzyme-catalyzed end product system is greater than or equal to 95.55% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0109] The twelfth aspect of the present invention provides a composition comprising rebaudioside D, which is prepared by the preparation method described in the sixth aspect of the present invention.

[0110] The thirteenth aspect of the present invention provides a composition comprising rebaudioside M, which is prepared by the preparation method described in the eighth aspect of the present invention.

[0111] The fourteenth aspect of the present invention provides a composition comprising rebaudioside D and rebaudioside M, which is prepared by the preparation method described in the eighth aspect of the present invention.

[0112] The fifteenth aspect of the present invention provides a composition comprising:

[0113] Rebadinoside A; Rebadinoside D; Rebadinoside M;

[0114] Among them, the content of rebaudioside A accounts for less than 1% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M; the content of rebaudioside D accounts for less than 9.5% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M; and the content of rebaudioside M accounts for more than 84% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0115] In some embodiments, the content of rebaudioside A in the composition is less than or equal to 0.88% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, the content of rebaudioside A in the composition is less than or equal to 0.36% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0116] In some embodiments, the content of rebaudioside D in the composition is less than or equal to 7.42% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, the content of rebaudioside D in the composition is less than or equal to 3.57% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0117] In some embodiments, the composition contains rebaudioside M at a percentage greater than or equal to 92.22% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, the composition contains rebaudioside M at a percentage greater than or equal to 95.55% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

[0118] The sixteenth aspect of the present invention provides a method for producing a target steviol glycoside (including rebaudioside D or rebaudioside M, or a composition of rebaudioside D and rebaudioside M), comprising the following steps:

[0119] (a) Provides a source of steviol glycosides;

[0120] (b) Provide glycosyltransferases;

[0121] (c) Providing a glycosyl donor or a glycosyl donor generation system;

[0122] (d) Prepare the reaction mixture using (i) steviol glycosides, (ii) glycosyltransferases and (iii) glycosyl donors or glycosyl donor production systems, and react at a temperature between about 30°C and 90°C; wherein the steps (a)-(c) above are not in any particular order;

[0123] (e) To achieve a distribution change of more than 50% for the target steviol glycoside within 2 hours, so as to form a precipitate containing the target steviol glycoside, more preferably, to achieve more than 60%, 70%, 80% or 90% within 2 hours; optionally, it also includes:

[0124] (f) Separate and purify the precipitate containing the target steviol glycoside from the reaction mixture, wherein the target steviol glycoside is preferably rebaudioside D and / or rebaudioside M.

[0125] The target steviol glycoside refers to Reb D when the main product is Reb D; Reb M when the main product is Reb M; and Reb D and Reb M when the main product is a mixture of Reb D and Reb M.

[0126] In some embodiments, the source of the steviol glycoside is selected from one or more of the group consisting of: stevia leaf extract, stevia glycoside, steviol glycoside, rebaudioside A, and rebaudioside D.

[0127] In some embodiments, the steviol glycoside contains at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 97 wt%, 98 wt% or more of rebaudioside A.

[0128] In some embodiments, the glycosyltransferase includes one or more selected from the group consisting of β-1,2-glycosyltransferase and β-1,3-glycosyltransferase.

[0129] In some embodiments, the β-1,2-glycosyltransferase is a glycosyltransferase that β-1,2-glycosylates the C2′ of 13-O glucose, 19-O glucose, or 13-O glucose and 19-O glucose in steviol glycosides; and the β-1,3-glycosyltransferase is a glycosyltransferase that β-1,3-glycosylates the C3′ of 13-O glucose, 19-O glucose, or 13-O glucose and 19-O glucose in steviol glycosides.

[0130] In some embodiments, the glycosyltransferase is thermostable.

[0131] Thermostable means that glycosyltransferases can react at 55℃~65℃ for 4-24 hours.

[0132] In some embodiments, the glycosyl donor includes NDP-glucose; the NDP-glucose is selected from ADP-glucose, GDP-glucose, CDP-glucose, UDP-glucose, TDP-glucose, IDP-glucose, or combinations thereof.

[0133] In some embodiments, the glycosyl donor generation system includes sucrose, a glycosyl donor synthase, and NDP.

[0134] In some embodiments, the glycosyl donor synthase is thermostable.

[0135] In some embodiments, at the start of the reaction, the molar ratio of sucrose to steviol glycoside in the reaction mixture is 4:1 to 10:1, preferably 4.5:1 to 5:1;

[0136] The concentration of sucrose is 100 g / L to 200 g / L, preferably 130 g / L to 140 g / L;

[0137] The concentration of steviol glycosides is between 20 g / L and 120 g / L, preferably between 70 g / L and 80 g / L;

[0138] And / or the reaction temperature is 55℃~65℃, preferably 60℃;

[0139] And / or the reaction pH is 5.0~8.0, preferably 6.0~6.2.

[0140] In some implementations, the pH value is achieved by adding food-grade acid.

[0141] In some embodiments, the food-grade acid is selected from the group consisting of phosphoric acid, acetic acid, and lemon.

[0142] In some embodiments, the amino acid sequence of the β-1,3-glycosyltransferase has at least about 90% identity with SEQ ID NO: 24; and / or the β-1,2-glycosyltransferase is the β-1,2-glucosyltransferase described in the first aspect of the present invention.

