Preparation method of rebaudioside M and application of beta-1, 2-glucosyltransferase in rebaudioside M

Abstract

The invention discloses a preparation method of rebaudioside D and rebaudioside M and an application of beta-1, 2-glucosyltransferase in the rebaudioside D and rebaudioside M. The beta-1, 2-glucosyltransferase has an amino acid sequence as shown in SEQ ID NO: 4, or has a mutant amino acid sequence which has at least 99% sequence identity compared with the amino acid sequence as shown in SEQ ID NO: 4. According to the invention, the yield of an intermediate product rebaudioside D when the beta-1, 2-glucosyltransferase catalyzes rebaudioside A to generate rebaudioside M is improved, and the yield of a final product rebaudioside M is correspondingly improved.

Description

[0001] This application claims the priority of Chinese patent application No. 2023110745202 filed on August 24, 2023. This application cites the entire text of the above patent application. Technical Field

[0002] The invention relates to the field of biotechnology, and in particular to a method for preparing rebaudioside M and application of beta-1,2-glucosyltransferase therein. Background Art

[0003] Stevioside (Steviol glycosides, also known as steviol glycosides) is a sweetener extracted from Stevia rebaudiana Bertoni. It has the advantages of high sweetness (such as 250 to 450 times the sweetness of sucrose), low calories (such as its calories are only 1 / 300 of white sugar), economical use (only one-third of the price of sucrose), stability (heat-resistant, acid-resistant, alkali-resistant, and not easy to decompose), safety (no toxic side effects), and does not participate in lipid metabolism in the human body. It also has anti-hypertension, anti-inflammatory, anti-tumor and other therapeutic effects. Therefore, it has a wide range of uses and high commercial value.

[0004] Stevia leaves can accumulate up to 10-20% (on a dry weight basis) of steviol glycosides. The main glycosides found in stevia leaves are rebaudioside A (2-10%), stevioside (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 rubusoside, are present in significantly lower amounts in the leaves (approximately 0-0.2%) and can vary depending on the variety of the stevia plant and the growing conditions.

[0005] The structural formula of the stevioside compound is shown in Formula I below, and the specific compounds involved are shown in Table 1 below.

[0006]

[0007] Table 1 List of types of stevioside compounds

[0008]

[0009]

[0010] As shown in Table 1, different types of steviol glycosides have a common glycoside nucleus: steviol (Steviol), and the difference lies in the number and type of sugar groups connected at the C-13 and C-19 positions. They mainly include stevioside, rebaudioside A (Reb A), rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside I, dulcoside A, steviol diglycosides and other glycosides.

[0011] Although steviol glycosides are high-intensity sweeteners, the lingering sweet-bitter aftertaste severely limits the application of steviol glycosides in areas with special taste requirements such as food and beverages. The root cause of this aftertaste is the intrinsic molecular structure of steviol glycosides. The more sugar groups attached to the R1 and R2 groups, the better the taste. In general, the sweetness of steviosides is 110-270 times that of sucrose, and the sweetness of rebaudioside A is 250-300 times that of sucrose. However, even in highly purified form, steviol glycosides still have undesirable taste attributes such as bitterness, sweet aftertaste, lingering sweetness, licorice taste, etc.

[0012] The starting material containing steviol glycosides can be modified by adding more glycosyl groups at the R1 and / or R2 positions of the steviol glycoside molecule using glucosyltransferases that transfer only glucose groups in an enzymatic reaction. Its mechanism of action involves catalyzing the transfer of glucose residues from a glycosyl donor to a glycosyl acceptor molecule, thereby modulating the activity of the acceptor molecule.

[0013] Glycosyl donors include nucleoside diphosphate (NDP) sugars such as UDP-glucose. UDP-glucose is the abbreviation of uridine diphosphate glucose, also abbreviated as UDP-Glc or UDPG, and is a vitamin composed of uridine diphosphate and glucose. It can be considered as "active glucose" and is widely distributed in the cells of plants, animals and microorganisms. It acts 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 type of glucosyltransferase that uses UDP-glucose as a glycosyl donor.

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

[0015] "Nucleoside diphosphate" refers to a sugar amine containing a nucleobase (i.e., a nitrogenous base), a 5-carbon sugar (e.g., ribose or deoxyribose), and a diphosphate (i.e., pyrophosphate) fragment. "Nucleoside diphosphate" can be abbreviated as "NDP." Non-limiting 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] In addition to UDP-glucose, other NDP-glycosyl donors include ADP-glucose, TDP-glucose, dTDP (diphosphate deoxythymine)-glucose, CDP-glucose, IDP-glucose or GDP-glucose. These glycosyl donors are expensive and are not conducive to industrial-scale production.

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

[0018] Rebaudioside A is the most abundant steviol glycoside in commercial stevia leaves. It has high sweetness, about 250 to 300 times sweeter than sucrose, low calories, easy to dissolve, heat-resistant and stable. The content or purity of rebaudioside A is also the main indicator of the quality of stevia sweeteners. In addition, rebaudioside A is also an important intermediate for the synthesis of other steviol glycoside compounds, such as rebaudioside D (RebD, RD), rebaudioside E (RebE, RE), rebaudioside M (RebM, RM), rebaudioside I (RebI, RI), etc.

[0019] Rebaudioside D is another steviol glycoside with excellent taste quality and potential application prospects in the field of food and beverage. Compared with other steviol glycosides, it has a higher sweetness, about 300 to 350 times that of sucrose. It has a pure sweetness and a taste close to sucrose, without any bitterness or licorice taste, and good stability. It is an ideal natural high-intensity sweetener.

[0020] However, the content of rebaudioside D in stevia leaves is very low (less than 5%), so the production by extraction method requires a large amount of stevia raw materials. In addition, the purification process of rebaudioside D is relatively complicated, and after extraction, it needs to go through multiple chromatographic columns, desalination, decolorization, recrystallization and other processes. This process produces a lot of wastewater, resulting in high production costs and is not suitable for large-scale industrial production.

[0021] Rebaudioside D can be synthesized by bioenzymatic method, starting from stevia leaf extract, using glucose transferase. This biosynthesis requires the addition of expensive UDP-glucose as one of the substrates. The process involves the action of UDP-glucosyltransferase (UGT), which catalyzes the production of rebaudioside D using stevioside or rebaudioside A as substrates. However, due to the extremely high price of UDP-glucose, the feasibility of industrial preparation of rebaudioside D is almost completely restricted, with poor economic benefits and lack of market competitiveness.

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

[0023] Rebaudioside M (Reb M, RM for short) has attracted more attention due to its better taste characteristics and more expensive price. However, its content in the dry weight of leaves is even lower than that of rebaudioside D, usually less than about 0.1%, resulting in high separation cost and high price. The method of obtaining high-concentration rebaudioside M by biocatalysis has attracted the attention of scholars. It has been reported that stevioside-derived recombinant enzymes can catalyze rebaudioside D to synthesize rebaudioside M, but the yield is relatively low. Rebaudioside M is obtained by microbial enzyme catalysis using rebaudioside D as a substrate. Compared with traditional extraction methods, this method not only improves the production process, but also reduces environmental pollution and increases the yield of rebaudioside M.

[0024] At present, enzyme catalysis is often used to prepare rebaudioside M. For example, CN113186141B discloses a method for preparing rebaudioside M by enzyme method, which uses sucrose, stevioside (ST) and uridine diphosphate (UDP) as raw materials. First, sucrose and UDP are catalyzed by sucrose synthase from Arabidopsis to generate UDPG, and UDPG and ST are catalyzed by glycosyltransferase from stevia to generate rebaudioside A; secondly, UDPG and rebaudioside A are catalyzed by glycosyltransferase from rice to generate rebaudioside D; finally, UDPG and rebaudioside D are catalyzed by glycosyltransferase from stevia to generate rebaudioside M. The yield of rebaudioside M is only about 37.9%, which is low and difficult to meet the demand for the yield of rebaudioside M in industrial production. The current enzyme catalytic methods for rebaudioside M have the following main problems: (1) The cost of producing rebaudioside M from rebaudioside D using enzyme catalysis is high, and the enzyme catalytic yield needs to be further optimized; (2) The glycosyltransferase used for catalysis is difficult to separate and recover from the product, is easily inactivated, and needs to be supplemented, which increases the cost; (3) The content of rebaudioside A is very high, while the content of D is very low. It is also a challenge to directly convert rebaudioside A into rebaudioside D at low cost.

