Glycosyltransferase Mutants and Their Application in the Synthesis of Reb D

Abstract

The present invention provides a glycosyltransferase, and the amino acid sequence of the glycosyltransferase has one or more amino acid mutations at the 129th, 208th, 20th, 69th, 150th, 238th, 273rd, 287th, and 349th amino acids corresponding to the amino acid sequence of the wild-type glycosyltransferase OsUGT91C1. The glycosyltransferase of the present invention has high enzyme activity, so that it can more effectively catalyze the formation of Reb D from Reb A. When the glycosyltransferase of the present invention is used in combination with sucrose synthase SUS1, the addition of expensive UDPG can be avoided, thereby further reducing the production cost of Reb D. The present invention lays a new material foundation for the production of Reb D.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a glycosyltransferase mutant, a method for constructing the same, and an application thereof in the synthesis of rebaudioside D (Reb D). Background Art

[0002] Stevioside is an important natural sweetener mainly composed of steviol glycosides and has been widely used in Paraguay and Japan. Steviol glycosides have low calories and high sweetness. They not only have no side effects on the human body but also have effects such as lowering blood sugar levels and maintaining dental health. Due to their stability at high temperatures, they have been widely used in baked products. So far, 64 steviol glycosides have been identified from stevia leaves. Among them, stevioside has the highest content, accounting for 5-10% of the dry weight, followed by rebaudioside A, accounting for 2-4% of the dry weight, which is the most widely used steviol glycoside in the current food additive market. However, such products still have disadvantages such as bitter aftertaste, which limits their further commercial applications. Rebaudioside D is a member of the steviol glycoside family. Compared with other steviol glycosides, it has no bitter aftertaste and higher sweetness (about 250-350 times that of sucrose), so it has the potential to become a new generation of high-quality sweeteners.

[0003] However, since the content of rebaudioside D in stevia is very low, the cost of production by plant extraction and purification is huge, and the yield far cannot meet the market demand. Therefore, it is impossible to produce rebaudioside D by directly extracting from plants and only artificial synthesis can be carried out. The artificial synthesis of rebaudioside D requires glycosylation reactions, and there are two methods: chemical synthesis and enzymatic synthesis. Chemical glycosylation usually requires cumbersome protection and deprotection steps and has low specificity and yield. Enzymatic glycosylation shows high efficiency, stereospecificity and regioselectivity, can directly and rapidly generate glycosidic bonds, and is environmentally friendly. So far, many glycosyltransferases from different sources have been identified to have the activity of catalyzing the synthesis of rebaudioside D, but these glycosyltransferases generally have low activity and cannot tolerate high substrate concentrations. And some enzymes have side reactions, which will produce homologues of rebaudioside D, increasing the pressure of downstream purification.

[0004] In view of the problem of low activity of natural enzymes, there is an urgent need in the art for glycosyltransferases with improved catalytic activity and thermal stability for the production of rebaudioside D. Summary of the Invention

[0005] The object of the present invention is to provide a glycosyltransferase mutant with significantly improved activity for synthesizing rebaudioside D and simultaneously significantly improved thermal stability.

[0006] The object of the present invention is also to provide a preparation method of the glycosyltransferase mutant.

[0007] The present invention also aims to provide a method for synthesizing rebaudioside D using the glycosyltransferase mutant.

[0008] In a first aspect, the present invention provides a glycosyltransferase that has one or more mutations at the 129th, 208th, 20th, 69th, 150th, 238th, 273rd, 287th, and 349th amino acids corresponding to the amino acid sequence of the wild-type glycosyltransferase.

[0009] In a preferred embodiment, the wild-type glycosyltransferase is a glycosyltransferase derived from rice (Oryza sativa).

[0010] In a preferred embodiment, the wild-type glycosyltransferase is the glycosyltransferase OsUGT91C1 derived from rice.

[0011] In a preferred embodiment, the amino acid sequence of the glycosyltransferase OsUGT91C1 derived from rice is as shown in SEQ ID NO:3.

[0012] In a preferred embodiment, the glycosyltransferase has a mutation at the 129th position and has 1, preferably 2, more preferably 3, more preferably 4, and most preferably 5 mutations among the 208th, 20th, 69th, 150th, 238th, 273rd, 287th, and 349th positions.

[0013] In a specific embodiment, the glycosyltransferase has a mutation selected from the following group in the amino acid sequence corresponding to the wild-type glycosyltransferase OsUGT91C1:

[0014] 1) The valine at the 129th position is mutated to alanine, leucine, or isoleucine, preferably alanine;

[0015] 2) The phenylalanine at the 208th position is mutated to methionine, leucine, or isoleucine, preferably methionine;

[0016] 3) The cysteine at the 20th position is mutated to phenylalanine, leucine, valine, isoleucine, alanine, or tyrosine, preferably phenylalanine;

[0017] 4) The alanine at the 69th position is mutated to aspartic acid or glutamic acid;

[0018] 5) The leucine at the 150th position is mutated to tryptophan, tyrosine, or phenylalanine, preferably tryptophan;

[0019] 6) The arginine at the 238th position is mutated to glycine, proline, or alanine, preferably glycine;