[0143] In some embodiments, the nucleotide sequence of the gene encoding the β-1,3-glycosyltransferase has at least about 90% identity with SEQ ID NO: 23; and / or the gene encoding the β-1,2-glycosyltransferase is the nucleic acid molecule described in the second aspect of the present invention.

[0144] In some embodiments, the amino acid sequence of the glycosyl donor synthase has at least about 90% identity with SEQ ID NO: 2.

[0145] In some embodiments, the nucleotide sequence encoding the glycosyl donor synthase gene has at least 90% identity with SEQ ID NO: 1.

[0146] In some embodiments, the change in the distribution of steviol glycosides reaches more than 85% within 30 minutes to 4 hours after the preparation of the reaction mixture.

[0147] In a seventeenth aspect of the present invention, a consumable comprising rebaudioside D and / or rebaudioside M is provided, prepared by the method described in a sixteenth aspect of the present invention.

[0148] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0149] The reagents and raw materials used in this invention are all commercially available.

[0150] The significant advantages of this invention are as follows: This invention employs an enzymatic catalytic method to catalyze the reaction of rebaudioside A and a glycosyl donor to generate rebaudioside D, which in turn generates rebaudioside M. Because this invention optimizes β-1,2-glucosyltransferase, it increases the yield of the intermediate rebaudioside D during the catalytic conversion of rebaudioside A to rebaudioside M, thereby correspondingly improving the yield of the final product, rebaudioside M. Attached Figure Description

[0151] Figure 1 This is the Reb M synthesis reaction route diagram; where β-1,2-GT: β-1,2-glycosyltransferase; β-1,3-GT: β-1,3-glycosyltransferase; SUS: sucrose synthase.

[0152] Figure 2 This is a graph showing the distribution of RebA, RebD, and RebM over time during the reaction. Detailed Implementation

[0153] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0154] The following three enzymes are used in the enzyme conversion process in the following examples.

[0155] 1. In the presence of uridine diphosphate glucose (UDPG) or adenosine diphosphate glucose (ADPG), β-1,2-glycosyltransferases promote glucose transfer. Reb A (SvG1G3) is converted to Reb D (SvG2G3), in which an additional glucose unit is β-1,2-covalently linked to the C-19 position of the steviol aglycone;

[0156] 2. In the presence of glycosyl donors UDPG or ADPG, β-1,3-glycosyltransferase promotes the transfer of glucose to Reb D (SvG2G3) to generate Reb M (SvG3G3);

[0157] 3. In the presence of sucrose, glucose groups are transferred to uridine diphosphate (“UDP”) or adenosine diphosphate (“ADP”) by sucrose synthase, producing UDPG or ADPG. The reaction is reversible, and the UDP-UDPG cycle can be repeated.

[0158] Highly efficient production of Reb M can be achieved when β-1,3-glycosyltransferase and β-1,2-glycosyltransferase-mediated glycosyltransferase reactions are coupled with sucrase-synthase-mediated UDP-UDPG or ADP-ADPG cyclic reactions. The reaction pathway is as follows: Figure 1 As shown.

[0159] Changes in the distribution of steviol glycosides:

[0160] As used in this article, "change in steviol glycoside distribution" refers to the increase or decrease in the relative proportion of the target steviol glycoside in the final product relative to its relative proportion in the starting material. For example, if the proportion of RM in the starting material is 0 and the proportion of RebM in the final product is 95%, then the change is 95%.

[0161] When producing high Reb M products, a useful change in the distribution of steviol glycosides is achieved when the distribution of the desired steviol glycosides reaches 50% or more within 2 hours, preferably 60%, 70%, 80%, or 90% or more within 2 hours.

[0162] The target distribution may vary. In some embodiments, Reb M is at a level of >50% by peak area in the conversion product, more preferably ≥95% by peak area. In some cases, a Reb M concentration of ≥95% by peak area can be achieved in less than 8 hours, preferably less than 5 hours, and in some embodiments less than 3 hours.

[0163] Compared to conventional reactions, this invention aims to achieve the target distribution in a shorter time. In some cases, the target distribution of steviol glycosides is achieved in 8 hours or less, and more ideally within about 4 hours after the start of the reaction.

[0164] The reagents used in this invention are as follows:

[0165] Rebaddi glycoside A (RA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0166] RA60 was purchased from Zhucheng Haotian Pharmaceutical Co., Ltd.

[0167] The sucrose was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0168] E. coli Trans10 competent cells and E. coli BL21(DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0169] The composition of LB liquid medium is: 10 g / L trypsin, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0170] The composition of TB liquid culture medium is: 2% tryptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and 0.4% glycerol.

[0171] Both the pET28a plasmid and the recombinant plasmid were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0172] The HPLC detection method is shown below:

[0173] Chromatographic column: Diamonsil C18 (2) (4.6 mm × 250 mm, 5 μm); Mobile phase A: 1.5 g sodium dihydrogen phosphate (NaH2PO4) was dissolved in 1000 mL of water, and the pH was adjusted to 2.6 with phosphoric acid; Mobile phase B: acetonitrile; gradient elution was performed according to the conditions in Table 2. Detection wavelength: 210 nm; Flow rate: 0.8 mL / min; Injection volume: 20 μL; Column temperature: 40℃. RA retention time was 21.905 min; RD retention time was 12.614 min; RM retention time was 16.458 min.