[0025] At present, the enzymes used for preparing steviol glycosides by bioenzymatic method are mainly wild-type enzymes derived from plant cells. These wild-type enzymes usually have disadvantages such as low enzyme activity and poor stability, resulting in high industrial-scale production costs of steviol glycosides. Therefore, it is necessary to improve the glucosyltransferase to obtain modified enzymes with higher enzyme activity and better stability, so as to better serve industrial-scale production. Summary of the invention

[0026] In order to overcome the defects of slow conversion rate and low yield in the preparation of required rebaudioside products such as rebaudioside D and rebaudioside M by enzyme catalysis in the prior art, the present invention provides a preparation method for greatly improving the required rebaudioside products such as rebaudioside D and rebaudioside M and the application of β-1,2-glucosyltransferase therein. The present invention improves the yield of the intermediate product rebaudioside D when β-1,2-glucosyltransferase catalyzes rebaudioside A to generate rebaudioside M, and then β-1,2-glucosyltransferase, β-1,3-glucosyltransferase and sucrose synthase are used in combination, which is conducive to improving the yield of the final product rebaudioside M.

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

[0028] In a first aspect, 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 as shown in SEQ ID NO:4.

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

[0030] In some embodiments, compared with 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 group consisting of the following amino acid residue differences: M181L; F185L; E188F; A196V; A198F; G201P; R373K; L375M; L325F; V326A. For another example, compared with 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 comprises 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 comprises 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 comprises 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 comprises 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 shown as SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:22.

[0036] The second aspect of the present invention provides an isolated nucleic acid molecule encoding 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 in SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

[0038] The third aspect of the present invention provides a recombinant expression vector, which comprises a backbone vector, and the isolated 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 plasmid comprises pET28a or pET21a.

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

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

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

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

[0044] The fifth aspect of the present invention provides a method for preparing the 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 condition is: shaking culture at a temperature of 37±1°C.

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

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

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

[0050] In the present invention, wet bacteria refer to the precipitate obtained by centrifuging the culture fluid of bacteria expressing glycosyltransferase and discarding the supernatant. Bacterial powder refers to the powder obtained by drying the above wet bacteria to remove the solvent water and crushing it. Liquid enzyme (such as crude enzyme solution) refers to the enzyme solution after resuspending the above wet bacteria in a certain proportion with a solvent (such as water), preferably the supernatant obtained after the resuspended enzyme solution is homogenized at a certain pressure and centrifuged at a certain speed. Reaction enzyme solution refers to a liquid in which the liquid enzyme is kept at a constant temperature of 80±10°C for a period of time to remove impurities. Solid enzyme powder refers to the powder obtained after drying and crushing the above liquid enzyme. Immobilized enzyme refers to the enzyme component in the above liquid enzyme fixed on an immobilized carrier. Immobilized carriers include resins, such as epoxy resins, amino resins, and adsorption resins. More specifically, epoxy resins can be ECHFA, ReliZymeTMHFA403, ReliZymeTMEP113, ReliZymeTMEP403 and / or ECEP.

[0051] In some embodiments, the amount of rebaudioside 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 embodiments, 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 bacteria, and the added mass ratio of the wet bacteria to the rebaudioside A in the reaction system can be (0.1-0.8):1, or (0.12-0.6):1, or (0.24-0.3):1.

[0057] In the present invention, the added mass refers to the initial mass of components such as wet bacteria or substrates when added into the reaction system.

[0058] In the present invention, an example of preparing the crude enzyme solution is to resuspend the wet bacteria expressing the β-1,2-glucosyltransferase using a buffer at a ratio of 1:5 (g / mL), then homogenize the resuspension at 550 bar for 1.5 min (to break the bacteria), and after centrifugation at 12000 rpm for 2 min, take the supernatant to obtain the crude enzyme solution.

[0059] In the present invention, the buffer may be a phosphate buffer with a pH value of 6.0.

[0060] In some embodiments, the amount of wet bacteria 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 in the form of 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 bacteria expressing the β-1,2-glucosyltransferase; the reaction enzyme solution is obtained by keeping the crude enzyme solution at a constant temperature of 80±10°C for a period of time.

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

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

[0064] The seventh aspect of the present invention provides an enzyme combination system, which includes: the 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 produce 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 produce rebaudioside M.

[0065] In some embodiments, the β-1,2-glucosyltransferase is used in the form of wet cells, such as the second wet cells; the β-1,3-glucosyltransferase is used in the form of wet cells, such as the third wet cells. The relevant definitions of the wet cells are as described above.

[0066] In some embodiments, the enzyme combination system further comprises: a glycosyl donor synthetase, which can catalyze the reaction of sugars and NDP to generate the glycosyl donor. In some embodiments, the glycosyl donor synthetase is used in the form of wet bacteria, for example, called the first wet bacteria.

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

[0068] In some embodiments, the glycosyl donor synthetase is obtained by fermentation culture of a first transformant, wherein the first transformant is a genetically recombinant cell obtained by introducing the nucleotide sequence shown in SEQ ID NO: 1 into a host cell, or a genetically recombinant cell capable of expressing the amino acid sequence 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 genetically recombinant cell obtained by introducing the isolated nucleic acid molecule described in the second aspect or the recombinant expression vector 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 genetically recombinant cell obtained by introducing the nucleotide sequence shown in SEQ ID NO:23 into a host cell, or a genetically recombinant cell capable of expressing the amino acid sequence shown in SEQ ID NO:24.

[0071] In some embodiments, the first transformant is a strain numbered RMIS609 of Yikelai 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 embodiments, the third transformant is a strain numbered RMIS109 of Yikelai Biotechnology (Shanghai) Co., Ltd.

[0074] The eighth aspect of the present invention provides a method for preparing rebaudioside M, which comprises: a step of forming an enzyme catalytic system with rebaudioside A, a glycosyl donor and the 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 with rebaudioside 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 step-by-step 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 is used in the form of wet bacteria, bacterial powder, liquid enzyme, solid enzyme powder or immobilized enzyme.

[0077] In some embodiments, in the enzyme catalysis system, the added amount of rebaudioside A 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 embodiments, 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 bacteria. In the enzyme catalysis system, the added mass ratio of the wet bacteria to the rebaudioside A can be (0.1-0.8):1, (0.12-0.6):1, or (0.24-0.3):1.

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

[0084] In some embodiments, the β-1,2-glucosyltransferase is used in the form of 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 keeping the crude enzyme solution at a constant temperature of 80±10°C 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 bacteria with a solvent, and the ratio of the added mass of the wet bacteria to the added volume of the solvent is 1 g: (5-10) mL.

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

[0087] In some embodiments, the wet bacteria expressing the β-1,2-glucosyltransferase or the liquid enzyme containing the β-1,2-glucosyltransferase is 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 bacteria, and the added mass ratio of the wet bacteria to the rebaudioside A in the enzyme catalysis system can be (0.1-0.8):1, or (0.12-0.6):1, or (0.24-0.3):1.

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

[0090] In some embodiments, the β-1,3-glucosyltransferase is used in the form of 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 bacterial cell expressing the β-1,3-glucosyltransferase; the reaction enzyme solution is obtained by keeping the crude enzyme solution at a constant temperature of 80±10°C 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 bacteria with a solvent, and the ratio of the added mass of the wet bacteria to the added volume of the solvent is 1 g: (5-10) mL.

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

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

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

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

[0096] In some embodiments, the glycosyl donor synthetase is used in the form of 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 synthetase; the reaction enzyme solution is obtained by keeping the crude enzyme solution constant at 80±10° C. for a period of time. In some embodiments, the glycosyl donor synthetase is sucrose synthase.