[0020] 7) The lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine;

[0021] 8) The glycine at position 287 is mutated to serine or threonine, preferably serine;

[0022] 9) The alanine at position 349 is mutated to proline;

[0023] 10) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine;

[0024] 11) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine, valine, isoleucine, alanine or tyrosine, preferably phenylalanine;

[0025] 12) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0026] 13) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0027] 14) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan;

[0028] 15) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine;

[0029] 16) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine;

[0030] 17) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0031] 18) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 349 is mutated to proline;

[0032] 19) The alanine at position 69 is mutated to aspartic acid or glutamic acid; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine;

[0033] 20) The leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine;

[0034] 21) The phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine;

[0035] 22) The phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0036] 23) The phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the alanine at position 349 is mutated to proline;

[0037] 24) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0038] 25) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine;

[0039] 26) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine;

[0040] 27) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid; the leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan;

[0041] 28) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid; the lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine;

[0042] 29) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0043] 30) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the alanine at position 69 is mutated to aspartic acid or glutamic acid; the alanine at position 349 is mutated to proline;

[0044] 31) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine;

[0045] 32) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0046] 33) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the alanine at position 349 is mutated to proline;

[0047] 34) The cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0048] 35) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0049] 36) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan;

[0050] 37) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the arginine at position 238 is mutated to glycine, proline or alanine, preferably glycine;

[0051] 38) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine, proline or alanine, preferably glycine;

[0052] 39) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0053] 40) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the alanine at position 349 is mutated to proline;

[0054] 41) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0055] 42) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan;

[0056] 43) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the arginine at position 238 is mutated to glycine, proline or alanine, preferably glycine;

[0057] 44) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0058] 45) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the alanine at position 349 is mutated to proline;

[0059] 46) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the arginine at position 238 is mutated to glycine, proline or alanine, preferably glycine; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0060] 47) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the arginine at position 238 is mutated to glycine, proline or alanine, preferably glycine; the leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan;

[0061] 48) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the arginine at position 238 is mutated to glycine, proline or alanine, preferably glycine; the glycine at position 287 is mutated to serine or threonine, preferably serine;

[0062] 49) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the arginine at position 238 is mutated to glycine, proline or alanine, preferably glycine; the alanine at position 349 is mutated to proline;

[0063] 50) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the alanine at position 349 is mutated to proline; the alanine at position 69 is mutated to aspartic acid or glutamic acid;

[0064] 51) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the alanine at position 349 is mutated to proline; the leucine at position 150 is mutated to tryptophan, tyrosine or phenylalanine, preferably tryptophan;

[0065] 52) The valine at position 129 is mutated to alanine, leucine or isoleucine, preferably alanine; the cysteine at position 20 is mutated to phenylalanine, leucine or isoleucine, preferably alanine; the phenylalanine at position 208 is mutated to methionine, leucine or isoleucine, preferably methionine; the lysine at position 273 is mutated to glycine or alanine, preferably glycine; the alanine at position 349 is mutated to proline; the glycine at position 287 is mutated to serine or threonine, preferably serine.

[0066] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the glycosyltransferase of the first aspect.

[0067] In a third aspect, the present invention provides an expression vector, and the expression vector contains the nucleic acid molecule described in the second aspect.

[0068] In a preferred embodiment, the expression vector is a plasmid.

[0069] In a fourth aspect, the present invention provides a host cell, and the host cell contains the expression vector described in the third aspect, or the nucleic acid molecule described in the second aspect is integrated into the genome of the host cell.

[0070] In a preferred embodiment, the host cell further contains a coding nucleotide sequence of sucrose synthase SUS1.

[0071] In a preferred embodiment, the sucrose synthase SUS1 is derived from Arabidopsis thaliana.

[0072] In a preferred embodiment, the coding nucleotide sequence of the sucrose synthase SUS1 is as shown in SEQ ID NO:2.

[0073] In a preferred embodiment, the host cell is a production cell of rebaudioside D.

[0074] In a preferred embodiment, the host cell is from the genus Escherichia, Corynebacterium, Brevibacterium sp., Bacillus, Pichia, Saccharomyces, Candida, Serratia or Vibrio.

[0075] In a preferred embodiment, the host cell is Escherichia coli or Corynebacterium glutamicum.

[0076] In a fifth aspect, the present invention provides a combination of host cells, and the combination contains the host cell described in the fourth aspect and a host cell expressing sucrose synthase SUS1.

[0077] In a sixth aspect, the present invention provides a lyophilized powder, which is made from the host cell described in the fourth aspect or the combination of host cells described in the fifth aspect.

[0078] In a seventh aspect, the present invention provides the use of the glycosyltransferase described in the first aspect, or the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect, or the host cell described in the fourth aspect, the combination of the host cells described in the fifth aspect, or the lyophilized powder described in the sixth aspect in the production of rebaudioside D.

[0079] In an eighth aspect, the present invention provides a method for preparing rebaudioside D, the method comprising the following steps:

[0080] a. Culturing the host cell described in the fourth aspect or the combination of the host cells described in the fifth aspect or the lyophilized powder described in the sixth aspect in a culture system to produce rebaudioside D; and

[0081] b. Optionally separating rebaudioside D from the culture solution obtained in a.