[0174] Table 2 HPLC gradient elution conditions

[0175]

[0176] Among them, when RA is used as a substrate to generate the final product RM, the actual concentration of RM generated (C) RM ) Calculated based on external standard method and standard sample.

[0177] The yield of RM (RM%) is calculated as follows:

[0178] ;

[0179] .

[0180] If RA is a substrate that generates RD, the actual concentration of RD generated (C) RD The yield (RD%) of RD and RD is calculated in the same way.

[0181] Example 1: Preparation of GT609 reactive enzyme solution

[0182] This embodiment relates to a method for preparing GT609 reaction enzyme solution. GT609 belongs to the sucrose synthase (SUS, also abbreviated as SuSy / SS, EC2.4.1.13), which can catalyze the transfer of hexose groups. The reaction equation is: NDP + sucrose → NDP-glucose + D-fructose, thus it is also a type of glycosyltransferase. NDP is an abbreviation for nucleoside diphosphate, including adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), and inosine diphosphate (IDP). Correspondingly, NDP-glucose includes ADP-glucose, UDP-glucose, CDP-glucose, GDP-glucose, TDP-glucose, and IDP-glucose. NDP-glucose can serve as a glycosyl donor for subsequent reactions.

[0183] The GT609 in this embodiment is derived from enzyme 14 (Enz.14) described in paragraph 0128 of the invention patent application specification with publication number CN115678867A. The nucleotide sequence of the gene encoding GT609 is shown in SEQ ID NO: 1, and the amino acid sequence of GT609 is shown in SEQ ID NO: 2.

[0184] The method for obtaining GT609 reactive enzyme solution provided in this embodiment is as follows:

[0185] 1.1. The nucleotide sequence of GT609 (as shown in SEQ ID NO: 1) was synthesized by Bioengineering (Shanghai) Co., Ltd. and integrated into the pET28a plasmid vector to obtain the recombinant plasmid pET28a-GT609.

[0186] 1.2. The recombinant plasmid pET28a-GT609 was transformed into E. coli BL21(DE3) competent cells to obtain a genetically engineered strain containing the recombinant plasmid pET28a-GT609 (denoted as RMIS609). A single colony of this genetically engineered strain was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 °C with shaking for 4 h. Then, at an inoculation rate of 2 v / v% (volume percentage), it was transferred to 50 mL of freshly prepared TB liquid medium also containing 50 μg / mL kanamycin, and cultured at 37 °C with shaking until the OD600 reached approximately 0.8. IPTG was then added to a final concentration of 0.1 mM, and the culture was induced at 25 °C for 20 h. After the incubation period, the culture was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet RMIS609 cells. These were stored at -20 °C for later use.

[0187] 1.3. The collected RMIS609 wet cells were resuspended in PBS (50 mM, pH 6.0) at a ratio of 1 g:5 mL (mass-volume ratio, i.e., M / V, also known as the homogenization ratio) to obtain a suspension. The suspension was then homogenized using a high-pressure homogenizer to obtain a homogenized enzyme solution. The homogenization conditions were 550 MPa for 1.5 min. The homogenized enzyme solution was centrifuged at 12000 rpm for 2 min, the precipitate was discarded, and the supernatant was collected to obtain the crude GT609 enzyme solution.

[0188] 1.4. The crude GT609 enzyme solution was kept at 80℃ for 15 minutes to obtain the GT609 reaction enzyme solution.

[0189] Example 2: Preliminary screening of β-1,2-glucosyltransferase

[0190] β-1,2-glucosyltransferase (β-1,2-GT) catalyzes the reaction of rebaudioside A with a glycosyl donor to generate rebaudioside D (RD).

[0191] After screening enzymes from our proprietary enzyme library (the enzyme screening reaction system is shown in Table 3, where the preparation methods for the crude enzyme solution and reaction enzyme solution of β-1,2-glucosyltransferase are the same as in Example 1), β-1,2-glucosyltransferase GT708, which exhibits high catalytic activity in the reaction of rebaudioside A to rebaudioside D, was finally screened. The nucleotide sequence encoding the GT708 gene is shown in SEQ ID NO: 3, and its amino acid sequence is shown in SEQ ID NO: 4.

[0192] The preparation methods for the crude GT708 enzyme solution and the GT708 reactive enzyme solution provided in this embodiment are the same as in Example 1, except that the recombinant plasmid pET28a-GT708 is obtained. The genetically engineered strain containing the recombinant plasmid pET28a-GT708 is designated as RMIS708.

[0193] Table 3 Composition of the enzyme screening reaction system

[0194]

[0195] In Table 3, the mother liquor concentration refers to the initial concentration of each component at the start of its addition. The stock solution refers to the GT609 enzyme solution or β-1,2-glucosyltransferase enzyme solution added undiluted. The final concentration refers to the initial concentration of each component in the entire reaction system after addition, which is the concentration at 0 h of reaction. NDP (ADP or UDP) refers to the preparation of reaction systems using ADP and UDP respectively using different NDPs; therefore, Table 3 includes both types of reaction systems.