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

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

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

[0100] The ninth aspect of the present invention provides a 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 preparing 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, which includes: rebaudioside A, a glycosyl donor, rebaudioside D, and the β-1,2-glucosyltransferase described in the first aspect.

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

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

[0104] The eleventh aspect of the present invention provides an enzyme-catalyzed end product system, which includes: rebaudioside A; rebaudioside D; rebaudioside M; a β-1,2-glucosyltransferase capable of catalyzing rebaudioside A and a glycosyl donor to generate the rebaudioside D, the β-1,2-glucosyltransferase being the β-1,2-glucosyltransferase described in the present invention; and a β-1,3-glucosyltransferase capable of catalyzing rebaudioside D and the glycosyl donor to generate the rebaudioside M.

[0105] In some embodiments, in the enzyme-catalyzed final 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, in the enzyme-catalyzed final product system, the content of rebaudioside A accounts for less than or equal to 0.88% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, in the enzyme-catalyzed final product system, the content of rebaudioside A accounts for less than or equal to 0.36% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

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

[0108] In some embodiments, in the enzyme-catalyzed final product system, the content of rebaudioside M accounts for greater than or equal to 92.22% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, in the enzyme-catalyzed final product system, the content of rebaudioside M accounts for 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] A fifteenth aspect of the present invention provides a composition comprising:

[0113] Rebaudioside A; Rebaudioside D; Rebaudioside 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, in the composition, the content of rebaudioside A accounts for less than or equal to 0.88% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, in the composition, the content of rebaudioside A accounts for less than or equal to 0.36% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M.

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

[0117] In some embodiments, in the composition, the content of rebaudioside M accounts for greater than or equal to 92.22% of the total content of rebaudioside A, rebaudioside D, and rebaudioside M. Further, in the composition, the content of rebaudioside M accounts for 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) providing a source of steviol glycosides;

[0120] (b) providing a glycosyltransferase;

[0121] (c) providing a glycosyl donor or a glycosyl donor production system;

[0122] (d) preparing a reaction mixture using (i) steviol glycoside, (ii) glycosyltransferase and (iii) a glycosyl donor or a glycosyl donor production system, and reacting at a temperature between about 30° C. and 90° C.; wherein the above steps (a) to (c) are carried out in no particular order;

[0123] (e) causing the distribution change of the target steviol glycoside to reach 50% or more within 2 hours to form a precipitate containing the target steviol glycoside, more preferably, reaching 60%, 70%, 80% or 90% or more within 2 hours; optionally, further comprising:

[0124] (f) separating and purifying a 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 means that when the prepared product is mainly Reb D, the target steviol glycoside is Reb D; when the prepared product is mainly Reb M, the target steviol glycoside is Reb M; when the prepared product is mainly a mixture of Reb D and Reb M, the target steviol glycoside is Reb D and Reb M.

[0126] In some embodiments, the source of steviol glycosides is selected from one or more of the following groups: stevia leaf extract, rubusoside, stevioside, rebaudioside A and rebaudioside D.

[0127] In some embodiments, the steviol glycosides contain rebaudioside A in an amount of at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 97 wt%, 98 wt% or more.

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

[0129] In some embodiments, the β-1,2 glycosyltransferase is a glycosyltransferase that performs β-1,2 glycosylation on C2′ of 13-O glucose, 19-O glucose, or 13-O glucose and 19-O glucose in steviol glycosides; the β-1,3-glycosyltransferase is a glycosyltransferase that performs β-1,3-glycosylation on 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] In some embodiments, wherein the glycosyl donor comprises an NDP-sugar; the NDP-sugar is selected from ADP-glucose, GDP-glucose, CDP-glucose, UDP-glucose, TDP-glucose, IDP-glucose or a combination thereof.

[0132] In some embodiments, the glycosyl donor generating system comprises sucrose, a glycosyl donor synthetase, and an NDP.

[0133] In some embodiments, the glycosyl donor synthetase is thermostable.

[0134] In some embodiments, at the beginning 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;

[0135] and / or the concentration of sucrose is 100 g / L to 200 g / L, preferably 130 g / L to 140 g / L;

[0136] and / or the concentration of steviol glycosides is between 20 g / L and 120 g / L, preferably 70 g / L to 80 g / L;

[0137] and / or the reaction temperature is 55°C to 65°C, preferably 60°C;

[0138] And / or the reaction pH is 5.0 to 8.0, preferably 6.0 to 6.2.

[0139] In some embodiments, the pH is achieved by adding a food grade acid.

[0140] In some embodiments, the food grade acid is selected from the group consisting of phosphoric acid, acetic acid, and citric acid.

[0141] In some embodiments, the amino acid sequence of the β-1,3-glycosyltransferase is at least about 90% identical to 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.

[0142] 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.

[0143] In some embodiments, the amino acid sequence of the glycosyl donor synthetase is at least about 90% identical to SEQ ID NO:2.

[0144] In some embodiments, the nucleotide sequence encoding the glycosyl donor synthetase gene has at least 90% identity with SEQ ID NO:1.

[0145] In some embodiments, the change in steviol glycoside distribution reaches more than 85% within 30 minutes to 4 hours after preparing the reaction mixture.

[0146] In the seventeenth aspect of the present invention, there is provided a consumable comprising rebaudioside D and / or rebaudioside M, which is prepared by the method described in the sixteenth aspect of the present invention.

[0147] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0148] The reagents and raw materials used in the present invention are commercially available.

[0149] The positive and progressive effect of the present invention is that the present invention adopts an enzyme catalysis method to catalyze the reaction of rebaudioside A and a glycosyl donor to generate rebaudioside D, and then generate rebaudioside M. Since the present invention optimizes the β-1,2-glucosyltransferase, the yield of the intermediate product rebaudioside D when the enzyme catalyzes rebaudioside A to generate rebaudioside M is increased, which is conducive to increasing the yield of the final product rebaudioside M. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 This is the reaction route for the synthesis of Reb M; wherein, β-1,2-GT: β-1,2-glycosyltransferase; β-1,3-GT: β-1,3-glycosyltransferase; SUS: sucrose synthase.

[0151] Figure 2 Figure 2 is the distribution of RebA, RebD and RebM in the reaction over time. DETAILED DESCRIPTION

[0152] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0153] The following three enzymes were used for the enzymatic conversion processes in the following examples.

[0154] 1. In the presence of uridine diphosphate glucose (UDPG) or adenosine diphosphate glucose (ADPG), β-1,2-glycosyltransferase promotes the glucose transfer reaction. Reb A (SvG1G3) is converted to Reb D (SvG2G3), in which an additional glucose unit is covalently linked to the C-19 position of the steviol aglycone by a β-1,2-covalent bond;

[0155] 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);

[0156] 3. In the presence of sucrose, the glucose group is transferred to uridine diphosphate ("UDP") or adenosine diphosphate ("ADP") by sucrose synthase to produce UDPG or ADPG. The reaction is reversible and the UDP-UDPG cycle reaction can be repeated.

[0157] When the glycosyl transfer reactions mediated by β-1,3-glycosyltransferase and β-1,2-glycosyltransferase are coupled with the UDP-UDPG or ADP-ADPG cycle reactions mediated by sucrose synthase, efficient production of Reb M can be achieved. Figure 1 shown.

[0158] Changes in Stevioside Distribution:

[0159] As used herein, "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 the 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%.

[0160] When producing high Reb M products, a useful steviol glycoside distribution change is that the distribution of the target steviol glycosides reaches more than 50% within 2 hours, preferably, reaches more than 60%, 70%, 80%, 90% within 2 hours.

[0161] The target profile will 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.

[0162] Compared with conventional reactions, the present invention is intended to achieve target distribution in a shorter time. In some cases, the target distribution of steviol glycosides is achieved in 8 hours or less, more preferably in about 4 hours after the reaction starts.

[0163] The reagents used in the present invention are as follows:

[0164] Rebaudioside A (RA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0165] RA60 was purchased from Zhucheng Haotian Pharmaceutical Co., Ltd.

