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EP-4737564-A1 - GLYCOSYLTRANSFERASE UGTSL2 MUTANT, GLYCOSYLTRANSFERASE MUTANT, AND METHOD FOR SYNTHESIZING REBAUDIOSIDE M2 THEREWITH

EP4737564A1EP 4737564 A1EP4737564 A1EP 4737564A1EP-4737564-A1

Abstract

Provided is a glycosyltransferase UGTSL2 mutant . Said UGTSL2 mutant is obtained by means of any one or more of the following mutations on an amino acid sequence shown as SEQ ID NO .1:the 23rd amino acid is mutated from N to E ; the 41st amino acid is mutated from R to P ; the 91st amino acid is mutated from H to K : the 95th amino acid is mutated from K to D ; the 123rd amino acid is mutated from E to P ; the 136th amino acid is mutated from L to F : the 151st amino acid is mutated from R to F ; the 124th amino acid is mutated from H to E ; the 168th amino acid is mutated from V to Y ;the 198th amino acid is mutated from C to K ; the 202nd amino acid is mutated from T to E ; the 217th amino acid is mutated from W to K : the 225th amino acid is mutated from P to L ; the 226th amino acid is mutated from F to V ; the 285th amino acid is mutated from A to V ; the 333rd amino acid is mutated from I to V , etc . Also provided is a glycosyltransferase mutant , the glycosyltransferase mutant being following A1) or A2):A1) being a protein which is obtained by means of substitution of an amino acid residue on an amino acid sequence shown as SEQ ID NO .66, and which has 90% or higher identity with and same functions as the original protein , and A2) being a fusion protein obtained by linking a tag to the Nterminal and / or the Cterminal of A1.

Inventors

  • ZHU, LIPING
  • JIA, Honghua
  • LI, YAN
  • CHEN, Kecai
  • PAN, Huayi
  • SONG, Weicai
  • CHEN, KAI
  • XU, Liangping
  • SHEN, Yingying

Assignees

  • Dongtai Haorui Biotechnology Co., Ltd

Dates

Publication Date
20260506
Application Date
20240426

Claims (20)