[0196] The reaction mechanisms of each substance in Table 3 are shown below. Sucrose and NDP are converted into NDP-glucose and D-fructose by sucrose synthase (SUS) in the GT609 reaction enzyme solution. NDP-glucose (as a glycosyl donor) and rebaudioside A (RA) are converted into rebaudioside D (RD) by β-1,2-glucosyltransferase (β-1,2-GT) in the β-1,2-glucosyltransferase reaction enzyme solution.

[0197] The enzyme screening method in this embodiment includes the following steps:

[0198] The reaction system was prepared according to Table 3. The reaction system was placed in a metal bath (heating temperature set at 60℃) and reacted at 60℃ for 30 min to obtain the reaction solution. The reaction solution was diluted 50 times and a sample was taken for HPLC analysis.

[0199] Based on HPLC results and dilution factors of the reaction solution, GT708 showed the best screening performance among β-1,2-glucosyltransferases, with an RD yield of 66.606% for the ADP-based reaction system and 28.842% for the UDP-based reaction system.

[0200] Example 3: Mutation of β-1,2-glucosyltransferase GT708

[0201] As shown in the results of Example 2, when sucrose and NDP are used as reaction starters, the content of RD in the reaction solution is relatively low when GT708 and GT609 reaction enzyme solutions are used together to prepare RD. To further improve the yield of RD, this example involves mutating GT708. The mutation method includes the following steps:

[0202] 3.1. Primers as shown in Table 4 were designed using the nucleotide sequence of GT708 as a template.

[0203] Table 4. Mutant Primers for GT708

[0204]

[0205] In Table 4 above, F represents the forward primer and R represents the reverse primer. The numbers following F and R are the primer numbers, used to distinguish different primers. The enzyme number indicates the number of the mutant enzyme obtained after mutation.

[0206] 3.2. PCR amplification was performed using primers F10~13 / R9 and F9 / R10~13, yielding two fragments per primer pair. Recombination was performed using the two-fragment homologous recombinase from Nanjing Novizan Biotechnology Co., Ltd., and the resulting fragments were transformed into E. coli Trans 10 competent cells. The transformed E. coli Trans 10 competent cells were plated onto LB medium with the appropriate antibiotic resistance and incubated overnight at 37°C. Single colonies were picked and transferred to LB tubes (Km resistant), incubated for 8 to 10 h, and plasmids were extracted for sequencing identification. The sequencing results are shown in Table 5 below.

[0207] Table 5. Sequencing results of four enzyme mutants based on GT708.

[0208]

[0209] Table 5 shows the mutation sites of the four enzyme mutants compared to the amino acid sequence of GT708. Here, D323N means that in the amino acid sequence of GT828, the amino acid at position 323 changes from D (aspartic acid) to N (asparagine) compared to GT708. The other mutation sites are represented similarly. The hyphen “-” separates the different mutation sites.

[0210] 3.3. The four correctly sequenced plasmids were transformed into E. coli BL21(DE3) competent cells to obtain four genetically engineered strains containing point mutations as shown in Table 5. Single colonies of each genetically engineered strain were picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C with shaking for 4 h. The inoculum was then transferred to 50 mL of freshly prepared TB liquid medium containing 50 μg / mL kanamycin at a 2 v / v (volume ratio) inoculation rate and cultured at 37 °C with shaking until OD (occurrence limit). 600 When the concentration reaches approximately 0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 25 ℃ for 20 h. After the culture is completed, centrifuge the culture medium at 4000 rpm for 20 min, discard the supernatant, collect the precipitate to obtain wet cells, and store at -20 ℃ for later use.

[0211] 3.4. The collected wet bacterial cells were resuspended in PBS (50 mM, pH 6.0) at a ratio of 1:5 g / mL (mass-to-volume ratio, e.g., m / v) to obtain four suspensions. Then, the four suspensions were homogenized using a high-pressure homogenizer (550 Mbar for 1.5 min) to obtain four homogenized enzyme solutions. The four homogenized enzyme solutions were centrifuged at 12000 rpm for 2 min, the precipitates were discarded, and the supernatants were collected to obtain four crude enzyme solutions: GT828 crude enzyme solution, GT829 crude enzyme solution, GT830 crude enzyme solution, and GT831 crude enzyme solution.

[0212] 3.5. The four crude enzyme solutions were incubated at 80℃ for 15 min to obtain GT828, GT829, GT830, and GT831 enzyme solutions, respectively. All four enzyme solutions contained β-1,2-glucosyltransferase.

[0213] Example 4: Preparation of Rebaudioside D using four types of enzyme solutions and GT609 enzyme solution

[0214] In this embodiment, reaction systems for preparing rebaudioside D were constructed using reaction enzyme solutions of four enzyme mutants related to GT708 (containing β-1,2-GT) and GT609 reaction enzyme solution (containing SUS). The contents of each component in the four reaction systems are shown in Table 6.