[0166] Sucrose was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0167] E. coli Trans10 competent cells and E. coli BL21 (DE3) competent cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0168] The composition of LB liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0169] The composition of TB liquid medium: 2% tryptone, 2.4% yeast extract, 72mM K 2 HPO 4 , 17mMKH 2 PO 4, 0.4% glycerol.

[0170] pET28a plasmid and recombinant plasmid were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0171] The HPLC detection method is as follows:

[0172] Chromatographic column: Diamonsil C18 (2) (4.6 mm × 250 mm, 5 μm); Mobile phase A: weigh 1.5 g of sodium dihydrogen phosphate (NaH 2 PO 4 ), dissolved in 1000 mL of water, adjusted to pH 2.6 with phosphoric acid; mobile phase B: acetonitrile; gradient elution according to the conditions in Table 2. Detection wavelength: 210 nm; flow rate: 0.8 mL / min; injection volume: 20 μL; column temperature: 40°C. RA retention time: 21.905 min; RD retention time: 12.614 min; RM retention time: 16.458 min.

[0173] Table 2 HPLC gradient elution conditions

[0174] Time (minutes) Mobile phase A% Mobile phase B% 0.00 75 25 13.0 75 25 15.0 68 32 27.0 68 32 40.0 30 70 45.0 30 70 45.5 75 25 60.0 75 25

[0175] Among them, when RA is used as substrate to generate the final product RM, the actual concentration of RM generated (C RM ) was calculated based on the external standard method and standards.

[0176] The calculation method of RM yield (RM%) is:

[0177]

[0178] If RA is used as a substrate to generate RD, the actual concentration of RD generated (C RD ) and the yield of RD (RD%) were calculated similarly.

[0179] Example 1: Preparation of GT609 reaction enzyme solution

[0180] The present embodiment relates to the preparation method of GT609 reaction enzyme liquid.GT609 belongs to sucrose synthase (Sucrosesynthase, SUS, also referred to as SuSy / SS, EC2.4.1.13), which can catalyze the transfer of hexose groups, and the reaction equation is: NDP+sucrose→NDP-glucose+D-fructose, so it belongs to one of glycosyltransferases.NDP is the abbreviation form of nucleoside diphosphate (Nucleoside diphosphate), including adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP) and inosine diphosphate (IDP) etc.Correspondingly, NDP-glucose includes ADP-glucose, UDP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and IDP-glucose etc.NDP-glucose can be used as the glycosyl donor of subsequent reaction.

[0181] GT609 of this example comes from enzyme 14 (Enz.14) described in paragraph 0128 of the invention patent 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.

[0182] The method for obtaining the GT609 reaction enzyme solution provided in this embodiment is as follows:

[0183] 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.

[0184] 1.2. The recombinant plasmid pET28a-GT609 was transformed into the host 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 the genetically engineered strain was picked and inoculated into 5 mL LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C for 4 hours. Then, the inoculation amount was transferred to 50 mL of freshly prepared TB liquid culture medium containing 50 μg / mL kanamycin at 2 v / v% (volume percentage), and cultured at 37°C with shaking until OD600 reached about 0.8, IPTG was added to a final concentration of 0.1 mM, and induced culture was performed at 25°C for 20 hours. After the culture was completed, the culture solution was centrifuged at 4000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was collected to obtain RMIS609 wet bacteria. It was stored at -20°C for use.

[0185] 1.3. The collected RMIS609 wet cells were suspended in PBS (50mM, pH 6.0) at 1g:5mL (mass-to-volume ratio, i.e., M / V, also known as homogenization ratio) to obtain a suspension. The suspension was then homogenized using a high-pressure homogenizer to obtain a homogenized enzyme solution. The homogenization condition was homogenization at a pressure of 550MPa for 1.5min. The homogenized enzyme solution was centrifuged at 12000rpm for 2min, the precipitate was discarded, and the supernatant was taken to obtain a GT609 crude enzyme solution.

[0186] 1.4. The GT609 crude enzyme solution was kept at a constant temperature of 80°C for 15 minutes to obtain the GT609 reaction enzyme solution.

[0187] Example 2: Preliminary screening of β-1,2-glucosyltransferase

[0188] β-1,2-glucosyltransferase (β-1,2-GT) can catalyze the reaction between rebaudioside A and glycosyl donor to produce rebaudioside D (RD).

[0189] After screening the enzymes in the own enzyme library (the enzyme screening reaction system is shown in Table 3, wherein the preparation method of the crude enzyme solution of β-1,2-glucosyltransferase and the reaction enzyme solution is the same as in Example 1), β-1,2-glucosyltransferase GT708 with high catalytic activity for the reaction of rebaudioside A to generate rebaudioside D was finally screened out. 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.

[0190] The preparation methods of the crude GT708 enzyme solution and the GT708 reaction enzyme solution provided in this example are the same as those in Example 1, except that the obtained recombinant plasmid pET28a-GT708 is obtained. The obtained genetic engineering strain containing the recombinant plasmid pET28a-GT708 is recorded as RMIS708.

[0191] Table 3 Composition of enzyme screening reaction system

[0192]

[0193] In Table 3, the mother solution concentration refers to the initial concentration of each component at the beginning of addition. The stock solution refers to the GT609 reaction enzyme solution or the β-1,2-glucosyltransferase reaction enzyme solution added in an undiluted state. The final concentration refers to the initial concentration of each component in the entire reaction system after addition, which is the concentration when the reaction is 0h. NDP (ADP or UDP) refers to the reaction system using ADP and the reaction system using UDP prepared using different NDPs, so Table 3 includes two reaction systems.

[0194] The reaction mechanism of each substance in Table 3 is as follows. Sucrose and NDP generate NDP-glucose and D-fructose under the action of sucrose synthase (SUS) in the GT609 reaction enzyme solution. NDP-glucose (as a glycosyl donor) and rebaudioside A (RA) generate rebaudioside D (RD) under the action of β-1,2-glucosyltransferase (β-1,2-GT) in the β-1,2-glucosyltransferase reaction enzyme solution.

[0195] The enzyme screening method in this embodiment comprises the following steps:

[0196] The reaction system was prepared according to Table 3. The reaction system was placed in a metal bath (the heating temperature was set to 60° C.) and reacted at 60° C. for 30 min to obtain a reaction solution. The reaction solution was diluted 50 times and then sampled for HPLC analysis.

[0197] According to the HPLC test results and the dilution multiple of the reaction solution, GT708 was the best among the β-1,2-glucosyltransferases. For the reaction system using ADP, the yield of RD was 66.606%. For the reaction system using UDP, the yield of RD was 28.842%.

[0198] Example 3: Mutation of β-1,2-glucosyltransferase GT708

[0199] According to the results of Example 2, when sucrose and NDP are used as the reaction starting materials, when RD is prepared by using GT708 reaction enzyme solution and GT609 reaction enzyme solution together, the content of RD in the reaction solution is low. In order to further improve the yield of RD, this example mutates GT708, and the mutation method includes the following steps:

[0200] 3.1. The primers shown in Table 4 were designed using the nucleotide sequence of GT708 as a template.

[0201] Table 4 Mutation primers for GT708

[0202]

[0203] In Table 4 above, F represents a forward primer and R represents a reverse primer. The numbers after F and R are primer numbers, which are used to distinguish different primers. The enzyme number represents the number of the mutant enzyme obtained after mutation.

[0204] 3.2. PCR amplification was performed using F10-13 / R9 and F9 / R10-13 as primers, and two fragments were obtained for each pair of primers. The two-fragment homologous recombinase of Nanjing Novozyme Biotechnology Co., Ltd. was used for recombination and transformed into E. coli Trans 10 competent cells. The transformed E. coli Trans 10 competent cells were spread on the LB medium of the corresponding resistance and inverted and cultured at 37°C overnight; a single colony was picked into an LB test tube (Km resistance), cultured for 8 to 10 hours, and the plasmid was extracted for sequencing identification. The sequencing results are shown in Table 5 below.