[0082] In a preferred embodiment, the culture system in step a further comprises sucrose, Reb A and UDP.

[0083] In a ninth aspect, the present invention provides a method for preparing Reb D, the method comprising the following steps:

[0084] a. Using the glycosyltransferase described in the first aspect to catalyze the formation of Reb D from Reb A in a reaction system; and

[0085] b. Optionally separating Reb D from the above reaction system.

[0086] In a preferred embodiment, the reaction system in step a further comprises UDPG and Reb A.

[0087] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 Shows the reaction process of the glycosyltransferase OsUGT91C1 catalyzing the formation of rebaudioside D from rebaudioside A:

[0089] Figure 2 Shows the relative activity of the mutant (single mutant) of the glycosyltransferase OsUGT91C1;

[0090] Figure 3 Shows the relative activity of the mutant (single mutant and multiple mutants) of the glycosyltransferase OsUGT91C1;

[0091] Figure 4The results of melting temperature determination of the glycosyltransferase OsUGT91C1 mutant are shown;

[0092] Figure 5 The conversion rate of the glycosyltransferase OsUGT91C1 mutant at different rebaudioside A concentrations is shown. DETAILED DESCRIPTION

[0093] After extensive and in-depth research, the inventor unexpectedly discovered a variety of mutants of glycosyltransferase OsUGT91C1. These mutants have one or more mutations at the 129th, 208th, 20th, 69th, 150th, 238th, 273rd, 287th, and 349th amino acids corresponding to the amino acid sequence of the wild-type glycosyltransferase. The efficiency of catalytic synthesis of rebaudioside D by these mutants is significantly improved, and they have excellent thermal stability and significantly improved tolerance to substrates, thereby solving the problem of insufficient yield of rebaudioside D catalyzed by the current enzymatic method, and further providing a method for catalytic synthesis of rebaudioside D by glycosyltransferase mutants. The present invention was completed on this basis.

[0094] Glycosyltransferase of the present invention

[0095] The glycosyltransferase of the present invention is a mutant of OsUGT91C1. OsUGT91C1 is a glycosyltransferase derived from rice (Oryza sativa), and has typical structural characteristics of GT-B folding family glycosyltransferases, including an N-terminal domain and a C-terminal domain composed of a Rossman domain (β / α / β), and the catalytic active sites are H27 and D128. OsUGT91C1 can catalyze the glycosylation reaction of the glucosyl at the C-13 or C-19 position of stevioside, respectively. During the catalytic process of OsUGT91C1, the active site H27 attacks the glucosyl hydroxyl at the C-13 or C-19 position of stevioside to capture a proton, making it a nucleophile and then attacking the glucosyl C-1 position of the glycosyl donor UDPG, thereby causing a glycosylation reaction to form a β-1,2 glycosidic bond. When rebaudioside A is used as a substrate, OsUGT91C1 only undergoes glycosylation reaction at the C-19 glucosyl group to form a β-1,2 glycosidic bond, generating a single product, rebaudioside D.

[0096] In a specific embodiment, the amino acid sequence of the wild-type glycosyltransferase OsUGT91C1 is shown in SEQ ID NO: 3. The amino acid sequence of the glycosyltransferase mutant of the present invention has one or more mutations at amino acids 129, 208, 20, 69, 150, 238, 273, 287, and 349 corresponding to the amino acid sequence of the wild-type glycosyltransferase OsUGT91C1.

[0097] Those skilled in the art know that for the mutation of wild-type polypeptides, it is more important to find the sites that can achieve the desired purpose. Therefore, based on the teachings of the present invention, those skilled in the art will mutate the amino acid residues at positions 129, 208, 20, 69, 150, 238, 273, 287, and / or 349 of the amino acid sequence of wild-type glycosyltransferase OsUGT91C1, or the amino acid residues corresponding to positions 129, 208, 20, 69, 150, 238, 273, 287, and / or 349 of the amino acid sequence of wild-type glycosyltransferase OsUGT91C1 in a certain amino acid sequence, and detect the relevant activities of the mutants. In a specific embodiment, the glycosyltransferase of the present invention mutates valine at position 129 corresponding to the amino acid sequence of wild-type glycosyltransferase OsUGT91C1 to (but not limited to) alanine, leucine, or isoleucine, preferably alanine; phenylalanine at position 208 is mutated to (but not limited to) methionine, leucine, or isoleucine, preferably methionine; cysteine at position 20 is mutated to (but not limited to) phenylalanine, leucine, valine, isoleucine, alanine, or tyrosine, preferably phenylalanine; alanine at position 69 is mutated to (but not limited to) aspartic acid or glutamic acid; leucine at position 150 is mutated to (but not limited to) tryptophan, tyrosine, or phenylalanine, preferably tryptophan; arginine at position 238 is mutated to (but not limited to) glycine, proline, or alanine, preferably glycine; lysine at position 273 is mutated to (but not limited to) glycine, proline, or alanine, preferably glycine; glycine at position 287 is mutated to (but not limited to) serine or threonine, preferably serine; alanine at position 349 is mutated to (but not limited to) proline.