[0215] Table 6. Composition of the reaction system for preparing rebaudioside D

[0216]

[0217] In Table 6, the mother liquor concentration refers to the initial concentration of each component when it is first added. The stock solution refers to the enzyme solutions added undiluted. The final concentration refers to the initial concentration of each component in the entire reaction system after addition, which is the concentration at 0 h of reaction time. NDP (ADP or UDP) refers to reaction systems prepared using ADP and UDP, respectively. Based on the four enzyme solutions (GT828, GT829, GT830, and GT831) and two NDPs, Table 6 includes a total of eight reaction systems.

[0218] The preparation method of rebaudioside D (RD) in this embodiment includes the following steps:

[0219] Eight reaction systems were prepared according to Table 6. Each reaction system was placed in a metal bath (heating temperature set at 60℃) and reacted at 60℃ for 30 min to obtain eight reaction solutions. Each of the eight reaction solutions was diluted 50-fold and samples were taken for HPLC analysis. A reaction system containing GT708 was also included as a control. The HPLC analysis results are shown in Table 7.

[0220] Table 7. HPLC results of reaction solutions obtained from different reaction systems containing β-1,2-GT

[0221]

[0222] In Table 7, the concentration of RD refers to the ratio (g / L) of the mass (g) of RD in the reaction solution after the reaction is terminated to the volume (L) of the reaction solution. The yield of RD (ADP) represents the yield of RD in the reaction system using ADP (without UDP) after the reaction is completed. The yield of RD (UDP) represents the yield of RD in the reaction system using UDP after the reaction is completed.

[0223] As shown in Table 7, the RD yield of the reaction system using ADP is higher than that of the reaction system using UDP. Furthermore, for GT829, the RD yield is significantly higher than that of other enzymes (GT828, GT830, GT831) regardless of whether it is used in the ADP-based or UDP-based reaction system. Therefore, GT829 was chosen for the preparation of RM.

[0224] Example 5: Preparation of GT109 reactive enzyme solution

[0225] This embodiment relates to a method for preparing GT109 reactive enzyme solution. GT109 belongs to the β-1,3-glucosyltransferase (β-1,3-GT). β-1,3-glucosyltransferase can transfer a glucosyl group (Glc) from a glycosyl donor to the glycosyl group at the C-19 position of rebaudioside D, thereby generating rebaudioside M (RM). The glycosyl donor can be NDP-glucose, including ADP-glucose, UDP-glucose, CDP-glucose, GDP-glucose, TDP-glucose, and IDP-glucose, etc.

[0226] The GT109 in this embodiment is derived from enzyme 24 (Enz.24) described in paragraph 0130 of the invention patent application publication number CN115418358A. The nucleotide sequence encoding the GT109 gene is shown in SEQ ID NO: 23, and the amino acid sequence of GT109 is shown in SEQ ID NO: 24.

[0227] The preparation method of the GT109 reactive enzyme solution provided in this embodiment is the same as that in Example 1, except that the recombinant plasmid pET28a-GT109 is obtained. The genetically engineered strain containing the recombinant plasmid pET28a-GT109 is designated as RMIS109.

[0228] In this embodiment, the homogenization ratio of GT109 wet cells used to generate GT109 crude enzyme solution is 1 g: 5 mL.

[0229] Example 6: Preparation of Rebaudioside M using GT109, GT829 and GT609 enzyme solutions

[0230] This embodiment utilizes GT109 enzyme solution (containing β-1,3-glucosyltransferase), GT829 enzyme solution (containing β-1,2-glucosyltransferase), and GT609 enzyme solution (containing sucrose synthase) to prepare rebaudioside M. The GT109 enzyme solution was obtained in Example 5, the GT829 enzyme solution in Example 3, and the GT609 enzyme solution in Example 1.

[0231] This embodiment describes the construction of a reaction system for preparing rebaudioside M. The content of each component in this reaction system is shown in Table 8.

[0232] Table 8. Composition of the reaction system for preparing lebodiin M

[0233]

[0234] The reaction mechanism of the reaction system shown in Table 8 is as follows: Sucrose and NDP are converted into NDP-glucose (ADP-glucose or UDP-glucose) and D-fructose by sucrose synthase (SUS) in GT609 enzyme solution. Rebaudidine A (RA) and NDP-glucose (as glycosyl donor) are converted into rebaudidine D (RD) by β-1,2-glucosyltransferase (β-1,2-GT) in GT829 enzyme solution. Rebaudidine D (RD) and NDP-glucose (as glycosyl donor) are converted into rebaudidine M (RM) by β-1,3-glucosyltransferase (β-1,3-GT) in GT109 enzyme solution.

[0235] In Table 8, NDP (ADP or UDP) refers to the reaction system prepared using ADP and the reaction system prepared using UDP, respectively. Therefore, Table 8 includes two types of reaction systems.

[0236] The preparation method of rebaudioside M in this embodiment includes the following steps:

[0237] Two reaction systems were prepared according to Table 8. Each reaction system was placed in a metal bath (heating temperature set at 60℃) and reacted at 60℃ for 6 h or 22 h. After terminating the reaction, two reaction solutions were obtained. The two reaction solutions were diluted 50 times and samples were taken for HPLC analysis. The processed HPLC analysis results are shown in Table 9.