  1. A glycosyltransferase UGTSL2 mutant, wherein the glycosyltransferase UGTSL2 mutant is any one selected from the group consisting of the following (A) to (C): (A) a protein produced through any one or more selected from the group consisting of the following mutations based on an amino acid sequence shown in SEQ ID NO: 1: a mutation of a 23rd amino acid from N to E; a mutation of a 41st amino acid from R to P; a mutation of a 91st amino acid from H to K; a mutation of a 95th amino acid from K to D; a mutation of a 123rd amino acid from E to P; a mutation of a 136th amino acid from L to F; a mutation of a 151st amino acid from R to F; a mutation of a 124th amino acid from H to E; a mutation of a 168th amino acid from V to Y; a mutation of a 198th amino acid from C to K; a mutation of a 202nd amino acid from T to E; a mutation of a 217th amino acid from W to K; a mutation of a 225th amino acid from P to L; a mutation of a 226th amino acid from F to V; a mutation of a 285th amino acid from A to V; a mutation of a 333rd amino acid from I to V; a mutation of a 358th amino acid from N to F; a mutation of a 392nd amino acid from T to V; and a mutation of a 419th amino acid from I to K; (B) a protein that has an identity of 95% or more with and the same function as an amino acid sequence defined in the (A); and (C) a fusion protein produced by linking a tag to a terminus of the protein defined in the (A) or the (B).
  2. The glycosyltransferase UGTSL2 mutant according to claim 1, wherein an amino acid sequence of the glycosyltransferase UGTSL2 mutant is shown in SEQ ID NO: 3.
  3. A biological material selected from the group consisting of the following: (A) an expression gene encoding the glycosyltransferase UGTSL2 mutant according to claim 1 or 2; (B) a recombinant plasmid carrying the expression gene in the (A); and (C) a recombinant cell carrying the recombinant plasmid or the expression gene encoding the glycosyltransferase UGTSL2 mutant.
  4. An enzyme composition, comprising: a glycosyltransferase UGTSL2 mutant, a glycosyltransferase Nt UGT_M, and a sucrose synthase (SuSy) AtSuSy, wherein the glycosyltransferase UGTSL2 mutant is the glycosyltransferase UGTSL2 mutant according to claim 1 or 2; the glycosyltransferase Nt UGT_M is the following (A1) or (A2) : (A1) an amino acid sequence shown in SEQ ID NO: 5; and (A2) a protein that has an identity of 95% or more with and the same function as the amino acid sequence defined in the (Al); and the SuSy AtSuSy is the following (B1) or (B2): (B1) an amino acid sequence shown in SEQ ID NO: 7; and (B2) a protein that has an identity of 95% or more with and the same function as the amino acid sequence defined in the (B1).
  5. The composition according to claim 4, wherein an enzyme activity ratio of the glycosyltransferase UGTSL2 mutant to the glycosyltransferase Nt UGT_M is (3-7):(7-3).
  6. A complete set of recombinant strains expressing the enzyme composition according to claim 4 or 5, comprising a recombinant strain A and a recombinant strain B, wherein the recombinant strain A comprises a recombinant plasmid A, and the recombinant plasmid A is produced by co-constructing a nucleic acid encoding the glycosyltransferase UGTSL2 mutant according to claim 1 or 2 and a nucleic acid encoding the SuSy At SuSy into an expression vector; the recombinant strain B comprises a recombinant plasmid B, and the recombinant plasmid B is produced by co-constructing a nucleic acid encoding the glycosyltransferase Nt UGT_M and a nucleic acid encoding the SuSy At SuSy into a plasmid; and an amino acid sequence of the glycosyltransferase Nt UGT_M is shown in SEQ ID NO: 5.
  7. The complete set of recombinant strains according to claim 6, wherein host strains comprise, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, or Corynebacterium glutamicum.
  8. A method for one-pot synthesis of rebaudioside M2 from rebaudioside A under biocatalysis, comprising the following steps: adding the rebaudioside A, sucrose, and the enzyme composition according to claim 4 or an induced expressed enzyme product of the complete set of recombinant strains according to claim 5 to a catalytic reaction system, allowing a reaction, and conducting enzyme inactivation and centrifugation to produce a supernatant, which is the rebaudioside M2, wherein the rebaudioside M2 is shown in the following formula (I):
  9. The method according to claim 8, wherein a process for acquiring the induced expressed enzyme product of the complete set of recombinant strains according to claim 5 or 6 comprises the following step: 1) activating the complete set of recombinant strains according to claim 5 or 6 to produce activated recombinant strains, transferring the activated recombinant strains to an induction medium, adding an inducer, and conducting an induction culture; conducting centrifugation to collect strain cells, resuspending the strain cells in an appropriate amount of a buffer, and conducting disruption; and conducting centrifugation to produce a supernatant, which is the induced expressed enzyme product; and the reaction is conducted for 5 h to 100 h.