[0205] Table 5 Sequencing results of four enzyme mutants based on GT708

[0206]

[0207]

[0208] Table 5 shows the mutation sites of the four enzyme mutants compared with the amino acid sequence of GT708. Among them, D323N means that the amino acid at position 323 of the amino acid sequence of GT828 changes from D (aspartic acid) to N (asparagine) relative to GT708, and the other mutation sites are represented similarly. The dash "-" is a separator that separates different mutation sites.

[0209] 3.3. The four plasmids with correct sequencing were transformed into host E. coli BL21 (DE3) competent cells to obtain four genetically engineered strains containing point mutations as shown in Table 5. Single colonies of genetically engineered strains were picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C for 4 h. The inoculation volume was transferred to 50 mL of freshly prepared TB liquid medium containing 50 μg / mL kanamycin at 2 v / v% (volume ratio), and cultured at 37°C with shaking until OD 600 When the pH value reached about 0.8, IPTG was added to a final concentration of 0.1 mM and induced for 20 h at 25°C. After the culture was completed, the culture solution was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet cells, which were stored at -20°C for later use.

[0210] 3.4. The collected wet cells were suspended in PBS (50mM, pH 6.0) at 1:5g / mL (mass-to-volume ratio, such as m / v) to obtain four suspensions. The four suspensions were then homogenized using a high-pressure homogenizer (550Mbar homogenization for 1.5min) to obtain four homogenized enzyme solutions. The four homogenized enzyme solutions were centrifuged at 12000rpm for 2min, the precipitate was discarded, and the supernatant was collected to obtain four crude enzyme solutions, namely GT828 crude enzyme solution, GT829 crude enzyme solution, GT830 crude enzyme solution, and GT831 crude enzyme solution.

[0211] 3.5. The above four crude enzyme solutions were kept at 80°C for 15 minutes to obtain GT828 reaction enzyme solution, GT829 reaction enzyme solution, GT830 reaction enzyme solution and GT831 reaction enzyme solution. All of these four reaction enzyme solutions contain β-1,2-glucosyltransferase.

[0212] Example 4: Preparation of rebaudioside D using four reaction enzyme solutions and GT609 reaction enzyme solution

[0213] In this example, the reaction enzyme solutions of four enzyme mutants related to GT708 (containing β-1,2-GT) and the reaction enzyme solution of GT609 (containing SUS) were used to construct reaction systems for preparing rebaudioside D. The contents of the components in the four reaction systems are shown in Table 6.

[0214] Table 6 Composition of the reaction system for preparing rebaudioside D

[0215]

[0216]

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

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

[0219] Eight reaction systems were prepared according to Table 6. Each reaction system was placed in a metal bath (the heating temperature was set to 60°C) and reacted at 60°C for 30 minutes to obtain eight reaction solutions. The eight reaction solutions were diluted 50 times and then sampled for HPLC analysis. At the same time, a reaction system containing GT708 was set 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 contained in the reaction solution obtained after the reaction is terminated to the volume (L) of the reaction solution. The yield (ADP) of RD refers to the yield of RD after the reaction is completed when the reaction system uses ADP (without UDP). The yield (UDP) of RD refers to the yield of RD after the reaction is completed when the reaction system uses UDP.

[0223] It can be seen from Table 7 that the yield of RD in the reaction system using ADP is higher than that in the reaction system using UDP. In addition, for GT829, whether in the reaction system using ADP or in the reaction system using UDP, the yield of RD is significantly higher than that of other enzymes (GT828, GT830, GT831), therefore, GT829 was selected for the preparation of RM.

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

[0225] This embodiment relates to a method for preparing a GT109 reaction enzyme solution. GT109 belongs to β-1,3-glucosyltransferase (β-1,3-GT for short). β-1,3-glucosyltransferase can transfer the glucosyl group (Glucosyl group, Glc for short) on the glycosyl donor to the glycosyl group at the C-19 position of rebaudioside D using rebaudioside D as a substrate, 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 of this example is derived from Enz.24 described in paragraph 0130 of the invention patent application specification with 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 reaction enzyme solution provided in this example is the same as that in Example 1, except that the obtained recombinant plasmid pET28a-GT109 is obtained. The obtained genetic engineering strain containing the recombinant plasmid pET28a-GT109 is recorded as RMIS109.

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

[0229] Example 6: Preparation of rebaudioside M using GT109 reaction enzyme solution, GT829 reaction enzyme solution and GT609 reaction enzyme solution

[0230] In this example, GT109 reaction enzyme solution (containing β-1,3-glucosyltransferase), GT829 reaction enzyme solution (containing β-1,2-glucosyltransferase) and GT609 reaction enzyme solution (containing sucrose synthase) were used to prepare rebaudioside M. GT109 reaction enzyme solution was obtained from Example 5, GT829 reaction enzyme solution was obtained from Example 3, and GT609 reaction enzyme solution was obtained from Example 1.

[0231] This example constructs a reaction system for preparing rebaudioside M. The contents of the components in the reaction system are shown in Table 8.

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

[0233]

[0234]

[0235] The reaction mechanism of the reaction system shown in Table 8 is as follows. Sucrose and NDP generate NDP-glucose (ADP-glucose or UDP-glucose) and D-fructose under the action of sucrose synthase (SUS) in the GT609 reaction enzyme solution. Rebaudioside A (RA) and NDP-glucose (as a glycosyl donor) generate rebaudioside D (RD) under the action of β-1,2-glucosyltransferase (β-1,2-GT) in the GT829 reaction enzyme solution. Rebaudioside D (RD) and NDP-glucose (as a glycosyl donor) generate rebaudioside M (RM) under the action of β-1,3-glucosyltransferase (β-1,3-GT) in the GT109 reaction enzyme solution.

[0236] In Table 8, NDP (ADP or UDP) refers to a reaction system using ADP and a reaction system using UDP, respectively. Therefore, Table 8 includes two reaction systems in total.

[0237] The preparation method of rebaudioside M in this embodiment comprises the following steps:

[0238] Two reaction systems were prepared according to Table 8. Each reaction system was placed in a metal bath (the heating temperature was set to 60°C) and reacted at 60°C for 6h or 22h. After the reaction was terminated, two reaction solutions were obtained. The two reaction solutions were diluted 50 times and then sampled for HPLC analysis. The HPLC analysis results were processed and are shown in Table 9.

[0239] Table 9 Content of each product in the reaction solution

[0240]

[0241] In Table 9, the reaction system using ADP refers to a reaction system in which one of the substrates of sucrose synthase is ADP (not UDP), and therefore, the glycosyl donor is ADP-glucose. The reaction system using UDP refers to a reaction system in which one of the substrates of sucrose synthase is UDP (not ADP), and therefore, the glycosyl donor is UDP-glucose. The concentration of RA refers to the ratio (g / L) of the mass (g) of RA contained 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 similar. The percentage content of RA refers to the percentage of the mass of RA contained in the enzyme-catalyzed end product system obtained after the reaction is terminated to the total mass of RA, RD and RM. The percentage content of RD and RM is similar.

[0242] According to the results in Table 9, in the reaction system using ADP, the yield of RM reached 94.77% after 6 hours of reaction. In the reaction system using UDP, the yield of RM% reached 91.12% after 22 hours of reaction, but it was still lower than the yield of RM after 6 hours of reaction in the reaction system using ADP. This shows that for GT829, the yield of RM when ADP-glucose is used as the glycosyl donor is higher than the yield of RM when UDP-glucose is used as the glycosyl donor.

[0243] The addition amounts of various embodiments of the present invention are shown in Table 10.

[0244] Table 10 Addition amount table of various embodiments of the present invention

[0245]

[0246] Example 7 Preparation of RebM by Enzymatic Amplification Reaction Using RA97 as Raw Material

[0247] 11.3g of β-1,3-glucosyltransferase GT109 bacterial sludge (produced by 5L scale fermentation) was homogenized with pH 7.0 PBS buffer to obtain 50g of enzyme solution; 7.7g of sucrose synthase GT609 bacterial sludge (produced by 5L scale fermentation) was homogenized with pH 7.0 PBS buffer to obtain 80g of enzyme solution; 6.4g of β-1,2-glucosyltransferase GT829 bacterial sludge (produced by 5L scale fermentation) was homogenized with pH 7.0 PBS buffer to obtain 61g of enzyme solution.