[0238] Table 9. Content of each product in the reaction solution

[0239]

[0240] In Table 9, a reaction system using ADP refers to a system where one of the substrates of sucrose synthase is ADP (not UDP), therefore, the glycosyl donor is ADP-glucose. A reaction system using UDP refers to a system where one of the substrates of sucrose synthase is UDP (not ADP), therefore, the glycosyl donor is UDP-glucose. The concentration of RA refers to the ratio (g / L) of the mass (g) of RA in the enzyme-catalyzed end product system obtained after the reaction is terminated to the volume (L) of the enzyme-catalyzed end product system. The concentrations of RD and RM are calculated similarly. The percentage content of RA refers to the percentage of the mass of RA in the enzyme-catalyzed end product system obtained after the reaction is terminated relative to the total mass of RA, RD, and RM. The percentage content of RD and RM is calculated similarly.

[0241] As shown in Table 9, in the reaction system using ADP, the yield of RM reached 94.77% after 6 hours of reaction. In contrast, in the reaction system using UDP, the yield of RM only reached 91.12% after 22 hours of reaction, which was still lower than the yield of RM after 6 hours in the ADP-based system. This indicates that, for GT829, the yield of RM is higher when ADP-glucose is used as the glycosyl donor than when UDP-glucose is used.

[0242] The amount of additives in various embodiments of the present invention is shown in Table 10.

[0243] Table 10 Dosage Table for Various Embodiments of the Invention

[0244]

[0245] Example 7: Preparation of RebM by enzyme-catalyzed scale-up reaction using RA97 as raw material

[0246] 11.3g of β-1,3-glucosyltransferase GT109 bacterial sludge (produced by 5L scale fermentation) was homogenized with PBS buffer at pH 7.0 to obtain 50g of enzyme solution; 7.7g of sucrose synthase GT609 bacterial sludge (produced by 5L scale fermentation) was homogenized with PBS buffer at pH 7.0 to obtain 80g of enzyme solution; 6.4g of β-1,2-glucosyltransferase GT829 bacterial sludge (produced by 5L scale fermentation) was homogenized with PBS buffer at pH 7.0 to obtain 61g of enzyme solution.

[0247] Add 200g of water to a 500mL reactor, start stirring, and heat to 60℃. Add 15g of RA97 and 26.5g of sucrose, stirring until dissolved, maintaining a temperature above 58℃ during the dissolution process. Then add 2.7g of GT109 enzyme solution, 1.5g of GT609 enzyme solution, 1.5g of GT829 enzyme solution, and 0.075g of ADP. Adjust the pH to 6.0-6.2 with approximately 1.5g of 25% dilute phosphoric acid, and then begin the reaction.

[0248] The distribution of steviol glycosides was detected by HPLC (JECFA, 2021) during the reaction. The product began to precipitate after approximately 1.5 hours of reaction.

[0249] This invention involved multiple batch reactions, with data obtained from two batches (SG00120240510 and SG00120240511) as shown in Table 11 below. For batch SG00120240510, after 1 hour of reaction, the distribution of RebA and steviol glycosides decreased by 91.9%, while the distribution of RebD and RebM increased by 90.45%; after 2 hours of reaction, the distribution of RebA and steviol glycosides decreased by 97.7%, while the distribution of RebD and RebM increased by 97.45%.

[0250] Data show that, as the enzyme reaction time progresses, the major conversions from Reb A to Reb D and Reb M occur within the first two hours of the reaction. Figure 2 The data shows that Reb A decreased from 98% to less than 1% after 2 hours, while Reb D initially increased to over 25% within 1 hour, then decreased to 2% after 2 hours. Therefore, after 2 hours of reaction, the Reb M distribution increased to over 95%, and the Reb D and Reb M distributions increased to over 97%. Data on the changes in steviol glycoside distribution are shown in Table 14.

[0251] Table 11 Distribution of steviol glycosides at different biotransformation times

[0252]

[0253] Example 8: Preparation of Reb D using RA97 as a raw material via catalysis

[0254] Add 100g of substrate RA97, 50g of sucrose, and 0.9L of water to a 2L three-necked flask. Stir to dissolve and heat to 60℃. After the materials are completely dissolved, add β-1,2-glucosyltransferase GT829 enzyme solution (6g of bacterial sludge produced by 5L scale fermentation homogenized with 24mL of pH7.0 PBS buffer), sucrose synthase GT609 enzyme solution (6g of bacterial sludge produced by 5L scale fermentation homogenized with 24mL of pH7.0 PBS buffer), and 0.3g of ADP. The total reaction volume is about 1L-1.2L. Adjust the pH to about 6.0-6.2 with 2% phosphate aqueous solution and start the reaction at 60℃.

[0255] The distribution of steviol glycosides was detected by HPLC (JECFA, 2021) during the reaction.

[0256] This invention involved multiple batch reactions, with data obtained from two batches (A319-156-1 and A319-158-1) as shown in Table 12 below. Taking A319-156-1 as an example, after 1 hour of reaction, the distribution of RebA decreased by 74.23%, while the distribution of RebD increased by 74.80%; after 2 hours of reaction, the distribution of RebA decreased by 86.13%, while the distributions of RebD and RebM increased by 86.68%. Data on the distribution changes of steviol glycosides are shown in Table 14.