[0098] The inventors further found that the above mutation sites can be combined to further improve the activity and substrate tolerance of the mutants of wild-type glycosyltransferase OsUGT91C1. In a preferred embodiment, the glycosyltransferase mutant of the present invention has 1, preferably 2 mutations, more preferably 3 mutations, more preferably 4 mutations, and most preferably 5 mutations at positions 208, 20, 69, 150, 238, 273, 287, 349 on the basis of having a mutation at position 129.

[0099] In addition, it is not difficult for those of ordinary skill in the art to know that changing a few amino acid residues in certain regions of a polypeptide, such as non-essential regions, will basically not change its biological activity. For example, a sequence obtained by appropriately replacing certain amino acids will not affect its activity (see Watson et al., Molecular Biology of The Gene, Fourth Edition, 1987, The Benjamin / Cummings Pub. Co. P224). Therefore, those of ordinary skill in the art can perform such replacements and ensure that the resulting molecule still has the desired biological activity.

[0100] Therefore, it is obvious to further mutate the glycosyltransferase of the present invention to obtain a further mutant that still has the function and activity of the glycosyltransferase, especially the function and activity of catalyzing the synthesis of Reb D from Reb A. For example, it is well known to those skilled in the art that adding or deleting several amino acid residues at either end of a polypeptide, such as preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue, will not affect the function of the resulting mutant. For example, for the convenience of purification, technicians often attach a 6×His tag to either end of the obtained protein, and this protein has the same function as the protein without the 6×His tag.

[0101] Therefore, the present invention should include conservative mutants of the glycosyltransferase of the present invention. These conservative mutants can be generated by amino acid substitutions according to, for example, the following table.

[0102]

[0103]

[0104] The present invention also provides a polynucleotide encoding the glycosyltransferase of the present invention. The term "polynucleotide encoding a glycosyltransferase" can be a polynucleotide including the polynucleotide encoding this glycosyltransferase, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0105] Therefore, as used herein, "comprising", "having" or "including" includes "containing", "consisting essentially of", "substantially consisting of", and "consisting of"; "consisting essentially of", "substantially consisting of" and "consisting of" are subordinate concepts of "comprising", "having" or "including".

[0106] "corresponding to"

[0107] As used herein, the term "corresponding to" has the meaning commonly understood by those of ordinary skill in the art. Specifically, "corresponding to" means the position in one sequence that corresponds to a specified position in another sequence after the two sequences are aligned by homology or sequence identity. Thus, with respect to "the amino acid residue corresponding to position 90 of the amino acid sequence of the wild-type glycosyltransferase", if a 6×His tag is added to one end of the amino acid sequence of the wild-type glycosyltransferase, then position 135 in the resulting mutant may correspond to position 129 of the amino acid sequence of the wild-type glycosyltransferase.

[0108] In a specific embodiment, the homology or sequence identity may be more than 90%, preferably more than 95%, more preferably 96%, 97%, 98%, 99% homology.

[0109] Methods for determining sequence homology or identity that are well known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, Lesk, A.M. ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W. ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G. eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J.M eds., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferred methods for determining identity obtain the maximum match between the sequences being tested. Methods for determining identity are compiled in computer programs that are publicly available. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0110] host cell

[0111] As used herein, the term "host cell" has the meaning commonly understood by those of ordinary skill in the art, i.e., a host cell capable of producing the glycosyltransferase mutant of the present invention. In other words, the present invention can utilize any host cell as long as the glycosyltransferase mutant of the present invention can be expressed in that host cell.

[0112] For example, the host cells applicable to the present invention are from (but not limited to) the genus Escherichia, the genus Corynebacterium, Brevibacterium sp., the genus Bacillus, the genus Pichia, the genus Saccharomyces, the genus Candida, the genus Serratia, or the genus Vibrio.

[0113] In a further preferred embodiment, the host cell is Escherichia coli or Corynebacterium glutamicum.

[0114] The host cells of the present invention can be used to prepare Reb D; that is, Reb D is synthesized by culturing the host cells of the present invention. To avoid the additional addition of expensive UDPG during the production of Reb D, the host cells of the present invention may further contain the coding nucleotide sequence of sucrose synthase SUS1 to establish a UDPG recycling system; alternatively, the host cells expressing the glycosyltransferase mutant of the present invention can be used in combination with the host cells expressing sucrose synthase SUS1 to prepare Reb D, that is, the host cells expressing the glycosyltransferase mutant of the present invention and the host cells expressing sucrose synthase SUS1 are cultured simultaneously. Therefore, the present invention also provides a combination of the host cells expressing the glycosyltransferase mutant of the present invention and the host cells expressing sucrose synthase SUS1. For ease of storage and use, the combination of the host cells of the present invention can be made into a freeze-dried powder.

[0115] Those skilled in the art can understand that when preparing Reb D using the host cells or the combination of host cells of the present invention, the culture system of the host cells or the combination of host cells may further contain sucrose, Reb A, and UDP.