  10. The method according to claim 9, wherein a final concentration of the inducer is 0.02 g/L to 1 g/L, and the induction culture is conducted for 4 h to 50 h.
  11. The method according to claim 9 or 10, wherein in the catalytic reaction system, a concentration of the rebaudioside A is 1 g/L to 1,000 g/L, a concentration of the sucrose is 1 g/L to 9,000 g/L, and an amount of the induced expressed enzyme product added is 1 MU/L to 100 MU/L; a pH of the catalytic reaction system is 7.0 to 8.5 and preferably 7.0; the reaction is conducted at 37°C to 40°C; and an enzyme activity ratio of the glycosyltransferase UGTSL2 mutant to the glycosyltransferase Nt UGT_M is (3-7):(7-3).
  12. A glycosyltransferase mutant, wherein the glycosyltransferase mutant is the following A1) or A2): A1) a protein produced through an amino acid residue substitution in an amino acid sequence shown in SEQ ID NO: 66, wherein the protein has an identity of 90% or more with and the same function as the amino acid sequence shown in SEQ ID NO: 66; and A2) a fusion protein produced by linking a tag to an N-terminus and/or a C-terminus of the protein in the A1).
  13. The glycosyltransferase mutant according to claim 12, wherein the glycosyltransferase mutant is a protein produced through the following one or more amino acid residue substitutions in the amino acid sequence shown in SEQ ID NO: 66: B1) a mutation of a 10th amino acid from alanine (A) to valine (V); B2) a mutation of a 51st amino acid from glutamine (Q) to lysine (K); B3) a mutation of a 72nd amino acid from phenylalanine (F) to leucine (L); B4) a mutation of a 87th amino acid from leucine (L) to proline (P); B5) a mutation of a 123rd amino acid from leucine (L) to proline (P); B6) a mutation of a 157th amino acid from leucine (L) to proline (P); B7) a mutation of a 219th amino acid from asparagine (N) to aspartic acid (D); B8) a mutation of a 380th amino acid from glycine (G) to leucine (L); and B9) a mutation of a 400th amino acid from serine (S) to arginine (R).
  14. A biological material, wherein the biological material is any one selected from the group consisting of the following C1) to C5): C1) a nucleic acid encoding the protein according to claim 12 or 13; C2) an expression cassette carrying the nucleic acid in the C1); C3) a recombinant vector carrying the nucleic acid in the C1) or a recombinant vector carrying the expression cassette in the C2); C4) a recombinant microorganism carrying the nucleic acid in the C1), or a recombinant microorganism carrying the expression cassette in the C2), or a recombinant microorganism carrying the recombinant vector in the C3); and C5) a transgenic plant cell line carrying the nucleic acid in the C1) or a transgenic plant cell line carrying the expression cassette in the C2).
  15. A method for synthesizing rebaudioside M2 under catalysis of a glycosyltransferase mutant, comprising the following steps: 1) construction of a recombinant strain carrying a double-enzyme co-expression system: co-constructing a coding gene for the glycosyltransferase mutant according to any one of claims 12 to 13 and a SuSy gene into an expression vector to produce a recombinant plasmid, and transforming the recombinant plasmid into a host strain to produce the recombinant strain carrying the double-enzyme co-expression system; 2) inducing the recombinant strain to express the glycosyltransferase mutant and SuSy; and 3) using the two enzymes obtained in the step 2) to prepare rebaudioside M2 with rebaudioside D and sucrose as raw materials:
  16. The method according to claim 15, wherein the expression vector is pRSFDuet-1.
  17. The method according to claim 15, wherein for the induced expression in the step 2), an inducer is added at a final concentration of 0.02 g/L to 1 g/L, and induction is conducted for 4 h to 50 h.
  18. The method according to claim 15, wherein the step 2) comprises: strain collection, strain disruption, and centrifugation to collect a supernatant, which is a crude enzyme.
  19. The method according to claim 18, wherein in the step 3), a concentration of the rebaudioside D is 1 g/L to 500 g/L, a concentration of the sucrose is 1 g/L to 1,500 g/L, and an amount of the crude enzyme added is 1 g/L to 100 g/L.
  20. The method according to claim 15, wherein the host strain comprises, but is not limited to, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, or Corynebacterium glutamicum.