[0257] Table 12 Distribution of steviol glycosides at different biotransformation times

[0258]

[0259]

[0260] Example 9: Catalytic preparation of Reb M using RA60 as a raw material

[0261] Add 60ml of water to a 250ml three-necked flask, maintain the temperature at 60℃, add 10g of RA60 while stirring, followed by 20g of sucrose. After dissolving, add 0.3g of ADP, then adjust the pH to 6.0-6.2 with 10% sodium hydroxide. Add β-1,2-glucosyltransferase GT829 enzyme solution (3g of sludge produced by 5L scale fermentation homogenized with 15mL of pH7.0 PBS buffer), β-1,3-glucosyltransferase GT109 enzyme solution (6g of sludge produced by 5L scale fermentation homogenized with 30mL of pH7.0 PBS buffer), and sucrose synthase GT609 enzyme solution (3g of sludge produced by 5L scale fermentation homogenized with 15mL of pH7.0 PBS buffer). Adjust the pH to 6.0-6.2 with 25% dilute phosphoric acid to begin the reaction.

[0262] The distribution of steviol glycosides was detected by HPLC (JECFA, 2021) during the reaction. The results are shown in Table 13. After 1 hour of reaction, the distribution of RebA and steviol glycosides decreased by 39.77%, while the distribution of RebD and RebM reached 41.57%, an increase of 37.64%. After 2 hours of reaction, the distribution of RebA and steviol glycosides decreased by more than 63.54%, while the distribution of RebD and RebM increased by 62.98%. The data on the changes in the distribution of steviol glycosides are shown in Table 14.

[0263] Table 13 Distribution of steviol glycosides at different times during biotransformation

[0264]

[0265]

[0266] As can be seen from Examples 7, 8, and 9, using this process, a high dispensing rate of Reb D, Reb M, and their combinations can be achieved in just 1 or 2 hours, making the process highly efficient in industrial-scale production. The data from Examples 7, 8, and 9 are summarized in the table below.

[0267] Table 14. Distribution changes of steviol glycosides at 1h, 2h, and 4h during biotransformation.

[0268]

[0269] The sequence used in this invention

[0270] The nucleotide sequence of GT609 (SEQ ID NO: 1):

[0271]

[0272] Amino acid sequence of GT609 (SEQ ID NO: 2):

[0273] MSERARSDDLIEQLRGFLDACPSVGHRVLHQIKKLERSFLQRSELCDAFADVCAAEDVPDGLRQSPLGKVIRLTQEAAVNDAWVYLAVRVRIASWRYVRIALEGMAVEEVTVRDFLRFKECLALGQHDFDEWLLEIDLGPFSREFPKLLEARSIGRGVEFLNKHLSLQLFDELGEGGERILNFLRIHSFRGQTLMLNEQIKSVSDLRRALRRADEVLAGHEPTATWEDVAAPLRALGFEVGWGREVARIRDTMSLLRDLLEAPDPRGLETFLARLPMVFSLAIISPHGYFGQANVLGRPDTGGQVVYILDQVRALETEMRSRLFEQGLDIEPQIVVLTRLIPQAEGTTCDQRLEPISGTRNARILRVPFRNASGEIVTHWISRFEVWPYLERYTLDSERELLAELGGRPDLIVGNYSDGNLVATLLSQRLGVTQCNIAHALEKTKYPHADLFWQENEAQYHFSCQFTADLIAMNAADFIITSTYQEIAGTRESVGQYESHTAFTMPKLFRVVNGIDVYDPKFNIVSPGADAAAYFPYTAVERRLPHLHTEIEQLVFGVDERVDARGVLTERDKPLLFTMARLDRIKNIVGLVEWFGACEALRKEANLLVISGHVDPERSSDTEELEQIRCMHALFNRYDLDRQVRWLGLRLPKDLAGEFYRYVADGRGAFVQPALFEAFGLTVIEAMASGLPCFATCFGGPSEIIEDGVSGFHIDPNHGDAAAERIARFFERTRQDPEYWNRISEGALKRVAERYTWQHYAERMMTLSRVYGFWRHVTDLERRETQRYLQALYSLQFRRLAQAMA

[0274] Nucleotide sequence of GT708 (SEQ ID NO: 3):

[0275]

[0276] Amino acid sequence of GT708 (SEQ ID NO: 4):

[0277] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLDHLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0278] Nucleotide sequence of GT828 (SEQ ID NO:15):

[0279]

[0280] Amino acid sequence of GT828 (SEQ ID NO: 16):

[0281] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKLREMLGTFPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0282] Nucleotide sequence of GT829 (SEQ ID NO: 17):

[0283]

[0284] Amino acid sequence of GT829 (SEQ ID NO: 18):

[0285] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLVPFQAPIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0286] Nucleotide sequence of GT830 (SEQ ID NO:19):

[0287]

[0288] Amino acid sequence of GT830 (SEQ ID NO:20):

[0289] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINAKLMVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0290] Nucleotide sequence of GT831 (SEQ ID NO: 21):

[0291]

[0292] Amino acid sequence of GT831 (SEQ ID NO: 22):

[0293] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKFAPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0294] Nucleotide sequence of GT109 (SEQ ID NO: 23):