[0116] Immobilized enzyme

[0117] As used herein, the term "immobilized enzyme" has the meaning commonly understood by those of ordinary skill in the art. Specifically, this term means that after a water-soluble enzyme is treated by physical or chemical methods, the enzyme is bound to a water-insoluble macromolecular carrier or the enzyme is entrapped therein, such that the enzyme forms a soluble gel or a microcapsule of a semipermeable membrane in water, thereby resulting in a decrease in fluidity.

[0118] The immobilized enzyme still has enzyme activity and acts on the substrate in a solid state during the catalytic reaction. After immobilization, the enzyme generally has increased stability, is easily separated from the reaction system, is easy to control, and can be used repeatedly. It is convenient for transportation and storage and is conducive to automated production. Immobilized enzymes are an enzyme application technology developed in the past decade or so and have attractive application prospects in industrial production, chemical analysis, medicine, etc.

[0119] Based on the teachings herein, those of ordinary skill in the art can easily process the glycosyltransferase mutant of the present invention into an immobilized enzyme for use in catalyzing the synthesis of Reb D from Reb A. Those skilled in the art will understand that when preparing Reb D using the immobilized enzyme prepared from the glycosyltransferase mutant of the present invention, the enzyme reaction system may contain UDPG and Reb A.

[0120] The beneficial effects of the present invention are mainly reflected in:

[0121] 1. Through semi-rational design of the enzyme, the present invention provides a glycosyltransferase OsUGT91C1 mutant with excellent activity and thermal stability, thus being able to adapt to the reaction under high-concentration rebaudioside A conditions;

[0122] 2. The glycosyltransferase OsUGT91C1 mutant of the present invention has the advantages of high catalytic activity, high tolerance to substrate concentration, and no other by-products, and can catalyze the substrate conversion of rebaudioside A with a concentration up to 100 mM, with a conversion rate reaching 80%, and no by-products are generated;

[0123] 3. The method for catalyzing the production of rebaudioside D from rebaudioside A by the glycosyltransferase OsUGT91C1 mutant of the present invention has a simple catalytic process, no by-products are generated in the reaction, and the subsequent separation is simple. Therefore, it has the advantages of high atom economy, mild reaction conditions, environmental friendliness, and simple product post-treatment;

[0124] 4. The glycosyltransferase OsUGT91C1 mutant of the present invention shows good industrial application prospects in the synthesis of rebaudioside D.

[0125] The following further describes the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional operations in the art or the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial sources unless otherwise specified.

[0126] Example 1: Strain construction

[0127] The sequence of the glycosyltransferase OsUGT91C1 derived from Oryza sativa was cloned, and after codon optimization, it was inserted into the expression plasmid pET-28a to obtain pET28a-OsUGT91C1. The sequence of the sucrose synthase AtSUS1 derived from Arabidopsis thaliana was cloned, and after codon optimization, it was inserted into the expression plasmid pET-21a to obtain pET21a-AtSUS1. The gene sequence of the glycosyltransferase OsUGT91C1 is shown in SEQ ID NO:1, and the gene sequence of the sucrose synthase AtSUS1 is shown in SEQ ID NO:2. After sequencing verification, pET28a-OsUGT91C1 and pET21a-AtSUS1 were transferred into the expression host Escherichia coli BL21(DE3) for subsequent expression of recombinant enzymes.

[0128] SEQ ID NO:1; Coding nucleotide sequence of wild-type OsUGT91C1 DNA

[0129]

[0130] SEQ ID NO:3; Amino acid sequence of wild-type OsUGT91C1

[0131] MDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALEHKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVARMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIVADMACHERYIDGFIQQLRSYKDLEHHHHHH*

[0132] SEQ ID NO:2; Coding nucleotide sequence of sucrose synthase SUS1

[0133]

[0134] MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDDLEHHHHHH*

[0135] Example 2: Selection of single mutants

[0136] Molecular docking was performed using the crystal structure of glycosyltransferase OsUGT91C1 and Reb A. Based on the docking results, at a distance from the glycosyl acceptor substrate Reb A binding site Fourteen residues were selected for alanine scanning mutagenesis within the range. Meanwhile, 15 UGTs (identity: 40%-60%) with similar catalytic glycosylation characteristics were collected from different sources using the Basic Local Alignment Search Tool (blast). According to the results of multiple sequence alignment, different sites on the glycosyltransferase OsUGT91C1 were further mutated into amino acids with higher conservation. The mutants obtained by screening were: C20F, A69D, A69E, V129A, L150W, R238G, F208M, K273G, G287S, A349P.

[0137] Example 3: Construction of single-point mutants of glycosyltransferase OsUGT91C1

[0138] Using the recombinant plasmid pET28a-OsUGT91C1 as the parent, primers were designed for the sites to be mutated respectively, and the upstream and downstream primer sequences are shown in Table 1. PCR system: 10 μL of DNA polymerase P515, 1 μL of each of the forward and reverse primers, 1 μL of the template, 7 μL of double-distilled water, and the total system was 20 μL. PCR program: First, pre-denature at 95 °C for 5 min; then cycle 30 times according to the following program: denature at 95 °C for 30 s, anneal at 58 °C for 20 s, extend at 72 °C for 5.5 min; finally extend at 72 °C for 3 min and store at 4 °C.