[0248] Add 200g water to a 500mL reactor, start stirring, and heat to 60°C. Add 15g RA97 and 26.5g sucrose respectively, stir until dissolved, and keep the temperature above 58°C during the dissolution process. Then add 2.7g GT109 enzyme solution, 1.5g GT609 enzyme solution and 1.5g GT829 enzyme solution, and 0.075g ADP. Adjust the pH to 6.0-6.2 with 25% dilute phosphoric acid, using about 1.5g, and then start the reaction.

[0249] During the reaction, the distribution of steviol glycoside components was detected by HPLC (JECFA, 2021). The product began to precipitate after about 1.5 h of reaction.

[0250] The present invention conducted multiple batches of reactions, and two batches (SG00120240510 and SG00120240511) respectively obtained the data in Table 11 below. In batch SG00120240510, after 1 hour of reaction, the distribution of RebA and steviol glycosides decreased by 91.9%, and 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%, and the distribution of RebD and RebM increased by 97.45%.

[0251] The data showed that as the enzyme reaction time progressed, the major conversion from Reb A to Reb D and Reb M occurred in the first two hours of the reaction time. Figure 2 It shows that Reb A decreased from 98% to less than 1% after 2 hours, and Reb D first increased to more than 25% within 1 hour and then decreased to 2% after 2 hours. Therefore, after 2 hours of reaction, the distribution of Reb M increased to more than 95%, and the distribution of Reb D and Reb M increased to more than 97%. The data on the distribution change of steviol glycosides are shown in Table 14.

[0252] Table 11 Distribution of steviol glycosides at different biotransformation times

[0253]

[0254]

[0255] Example 8 Preparation of Reb D using RA97 as a raw material

[0256] Add 100 g of substrate RA97, 50 g of sucrose, and 0.9 L of water to a 2 L three-necked bottle, stir to dissolve, heat to 60 ° C, and after the materials are completely dissolved, add β-1,2-glucosyltransferase GT829 enzyme solution (6 g of the sludge produced by 5 L scale fermentation is homogenized with 24 mL of pH 7.0 PBS buffer), sucrose synthase GT609 enzyme solution (6 g of the sludge produced by 5 L scale fermentation is homogenized with 24 mL of pH 7.0 PBS buffer), add 0.3 g of ADP, the total reaction volume is about 1 L-1.2 L, adjust the pH to about 6.0-6.2 with 2% phosphoric acid aqueous solution, and start the reaction at 60 ° C.

[0257] During the reaction, the distribution of steviol glycoside components was detected by HPLC (JECFA, 2021).

[0258] The present invention carried out multiple batches of reactions, and two batches (A319-156-1, A319-158-1) respectively obtained the data in the following Table 12. Taking A319-156-1 as an example, after 1 hour of reaction, the distribution of RebA decreased by 74.23%, and the distribution of RebD increased by 74.80%; after 2 hours of reaction, the distribution of RebA decreased by 86.13%, and the distribution of RebD and RebM increased by 86.68%. The data on the change in the distribution of steviol glycosides are shown in Table 14.

[0259] Table 12 Distribution of steviol glycosides at different biotransformation times

[0260]

[0261]

[0262] Example 9 Catalytic preparation of Reb M using RA60 as raw material

[0263] Add 60 ml of water to a 250 ml three-necked flask, control the temperature at 60 ° C, add 10 g of RA60 while stirring, then add 20 g of sucrose, add 0.3 g of ADP after it is dissolved, then adjust the pH to 6.0-6.2 with 10% sodium hydroxide, add β-1,2-glucosyltransferase GT829 enzyme solution (3 g of the sludge produced by 5L scale fermentation is homogenized with 15 mL of pH7.0 PBS buffer), β-1,3-glucosyltransferase GT109 enzyme solution (6 g of the sludge produced by 5L scale fermentation is homogenized with 30 mL of pH7.0 PBS buffer), sucrose synthase GT609 enzyme solution (3 g of the sludge produced by 5L scale fermentation is homogenized with 15 mL of pH7.0 PBS buffer), and adjust the pH to 6.0-6.2 with 25% dilute phosphoric acid to start the reaction.

[0264] During the reaction, the distribution of steviol glycoside components was detected by HPLC (JECFA, 2021). The results are shown in Table 13. After 1 hour of reaction, the distribution of RebA and steviol glycosides decreased by 39.77%, and 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%, and the distribution of RebD and RebM increased by 62.98%. The distribution change data of steviol glycosides is shown in Table 14.

[0265] Table 13 Distribution of steviol glycosides at different biotransformation times

[0266]

[0267]

[0268] As can be seen from Examples 7, 8, and 9, using this process, a high distribution of Reb D, Reb M, and combinations thereof can be achieved in just 1 or 2 hours, making this process very efficient for industrial scale production. The data in Examples 7, 8, and 9 are summarized in the following table.

[0269] Table 14 Distribution changes of steviol glycosides after 1h, 2h and 4h biotransformation

[0270]

[0271]

[0272] Sequences used in the present invention

[0273] Nucleotide sequence of GT609 (SEQ ID NO: 1):

[0274]

[0275] Amino acid sequence of GT609 (SEQ ID NO:2):

[0276] MSERARSDDLIEQLRGFLDACPSVGHRVLHQIKKLERSFLQRSELCDAFADVCAAEDVPDGLRQSPLGKVIRLTQEAAVNDAWVYLAVRVRIASWRYVRIALEGMAVEEVTVRDFLRFKECLALGQHDFDEWLLEIDLGPFSREFPKLLEARSIGRGVEFLNKHLSLQLFDELGEGGERILNFLRIHSFRGQTLMLNEQIKSVSDLRRALRRADEVLAGHEPTATWEDVAAPLRALGFEVGWGREVARIRDTMSLLRDLLEAPDPRGLETFLARLPMVFSLAIISPHGYFGQANVLGRPDTGGQVVYILDQVRALETEMRSRLFEQGLDIEPQIVVLTRLIPQAEGTTCDQRLEPISGTRNARILRVPFRNASGEIVTHWISRFEVWPYLERYTLDSERELLAELGGRPDLIVGNYSDGNLVATLLSQRLGVTQCNIAHALEKTKYPHADLFWQENEAQYHFSCQFTADLIAMNAADFIITSTYQEIAGTRESVGQYESHTAFTMPKLFRVVNGIDVYDPKFNIVSPGADAAAYFPYTAVERRLPHLHTEIEQLVFGVDERVDARGVLTERDKPLLFTMARLDRIKNIVGLVEWFGACEALRKEANLLVISGHVDPERSSDTEELEQIRCMHALFNRYDLDRQVRWLGLRLPKDLAGEFYRYVADGRGAFVQPALFEAFGLTVIEAMASGLPCFATCFGGPSEIIEDGVSGFHIDPNHGDAAAERIARFFERTRQDPEYWNRISEGALKRVAERYTWQHYAERMMTLSRVYGFWRHVTDLERRETQRYLQALYSLQFRRLAQAMA

[0277] Nucleotide sequence of GT708 (SEQ ID NO:3):

[0278]

[0279] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLDHLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0280] The nucleotide sequence of GT828 (SEQ ID NO:15):

[0281]

[0282] Amino acid sequence of GT828 (SEQ ID NO:16):

[0283] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKLREMLGTFPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0284] Nucleotide sequence of GT829 (SEQ ID NO:17):

[0285]

[0286] Amino acid sequence of GT829 (SEQ ID NO:18):

[0287] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLVPFQAPIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0288] Nucleotide sequence of GT830 (SEQ ID NO:19):

[0289]

[0290] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKLVPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINAKLMVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

[0291] The nucleotide sequence of GT831 (SEQ ID NO: 21):

[0292]