[0295]

[0296] Amino acid sequence of GT109 (SEQ ID NO: 24):

[0297] MPNTNPTTVRRRRVIMFPVPFPGHLNPMLQLANVLYRRGFEITILHTNFNAPKTSLYPHFQFRFILDNDPQPEWLRNLPTTGPGVGARIPVINKHGADEFRKELEICMRDTPSDEEVACVITDALWYFAQPVADSLNLKRLVLQTGSLFNFHCLVCLPKFLELGYLDPETKHRPDEPVVGFPMLKVKDIRRAYSHIQESKPILMKMVEETRASSGVIWNSAKELEESELETIQREIPAPSFLLPLPKHYRASSTSLLDTDPSTAQWLDQQPPSSVLYVGFGSQSSLDPADFLEIARGLVASKQSFLWNVRPGFVKGYEWIELLPDGFLGEKGRIVKSAPQQEVLAHKAIGAFWTHGGWNGTMEAVCEGVPMIFSDFGLDQPLNARYMSEVLHVGVYLENGFIRGEIINAVRRVMVDPEGEVMRQNARKLKDKLDRSIAPGGSSYESLERLESYISS。

Claims

1. A method for producing target steviol glycosides, characterized in that, The method includes the following steps: (a) Provides a source of steviol glycosides; (b) Provide glycosyltransferases; (c) Providing a glycosyl donor or a glycosyl donor generation system; (d) Prepare the reaction mixture using (i) steviol glycosides, (ii) glycosyltransferases and (iii) glycosyl donors or glycosyl donor production systems, and react at a temperature of about 30°C to 90°C; wherein the steps (a)-(c) above are not in any particular order; (e) To achieve a distribution change of more than 50% for the target steviol glycoside within 2 hours, so as to form a precipitate containing the target steviol glycoside, more preferably, to achieve more than 60%, 70%, 80% or 90% within 2 hours; optionally, it further includes: (f) Separate and purify the precipitate containing the target steviol glycoside from the reaction mixture, wherein the target steviol glycoside is preferably rebaudioside D and / or rebaudioside M.

2. The method as described in claim 1, characterized in that, The source of the steviol glycoside is selected from one or more of the following groups: stevia leaf extract, stevia glycoside, steviol glycoside, rebaudioside A and rebaudioside D; Preferably, the steviol glycoside contains at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 97 wt%, 98 wt% or more of rebaudioside A.

3. The method as described in claim 1, characterized in that, The glycosyltransferase is selected from one or more of the following group: β-1,2-glycosyltransferase and β-1,3-glycosyltransferase; Preferably, the β-1,2-glycosyltransferase is a glycosyltransferase that β-1,2-glycosylates the C2′ of 13-O glucose, 19-O glucose, or 13-O glucose and 19-O glucose in steviol glycosides; and the β-1,3-glycosyltransferase is a glycosyltransferase that β-1,3-glycosylates the C3′ of 13-O glucose, 19-O glucose, or 13-O glucose and 19-O glucose in steviol glycosides. More preferably, the glycosyltransferase is thermostable.

4. The method as described in claim 1, characterized in that, The glycosyl donor includes NDP-sugar; Preferably, the NDP-glucose is selected from ADP-glucose, GDP-glucose, CDP-glucose, UDP-glucose, TDP-glucose, IDP-glucose, or combinations thereof.

5. The method as described in claim 1, characterized in that, The glycosyl donor generation system includes sucrose, a glycosyl donor synthase, and NDP; preferably, the glycosyl donor synthase is thermally stable.

6. The method as described in claim 5, characterized in that, At the start of the reaction, the molar ratio of sucrose to steviol glycoside in the reaction mixture is 4:1 to 10:1, preferably 4.5:1 to 5:1; The concentration of sucrose is 100 g / L to 200 g / L, preferably 130 g / L to 140 g / L; The concentration of steviol glycosides is between 20 g / L and 120 g / L, preferably between 70 g / L and 80 g / L; And / or the reaction temperature is 55℃~65℃, preferably 60℃; And / or the reaction pH is 5.0~8.0, preferably 6.0~6.

2.

7. The method as described in claim 6, characterized in that, The pH is achieved by adding food-grade acid; preferably, the food-grade acid is selected from the group consisting of phosphoric acid, acetic acid, and citric acid.

8. The method as described in claim 3, characterized in that, The amino acid sequence of the β-1,3-glycosyltransferase is shown in SEQ ID NO: 24; and / or the amino acid sequence of the β-1,2-glycosyltransferase is shown in SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22; Preferably, the nucleotide sequence of the gene encoding the β-1,3-glycosyltransferase is shown in SEQ ID NO: 23; and / or the nucleotide sequence of the gene encoding the β-1,2-glycosyltransferase is shown in SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:

21.

9. The method as described in claim 5, characterized in that, The amino acid sequence of the glycosyl donor synthase has at least about 90% identity with SEQ ID NO: 2; Preferably, the nucleotide sequence of the gene encoding the glycosyl donor synthase has at least 90% identity with SEQ ID NO:

1.

10. The method as described in claim 1, characterized in that, The distribution change of the steviol glycosides reached more than 85% within 30 minutes to 4 hours.

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