Description

CROSS REFERENCE TO RELATED APPLICATIONS The present application claims priority to the Chinese Patent 202310795002.3 filed to the China National Intellectual Property Administration (CNIPA) on June 30, 2023 and entitled "GLYCOSYLTRANSFERASE MUTANT AND METHOD FOR SYNTHESIZING REBAUDIOSIDE M2 UNDER CATALYSIS OF MUTANT" and the Chinese Patent 202310813254.4 filed to the China National Intellectual Property Administration (CNIPA) on June 30, 2023 and entitled "GLYCOSYLTRANSFERASE UGTSL2 MUTANT AND METHOD FOR SYNTHESIZING REBAUDIOSIDE M2", which are incorporated herein by reference in their entireties. TECHNICAL FIELD The present disclosure relates to the technical field of biocatalytic synthesis, and specifically to a glycosyltransferase UGTSL2 mutant, a glycosyltransferase mutant, and a method for synthesizing rebaudioside M2 under catalysis of the mutants. BACKGROUND There is a growing demand for zero-calorie natural sugar substitutes, especially by diabetics. Steviol glycosides (SGs) are a group of natural calorie-free ingredients extracted from the leaves of the stevia plant. SGs have been approved as safe food additives in countries such as the United States, European countries, and Asian countries. In 2014, the use of SGs as food additives in products such as food, beverages, and nutritional supplements was approved in China. Therefore, SGs have received extensive attention. Rebaudioside D is a very promising SG with sweetness 200 times to 300 times higher than sucrose. However, the short-term lingering bitterness carried by rebaudioside D affects the application and promotion of rebaudioside D on the market. Rebaudioside M2 is produced by adding a glucosyl group at C-6' of the first glucosyl group linked to C19 of rebaudioside D. Rebaudioside M2 exhibits increased sweetness and no bitterness compared with rebaudioside D. Thus, rebaudioside M2 has better sugar properties and a more desirable taste than rebaudioside D. Currently, rebaudioside M2 is a monoglycosylated derivative of rebaudioside D, and is prepared through biotransformation. NtUGT derived from Nicotiana tomentosiformis can catalyze the C-6' glycosylation of the first glucosyl group linked to C19 of an SG compound, such that the high-value sweetener rebaudioside M2 can be produced by catalyzing rebaudioside D with NtUGT. NtUGT is a Leloir glycosyltransferase belonging to the GT1 family. Glycosides are generally synthesized with uridine diphosphate glucose (UDPG) as a glycosyl donor. NtUGT can be produced in large quantities through the heterologous expression in Escherichia coli or Saccharomyces cerevisiae. As a result, NtUGT can be used to catalyze the synthesis of rebaudioside M2 from rebaudioside D. With the continuous improvement of UDPG production methods, UDPG can be synthesized in large quantities with enzymes such as sucrose synthase (SuSy). On this basis, the SuSy-UGT cascade reaction system can be adopted. In the SuSy-UGT cascade reaction system, SuSy provides as a glycosyl donor for NtUGT to achieve the recycling of UDPG, which greatly reduces the application cost. In the NtUGT-SuSy cascade reaction system, SuSy provides a glycosyl donor for the smooth glycosylation. When a regeneration rate of UDPG is not limited (for example, the regeneration rate of UDPG can be increased by increasing a sucrose concentration), NtUGT is the key to the synthesis efficiency of rebaudioside M2. The tobacco-derived NtUGT exhibits poor thermal stability and is basically inactivated after 10 h of catalysis, which limits the efficient application of the glycosyltransferase. SUMMARY Therefore, a technical problem to be solved by the present disclosure is to overcome the defects of a low enzyme activity and a short half-life period of the glycosyltransferase UGTSL2 in the prior art and the defect of poor thermal stability of NtUGT and thus provide a glycosyltransferase UGTSL2 mutant with high enzyme activity and long half-life and a glycosyltransferase mutant with high M2 yield and strong thermal stability. In a first aspect, the present disclosure provides a glycosyltransferase UGTSL2 mutant, where the glycosyltransferase UGTSL2 mutant is any one selected from the group consisting of the following (A) to (C): (A) a protein produced through any one or more selected from the group consisting of the following mutations based on an amino acid sequence shown in SEQ ID NO: 1: a mutation of a 23rd amino acid from N to E;a mutation of a 41st amino acid from R to P;a mutation of a 91st amino acid from H to K;a mutation of a 95th amino acid from K to D;a mutation of a 123rd amino acid from E to P;a mutation of a 136th amino acid from L to F;a mutation of a 151st amino acid from R to F;a mutation of a 124th amino acid from H to E;a mutation of a 168th amino acid from V to Y;a mutation of a 198th amino acid from C to K;a mutation of a 202nd amino acid from T to E;a mutation of a 217th amino acid from W to K;a mutation of a 225th amino acid from P to L;a mutation of a