[0293] MHHHHEGVSDQTLRVTMFPWLGLGHVNPFLRIAKQLADRGFVIYLVSTAINLEMIKKRIPEKYSNSIHLVELRLPELPELPPHYHTTNGLPPHLNKTLHKALKMSAPNFSKILQNIKPDLVLYDFLVPWAEKVALEQGIPAVPLLTSGAALFSYFFNFLKRPGEEFPFEAIRLSKREQDKMREMFGTEPPEEDFLAPAQAGIMLMCTSRVIEAKYLDYCTELTNVKVVPVGPPFQDPLTEDIDDPELMDWLDTKPEHSVVYVSFGSEAFLSREDMEEVAFGLELSGVNFIWVARFPKGEEQRLEDVLPKGFLERVGDRGRVLNKFAPQAHILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPMQWDQPINARLLVELGVAVEIPRDEDGRVHRAEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

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

[0295]

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

[0297] MPNTNPTTVRRRRVIMFPVPFPGHLNPMLQLANVLYRRGFEITILHTNFNAPKTSLYPHFQFRFILDNDPQPEWLRNLPTTGPGVGARIPVINKHGADEFRKELEICMRDTPSDEEVACVITDALWYFAQPVADSLNLKRLVLQTGSLFNFHCLVCLPKFLELGYLDPETKHRPDEPVVGFPMLKVKDIRRAYSHIQESKPILMKMVEETRASSGVIWNSAKELEESELETIQREIPAPSFLLPLPKHYRASSTSLLDTDPSTAQWLDQQPPSSVLYVGFGSQSSLDPADFLEIARGLVASKQSFLWNVRPGFVKGYEWIELLPDGFLGEKGRIVKSAPQQEVLAHKAIGAFWTHGGWNGTMEAVCEGVPMIFSDFGLDQPLNARYMSEVLHVGVYLENGFIRGEIINAVRRVMVDPEGEVMRQNARKLKDKLDRSIAPGGSSYESLERLESYISS。

Claims

1. A β-1,2-glucosyltransferase, characterized in that The β-1,2-glucosyltransferase has an amino acid sequence as shown in SEQ ID NO: 4, or has a mutant amino acid sequence having at least 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:

4.

2. The β-1,2-glucosyltransferase according to claim 1, characterized in that Compared with 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; Preferably, 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.

3. The β-1,2-glucosyltransferase according to claim 1, characterized in that The mutant amino acid sequence comprises the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, M181L, F185L and E188F; or The mutant amino acid sequence comprises the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, A196V, A198F and G201P; or The mutant amino acid sequence comprises the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, R373K and L375M; or The mutant amino acid sequence comprises the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 4: D323N, H324K, L325F and V326A; or The mutant amino acid sequence is shown in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:

22.

4. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the β-1,2-glucosyltransferase according to any one of claims 1 to 3; Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:

21.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a backbone vector and the isolated nucleic acid molecule according to claim 4; Preferably, the backbone vector comprises a phage vector, a plasmid vector or a viral vector; More preferably, the plasmid vector comprises pET28a or pET21a.

6. A transformant, characterized in that The transformant is a genetically recombinant cell obtained by introducing the isolated nucleic acid molecule according to claim 4 or the recombinant expression vector according to claim 5 into a host cell; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; More preferably, the prokaryotic cell is Escherichia coli, preferably BL21 (DE3); Further preferably, the transformant is selected from any one or more of RMIS708, RMIS828, RMIS829, RMIS830 and RMIS831 of Yikelai Biotechnology (Shanghai) Co., Ltd.

7. A method for preparing the β-1,2-glucosyltransferase according to any one of claims 1 to 3, characterized in that: The method comprises culturing the transformant according to claim 6 in a culture medium, thereby obtaining the β-1,2-glucosyltransferase; Preferably, the culture medium is selected from LB liquid culture medium and / or TB liquid culture medium; and / or The culture conditions are: shaking culture at a temperature of 37±1°C.

8. A method for preparing rebaudioside D, characterized in that: The preparation method comprises the steps of at least forming a reaction system with rebaudioside A, a glycosyl donor and the β-1,2-glucosyltransferase according to any one of claims 1 to 3 and reacting them.

9. The preparation method according to claim 8, characterized in that: The β-1,2-glucosyltransferase is used in the form of wet bacteria, bacterial powder, liquid enzyme, solid enzyme powder or immobilized enzyme; and / or, The amount of rebaudioside A added in the reaction system is 20-120 g / L, preferably 40-110 g / L, more preferably 50-100 g / L; and / or, The glycosyl donor is NDP-glucose; NDP is preferably selected from any one or more of adenosine diphosphate, uridine diphosphate, cytidine diphosphate, guanosine diphosphate, thymidine diphosphate and inosine diphosphate; and / or, the pH of the reaction is 5.0-8.0, preferably 6.0; And / or, the reaction temperature is 20-90°C, preferably 60°C; And / or, the reaction time is 10 minutes to 24 hours, preferably 30 minutes.

10. The preparation method according to claim 9, characterized in that: The β-1,2-glucosyltransferase is used in the form of wet bacteria, and the added mass ratio of the wet bacteria to the rebaudioside A in the reaction system can be (0.1-0.8):1, can be (0.12-0.6):1, can be (0.24-0.3):1; Preferably: The amount of wet cells added to the reaction system may be 6-30 g / L, or 12-24 g / L; and / or, The β-1,2-glucosyltransferase is used in the form of 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 bacteria expressing the β-1,2-glucosyltransferase; the reaction enzyme solution is obtained by keeping the crude enzyme solution at a constant temperature of 80±10°C for a period of time; Further, The crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacteria with a solvent, and the ratio of the added mass of the wet bacteria to the added volume of the solvent is 1 g: (5-10) mL; And / or, the ratio of the added volume of the crude enzyme solution to the added mass of rebaudioside A in the reaction system can be (0.5-5):1, or (0.6-3):1, or (1.2-1.5):1, and the unit is mL / g.

11. An enzyme combination system, characterized in that: The enzyme combination system comprises: The β-1,2-glucosyltransferase according to any one of claims 1 to 3, wherein the β-1,2-glucosyltransferase is capable of catalyzing the reaction of rebaudioside A with a glycosyl donor to produce rebaudioside D; and β-1,3-glucosyltransferase, wherein the β-1,3-glucosyltransferase is capable of catalyzing the reaction of the rebaudioside D with a glycosyl donor to generate rebaudioside M; Preferably, the enzyme combination system also includes: A glycosyl donor synthetase, wherein the glycosyl donor synthetase is capable of catalyzing the reaction of a carbohydrate with an NDP to generate the glycosyl donor; More preferably, the sugar is sucrose, the glycosyl donor is NDP-glucose, and the glycosyl donor synthase is sucrose synthase; Further, The glycosyl donor synthetase is obtained by fermentation culture of a first transformant, wherein the first transformant is a genetically recombinant cell obtained by introducing the nucleotide sequence shown in SEQ ID NO: 1 into a host cell, or a genetically recombinant cell capable of expressing the amino acid sequence shown in SEQ ID NO: 2; and / or The β-1,2-glucosyltransferase is obtained by fermentation culture of the second transformant; the second transformant is a genetically recombinant cell obtained by introducing the isolated nucleic acid molecule according to claim 4 or the recombinant expression vector according to claim 5 into a host cell; and / or The β-1,3-glucosyltransferase is obtained by fermentation culture of a third transformant; the third transformant is a genetically recombinant cell obtained by introducing the nucleotide sequence shown in SEQ ID NO: 23 into a host cell, or a genetically recombinant cell capable of expressing the amino acid sequence shown in SEQ ID NO: 24; Furthermore, The first transformant is a strain numbered RMIS609 of Yikelai Biotechnology (Shanghai) Co., Ltd.; and / or 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.; and / or The third transformant is the strain numbered RMIS109 of Yikelai Biotechnology (Shanghai) Co., Ltd.