[0139] Table 1. Primers used for single-point mutants of glycosyltransferase OsUGT91C1

[0140]

[0141] Add 0.3 μL of DpnI enzyme to the PCR product and react at a constant temperature of 37 °C for 1.5 h for template plasmid digestion. Transfer 10 μL of the digested PCR product into competent cells of Escherichia coli BL21(DE3), place on ice for 30 min, heat shock at 42 °C for 90 s, place on ice for 2 min, add 600 μL of LB medium (formula: 10 g / L of NaCl, 5 g / L of yeast powder, 10 g / L of peptone), shake at 37 °C at 200 rpm for 45 min of resuscitation, take all the bacterial liquid and spread it evenly on an LB plate containing kanamycin resistance (Kan 50 μg / mL), and culture overnight at 37 °C. Pick 2 single colonies on the plate, inoculate them into 700 μL of LB liquid medium for 3 h and then sequence. Transfer the bacterial liquid with correct sequencing results into a test tube containing 5 mL of LB medium (Kan 50 μg / mL), shake at 37 °C at 200 rpm overnight for 12-16 h, then preserve the bacteria and carry out subsequent expression.

[0142] Example 4: Strain culture

[0143] Take 500 μL of the overnight cultured bacterial solution with the correct sequencing result and add it to a 250 mL shaking flask containing 50 mL of LB liquid medium (with resistance). Culture it in a constant temperature shaker at 37°C for about 2.5 h until the OD 600 reaches about 0.6. Add IPTG with a final concentration of 0.1 mM for induction, and place it in a constant temperature shaker at 20°C for induction expression for 18 - 20 h. After the induction expression is completed, pour the bacterial solution into a 50 mL centrifuge tube, balance it, and centrifuge at 8000 rpm at 4°C for 10 min. After centrifugation, discard the supernatant, add 15 mL of normal saline, resuspend the cells, balance it, and centrifuge at 8000 rpm at 4°C for 10 min. After centrifugation, discard the supernatant, perform an idling spin, stop centrifugation when the speed reaches 6000 rpm, use a pipette to aspirate the residual supernatant liquid, and store the collected cells in a -40°C refrigerator for later use. Subsequently, add a certain volume of buffer to the harvested recombinant cells for ultrasonic disruption, centrifuge to collect the supernatant, and thus obtain a crude enzyme solution; use a vacuum freeze dryer to freeze-dry it to obtain a freeze-dried enzyme powder.

[0144] Example 5: Construction of the combinatorial mutant of glycosyltransferase OsUGT91C1

[0145] Using plasmid pET28a-OsUGT91C1 (C20F) as a template, perform whole plasmid PCR amplification using the primers in Table 1 of Example 2 to obtain double point mutants C20F / A69D, C20F / A69E, C20F / V129A, C20F / L150W, C20F / F208M, C20F / K273G, C20F / G287S, C20F / A349P.

[0146] Using plasmid pET28a-OsUGT91C1 (A69D) as a template, perform whole plasmid PCR amplification using the primers in Table 1 of Example 2 to obtain double point mutants A69D / V129A and A69D / F208M.

[0147] Using plasmid pET28a-OsUGT91C1 (A69E) as a template, perform whole plasmid PCR amplification using the primers in Table 1 of Example 2 to obtain double point mutants A69E / V129A and A69E / F208M.

[0148] Using plasmid pET28a-OsUGT91C1 (F208M) as a template, perform whole plasmid PCR amplification using the primers in Table 1 of Example 2 to obtain double point mutants V129A / F208M, L150W / F208M, F208M / K273G, F208M / G287S, F208M / A349P.

[0149] Using plasmid pET28a-OsUGT91C1(C20F / A69D) as a template, full plasmid PCR amplification was carried out using the primers in Table 1 of Example 2 to obtain C20F / A69D / V129A, C20F / A69D / F208M, C20F / A69D / L150W, C20F / A69D / K273G, C20F / A69D / G287S, and C20F / A69D / A349P triple mutants.

[0150] Using plasmid pET28a-OsUGT91C1(C20F / A69E) as a template, full plasmid PCR amplification was carried out using the primers in Table 1 of Example 2 to obtain C20F / A69E / V129A, C20F / A69E / F208M, C20F / A69E / L150W, C20F / A69E / K273G, C20F / A69E / G287S, and C20F / A69E / A349P triple mutants.

[0151] Using plasmid pET28a-OsUGT91C1(C20F / F208M) as a template, full plasmid PCR amplification was carried out using the primers in Table 1 of Example 2 to obtain C20F / V129A / F208M, C20F / F208M / K273G, C20F / F208M / G287S, and C20F / F208M / A349P triple mutants.

[0152] Using plasmid pET28a-OsUGT91C1(C20F / K273G) as a template, full plasmid PCR amplification was carried out using the primers in Table 1 of Example 2 to obtain the C20F / K273G / G287S triple mutant.

[0153] Using plasmid pET28a-OsUGT91C1(C20F / V129A / F208M) as a template, full plasmid PCR amplification was carried out using the primers in Table 1 of Example 2 to obtain C20F / V129A / F208M / A69D, C20F / V129A / F208M / A69E, C20F / V129A / F208M / L150W, C20F / V129A / F208M / R238G, C20F / V129A / F208M / K273G, C20F / V129A / F208M / G287S, and C20F / V129A / F208M / A349P quadruple mutants.

[0154] Using plasmid pET28a-OsUGT91C1(C20F / V129A / F208M / K273G) as a template, full plasmid PCR amplification was performed using the primers in Table 1 of Example 2 to obtain the five-point mutants C20F / V129A / F208M / K273G / A69D, C20F / V129A / F208M / K273G / A69E, C20F / V129A / F208M / K273G / L150W, C20F / V129A / F208M / K273G / R238G, C20F / V129A / F208M / K273G / G287S, and C20F / V129A / F208M / K273G / A349P.

[0155] Using plasmid pET28a-OsUGT91C1(C20F / V129A / F208M / K273G / R238G) as a template, full plasmid PCR amplification was performed using the primers in Table 1 of Example 2 to obtain the six-point mutants C20F / V129A / F208M / K273G / R238G / A69D, C20F / V129A / F208M / K273G / R238G / A69E, C20F / V129A / F208M / K273G / R238G / L150W, C20F / V129A / F208M / K273G / R238G / G287S, and C20F / V129A / F208M / K273G / R238G / A349P.

[0156] Using plasmid pET28a-OsUGT91C1(C20F / V129A / F208M / K273G / A349P) as a template, full plasmid PCR amplification was performed using the primers in Table 1 of Example 2 to obtain the six-point mutants C20F / V129A / F208M / K273G / A349P / A69D, C20F / V129A / F208M / K273G / A349P / A69E, C20F / V129A / F208M / K273G / A349P / L150W, C20F / V129A / F208M / K273G / A349P / G287S.

[0157] Example 6: Activity Assay of Glycosyltransferase OsUGT91C1 and Its Mutants

[0158] The reaction system for enzyme activity detection was 1 ml, which contained 10 mg of freeze-dried cells of glycosyltransferase OsUGT91C1, 10 mg of freeze-dried cells of AtSus1, 20 mM Reb A, 2 mM UDP, 200 mM sucrose, 3 mM MgCl2 and 50 mM potassium phosphate buffer pH 7.0. The reaction was carried out at 30 °C for 2 h, and then the reaction was terminated by heating at 90 °C for 10 min. After adding 500 μL of DMSO to the reaction solution to dissolve the product Reb D, it was centrifuged at 14000 rpm for 10 min and filtered through a 0.22 μm filter. The sample was analyzed by HPLC using an SB-Aq analytical column at 30 °C. The detection wavelength was 210 nm. The results are as Figure 2 , Figure 3 shown.

[0159] Example 7: Purification of glycosyltransferase OsUGT91C1 and its mutant proteins

[0160] The wild-type glycosyltransferase OsUGT91C1 and mutants were cultured and expressed as shown in Example 4. The obtained crude enzyme solution of the supernatant was loaded onto a Ni-NTA column equilibrated with binding buffer a (50 mM potassium phosphate buffer, pH 7.0, containing 500 mM NaCl and 10 mM imidazole). Cellular proteins were removed with binding buffer B (50 mM potassium phosphate buffer, pH 7.0, containing 500 mM NaCl and 25 mM imidazole). Then the target protein was eluted with elution buffer (200 mM KH2PO4, pH 8.0, containing 500 mM NaCl and 200 mM imidazole). Subsequently, imidazole was removed and the target protein was concentrated by ultrafiltration. The purity of the target protein was determined by SDS-PAGE. Using bovine serum albumin as a standard reagent, the concentration of the purified enzyme was determined by the Bradford method.

[0161] Example 8: Determination of kinetic parameters of glycosyltransferase OsUGT91C1 and its mutants

[0162] The kinetic parameters of OsUGT91c1-WT and its variants were measured in a 400 μL reaction mixture containing 3 mM MgCl2, 1.5 mM UDPG, a certain amount of enzyme (15 - 30 μg) and different concentrations of Reb A (0.05 - 1.5 mM) (all prepared in 50 mM potassium phosphate buffer), and continued at 30 °C for 10 - 30 minutes. Then, the reaction was terminated by heating at 90 °C for 10 minutes. Sample preparation and analysis were carried out by the same method as above. The results are shown in Table 2.

[0163] Table 2. Kinetic parameters of glycosyltransferase OsUGT91C1 and its mutants

[0164]

[0165] Example 9: Determination of the stability parameters of glycosyltransferase OsUGT91C1 and its mutants

[0166] The melting temperature T of glycosyltransferase OsUGT91C1 and its mutants m was determined by differential scanning fluorimetry (DSF). 20 μL of 0.5 mg / mL pure enzyme and 5 μL of 5×SYPRO Orange protein gel stain were added to an IQ 96-well PCR plate (Bio-Rad, Hercules, CA, USA). Detection was performed using a CFX 96 real-time quantitative PCR detection system (Bio-Rad, Hercules, CA, USA). The melting curve program for the enzyme: the temperature range was 20 - 99 °C, and the temperature gradient was 0.5 °C / min. The results are as Figure 4 shown. The T of the glycosyltransferase OsUGT91C1 mutant (C20F / V129A / F208M / K273G / A349P / A69, M6) m increased threefold compared to the wild type.