12. A method for preparing rebaudioside M, characterized in that: The preparation method comprises: The step of forming an enzyme catalytic system with rebaudioside A, a glycosyl donor and the enzyme combination system as claimed in claim 11 to carry out a reaction; or The steps of forming an enzyme catalytic system with rebaudioside A, a glycosyl donor and β-1,2-glucosyltransferase and reacting them, and then adding β-1,3-glucosyltransferase and continuing the reaction; Further, The β-1,2-glucosyltransferase and / or the β-1,3-glucosyltransferase are used in the form of wet bacteria, bacterial powder, liquid enzyme, solid enzyme powder or immobilized enzyme; And / or, in the enzyme catalysis system, the added amount of rebaudioside A can be 20-120 g / L, 40-110 g / L, or 50-100 g / L; And / or, the glycosyl donor is NDP-glucose; NDP is preferably selected from any one or more of adenosine diphosphate, uridine diphosphate, cytidine diphosphate, guanosine diphosphate, thymidine diphosphate and inosine diphosphate; and / or, the pH of the reaction is 5.0-8.0, preferably 6.0; And / or, the reaction temperature is 20-90°C, preferably 60°C; And / or, the reaction time is 10 minutes to 24 hours, preferably 30 minutes to 6 hours; Furthermore, In the enzyme catalysis system, the added mass ratio of the wet bacteria to the rebaudioside A can be (0.1-0.8):1, can be (0.12-0.6):1, can be (0.24-0.3):1; or, In the enzyme catalysis system, the addition amount of the wet bacteria can be 6-30 g / L, or 12-24 g / L; or, The liquid enzyme is preferably a crude enzyme solution or a reaction enzyme solution; the crude enzyme solution is prepared from wet bacteria expressing the β-1,2-glucosyltransferase and / or the β-1,3-glucosyltransferase; the reaction enzyme solution is obtained by keeping the crude enzyme solution at a constant temperature of 80±10° C. for a period of time; Further, The crude enzyme solution is a homogenized enzyme solution obtained by resuspending and homogenizing the wet bacteria with a solvent, and the ratio of the added mass of the wet bacteria to the added volume of the solvent is 1 g: (5-10) mL; And / or, the ratio of the added volume of the crude enzyme solution to the added mass of rebaudioside A in the enzyme catalysis system can be (0.5-5):1, or (0.6-3):1, or (1.2-1.5):1, and the unit is mL / g.

13. The preparation method according to claim 12, characterized in that: The wet bacteria expressing the β-1,2-glucosyltransferase or the liquid enzyme containing the β-1,2-glucosyltransferase is 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.; and / or The wet bacteria expressing the β-1,3-glucosyltransferase or the liquid enzyme containing the β-1,3-glucosyltransferase is prepared from a third transformant capable of expressing the β-1,3-glucosyltransferase; further, the third transformant is a strain numbered RMIS109 of Yikelai Biotechnology (Shanghai) Co., Ltd.; and / or The wet bacteria expressing the glycosyl donor synthetase in the enzyme combination system or the liquid enzyme containing the glycosyl donor synthetase is prepared from a first transformant capable of expressing the glycosyl donor synthetase; further, the first transformant is a strain numbered RMIS609 of Yikelai Biotechnology (Shanghai) Co., Ltd.

14. Use of the β-1,2-glucosyltransferase according to any one of claims 1 to 3 or the enzyme combination system according to claim 11 in preparing rebaudioside D or rebaudioside M using rebaudioside A and a glycosyl donor as substrates.

15. An enzyme-catalyzed reaction solution, characterized in that: include: Rebaudioside A, a glycosyl donor, rebaudioside D, and the β-1,2-glucosyltransferase according to any one of claims 1 to 3; Preferably, the enzyme-catalyzed reaction solution further comprises β-1,3-glucosyltransferase and rebaudioside M; and / or, the enzyme-catalyzed reaction solution further comprises: sucrose synthase, NDP, sucrose, NDP-glucose and D-fructose.

16. An enzyme catalytic end product system, characterized in that: include: Rebaudioside A; Rebaudioside D; Rebaudioside M ； The β-1,2-glucosyltransferase according to any one of claims 1 to 3; and A β-1,3-glucosyltransferase capable of catalyzing the formation of rebaudioside M from rebaudioside D and a glycosyl donor; Preferably, in the enzyme-catalyzed final 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.

17. A composition, characterized in that include: Rebaudioside A; Rebaudioside D; Rebaudioside M; 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.

18. A method for producing a target steviol glycoside, characterized in that: The method comprises the following steps: (a) providing a source of steviol glycosides; (b) providing a glycosyltransferase; (c) providing a glycosyl donor or a glycosyl donor production system; (d) preparing a reaction mixture using (i) steviol glycoside, (ii) glycosyltransferase and (iii) a glycosyl donor or a glycosyl donor production system, and reacting at a temperature of about 30° C. to 90° C.; wherein the above steps (a) to (c) are carried out in no particular order; (e) making the distribution change of the target steviol glycoside reach 50% or more within 2 hours to form a precipitate containing the target steviol glycoside, more preferably, reaching 60%, 70%, 80% or 90% or more within 2 hours; optionally, further comprising: (f) separating and purifying a precipitate containing the target steviol glycoside from the reaction mixture, wherein the target steviol glycoside is preferably rebaudioside D and / or rebaudioside M.

19. The method according to claim 18, characterized in that The source of the steviol glycoside is selected from one or more of the following groups: stevia leaf extract, rubusoside, stevioside, rebaudioside A and rebaudioside D.

20. The method of claim 19, wherein: The steviol glycosides contain at least 60wt%, 70wt%, 80wt%, 90wt%, 95wt%, 97wt%, 98wt% or more of rebaudioside A.

21. The method of claim 18, wherein: The glycosyltransferase is selected from one or more of the following groups: β-1,2-glycosyltransferase and β-1,3-glycosyltransferase.

22. The method according to claim 21, characterized in that The β-1,2 glycosyltransferase is a glycosyltransferase that performs β-1,2 glycosylation on C2′ of 13-O glucose, 19-O glucose, or 13-O glucose and 19-O glucose in steviol glycosides; the β-1,3-glycosyltransferase is a glycosyltransferase that performs β-1,3-glycosylation on C3′ of 13-O-glucose, 19-O-glucose, or 13-O-glucose and 19-O-glucose in steviol glycosides.

23. The method of claim 21, wherein: The glycosyltransferase is thermostable.

24. The method of claim 18, wherein: The glycosyl donor includes an NDP-sugar.

25. The method of claim 24, wherein: The NDP-sugar is selected from ADP-glucose, GDP-glucose, CDP-glucose, UDP-glucose, TDP-glucose, IDP-glucose or a combination thereof.

26. The method of claim 18, wherein: The glycosyl donor generating system comprises sucrose, glycosyl donor synthetase and NDP.

27. The method of claim 26, wherein: The glycosyl donor synthetase is thermostable.

28. The method of claim 26, wherein: At the beginning 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; and / or the concentration of sucrose is 100 g / L to 200 g / L, preferably 130 g / L to 140 g / L; and / or the concentration of steviol glycoside 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°C to 65°C, preferably 60°C; And / or the reaction pH is 5.0 to 8.0, preferably 6.0 to 6.

2.

29. The method of claim 28, wherein: The pH is achieved by adding food grade acid.

30. The method of claim 29, wherein: The food grade acid is selected from the group consisting of phosphoric acid, acetic acid and citric acid.

31. The method of claim 21, wherein: The amino acid sequence of the β-1,3-glycosyltransferase is at least about 90% identical to SEQ ID NO: 24; and / or the β-1,2-glycosyltransferase is the β-1,2-glucosyltransferase according to any one of claims 1-3.

32. The method of claim 31, wherein: 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 of claim 4.

33. The method of claim 26, wherein: The amino acid sequence of the glycosyl donor synthetase is at least about 90% identical to SEQ ID NO:

2.

34. The method of claim 33, wherein: The nucleotide sequence of the gene encoding the glycosyl donor synthetase has at least 90% identity with SEQ ID NO:

1.

35. The method of claim 18, wherein: The steviol glycoside distribution change amount reaches more than 85% within 30 minutes to 4 hours.

36. A consumable comprising rebaudioside D and / or rebaudioside M, prepared by the method of any one of claims 18-35.

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