[0167] Example 10: Determination of the conversion rates of glycosyltransferase OsUGT91C1 and its mutants at different rebaudioside A concentrations

[0168] The reaction system was 1 mL and contained 10 mg of freeze-dried glycosyltransferase OsUGT91C1 cells, 10 mg of freeze-dried AtSus1 cells, 20 - 100 mM Reb A, 2 mM UDP, 300 mM sucrose, 3 mM MgCl2, and 50 mM potassium phosphate buffer pH 7.0. The reaction was carried out at 30 °C for 36 h and then terminated by heating at 90 °C for 10 min. After adding 500 μL of DMSO to dissolve the product Reb D, the reaction solution was diluted 10-fold again with DMSO, centrifuged at 14000 rpm for 10 min, and filtered through a 0.22 μm filter. The samples were analyzed by HPLC using an SB-Aq analytical column at 30 °C. The conversion rate results are as Figure 4 shown. The conversion rates of each concentration of the glycosyltransferase OsUGT91C1 mutant (C20F / V129A / F208M / K273G / A349P / A69, M6) were all above 80%, which was more than 4 times higher than that of the wild type.

[0169] The formula for calculating the rebaudioside A conversion rate is as follows:

[0170] Rebaudioside A conversion rate (Conversion rate of Reb A) (%) = C(RA) / C0(RA)

[0171] Wherein C0(RA) and C(RA) represent the initial content and the content at the end of the reaction of rebaudioside A, respectively, and C(RD) represents the content of rebaudioside D at the end of the reaction. The contents of rebaudioside A and rebaudioside D are calculated using their respective standard curves.

[0172] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A glycosyltransferase, wherein the following mutation occurs in the amino acid sequence of a wild-type glycosyltransferase OsUGT91C1, and the amino acid sequence of the wild-type glycosyltransferase OsUGT91C1 is shown in SEQ ID NO: 3: The valine at position 129 mutated to alanine; the cysteine ​​at position 20 mutated to phenylalanine; the phenylalanine at position 208 mutated to methionine; the lysine at position 273 mutated to glycine; the alanine at position 349 mutated to proline; and the alanine at position 69 mutated to aspartic acid or glutamic acid.

2. An isolated nucleic acid molecule encoding the glycosyltransferase of claim 1.

3. An expression vector comprising the nucleic acid molecule according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a plasmid.

5. A host cell, comprising the expression vector according to claim 3 or 4, or the nucleic acid molecule according to claim 2 is integrated into the genome of the host cell.

6. The host cell according to claim 5, characterized in that The host cell also contains a nucleotide sequence encoding sucrose synthase SUS1.

7. The host cell according to claim 6, characterized in that The sucrose synthase SUS1 is derived from Arabidopsis thaliana ( Arabidopsis thaliana ).

8. The host cell according to claim 7, wherein The encoding nucleotide sequence of the sucrose synthase SUS1 is shown in SEQ ID NO:

2.

9. The host cell according to claim 5, characterized in that The host cell is a rebaudioside D producing cell.

10. The host cell according to claim 9, characterized in that The host cell is from the genus Escherichia ( Escherichia ), Corynebacterium ( Corynebacterium ), Brevibacterium ( Brevibacterium sp . ), Bacillus ( Bacillus ), Pichia Pichia ), Saccharomyces cerevisiae ( Saccharomyces ), Candida spp. Candida ), Serratia spp. Serratia ) or Vibrio spp. ( Vibrio ).

11. The host cell according to claim 10, wherein The host cell is Escherichia coli ( E. coli ) or Corynebacterium glutamicum ( Corynebacterium glutamicum ). 12 . A combination of host cells, comprising the host cell according to any one of claims 5 to 11 and a host cell expressing sucrose synthase SUS1.

13. A lyophilized powder made from the host cell of any one of claims 5 to 11 or a combination of the host cells of claim 12.

14. Use of the glycosyltransferase according to claim 1, or the nucleic acid molecule according to claim 2, or the expression vector according to claim 3 or 4, or the host cell according to any one of claims 5 to 11, the combination of the host cells according to claim 12, or the lyophilized powder according to claim 13 in the production of rebaudioside D.

15. A method for preparing rebaudioside D, the method comprising the following steps: a. In a culture system, culturing the host cell according to any one of claims 5-11 or the combination of host cells according to claim 12 to produce rebaudioside D; and b. Optionally, isolating rebaudioside D from the culture broth obtained in a.

16. The method according to claim 15, characterized in that The culture system in step a also contains sucrose, Reb A and UDP.

17. A method for preparing Reb D, the method comprising the steps of: a. In a reaction system, using the glycosyltransferase according to claim 1 to catalyze Reb A to produce Reb D; and b. Optionally isolating Reb D from the above reaction system.

18. The method according to claim 17, wherein: The reaction system of step a also comprises UDPG and Reb A.

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