CN-122012436-A - Glycosyltransferase UGT94B1 mutant with improved thermostability and activity

Abstract

The invention discloses a glycosyltransferase UGT94B1 mutant with improved thermostability and activity, and belongs to the technical field of biocatalysis synthesis. According to the invention, through rational design, 11 th, 96 th, 219 th, 281 th and 327 th positions of glycosyltransferase are mutated, and compared with a starting enzyme, the half life of the obtained combined mutant UGT94B1 M3 at 40 ℃ is improved from 0.43 h to 8.88 h, the heat stability is improved by 20.47 times, and the enzyme activity is improved by 1.45 times. The invention expresses the mutant in escherichia coli, and constructs cascade reaction with sucrose synthase, so that the conversion rate of the Reb D reaches 84.67%, and a novel efficient way is provided for the production of the rebaudioside D.

Inventors

• RAO YIJIAN
• LUO ZHENGSHAN
• GUO XUPENG

Assignees

• 江南大学

Dates

Publication Date

20260512

Application Date

20260126

Claims (10)

1. 1. The glycosyltransferase mutant is characterized in that one or more amino acids at 11 th, 96 th, 219 th, 281 th and 327 th are mutated on the basis of a parent, and the amino acid sequence of the parent is shown as SEQ ID NO. 3.
2. 2. The glycosyltransferase mutant according to claim 1, wherein the mutant has any one of the mutations (a) - (c) based on the amino acid sequence shown in SEQ ID No. 3: (a) Mutating alanine at position 11 to leucine and threonine at position 219 to lysine; (b) Mutating alanine at position 11 to leucine and threonine at position 96 to methionine and threonine at position 219 to lysine and threonine at position 281 to asparagine; (c) Alanine 11 to leucine and threonine 96 to methionine and threonine 219 to lysine and threonine 281 to asparagine and serine 327 to proline.
3. 3. A gene encoding the glycosyltransferase mutant of claim 1 or 2.
4. 4. An expression vector carrying the gene of claim 3.
5. 5. A recombinant microbial cell of the glycosyltransferase mutant of claim 1 or 2.
6. 6. The recombinant escherichia coli is characterized in that escherichia coli BL21 (DE 3) is taken as a host, and pET-21b (+) is taken as a vector to express the glycosyltransferase mutant.
7. 7. The recombinant E.coli according to claim 6, wherein the glycosyltransferase mutant is further co-expressed with a sucrose synthase having an amino acid sequence as shown in SEQ ID NO. 5.
8. 8. A biocatalyst comprising the glycosyltransferase mutant of claim 1 or 2 and a sucrose synthase as set forth in SEQ ID No. 5.
9. 9. A method for catalytic synthesis of Reb D is characterized in that Reb A is used as a substrate, a biocatalyst according to claim 8 is used for catalytic reaction, optionally, the concentration of sucrose in a reaction system is more than or equal to 400 mM, 0.5-3.0 mM UDP is added in the reaction system, and the catalytic reaction is carried out at 30-40 ℃.
10. 10. Use of a glycosyltransferase mutant according to claim 1 or 2, or a gene according to claim 3, or a recombinant microbial cell according to claim 5, or a recombinant escherichia coli according to any one of claims 6 to 7, or a biocatalyst according to claim 8, or a method according to claim 9, for the preparation of a rebaudioside D containing product.

Description

Glycosyltransferase UGT94B1 mutant with improved thermostability and activity Technical Field The invention relates to a glycosyltransferase UGT94B1 mutant with improved thermostability and activity, belonging to the technical field of biocatalysis synthesis. Background Steviol glycosides are a class of natural sweeteners extracted from stevia rebaudiana, over 60 of which have been identified at present, with the main differences in the number and location of glucose units attached to the side chains. Among them, rare rebaudioside D (Reb D) is 250 to 400 times sweeter than sucrose and has much less bitter taste residual than rebaudioside a (Reb a), which makes it especially favored in the food and beverage industry, helping to balance the dual needs of flavor and health. Unfortunately, the very low content of Reb D in plants (only 0.1% to 0.5% by weight of dry leaf) results in the traditional extraction method being both expensive and inefficient. Therefore, enzymatic conversion of enriched Reb a to Reb D by uridine diphosphate glycosyltransferases (UGTs) has become a promising alternative. Several UGTs capable of catalyzing this transformation have been reported, including YojK, osUGT91C1, UGT94B1 and UGTSL, whose performance is further enhanced by directed evolution or rational design methods. For example, shoji et al increased their activity on Reb a by engineering UGT91D2, whereas inventors have earlier designed UGT94B1 rationally to obtain mutant UGT94B1-I146C/Y164G with a 5.3-fold increase in catalytic efficiency. Although its catalytic activity is improved, the mutant is found to have poor thermostability in the cascade, resulting in reduced efficiency and limiting the expansibility of the enzyme. Thus, increasing the thermal stability of UGT94B1-I146C/Y164G is a crucial step in achieving efficient and sustainable biocatalytic production of Reb D. Disclosure of Invention The invention successfully obtains a combined mutant with improved activity and thermal stability based on UGT94B1-I146C/Y164G, and the mutant can efficiently catalyze Reb A to generate Reb D, thereby meeting the requirement of industrial production. The invention provides glycosyltransferase mutants, wherein one or more amino acids at 11 th, 96 th, 219 th, 281 th and 327 th are mutated on the basis of glycosyltransferase UGT94B1-I146C/Y164G (abbreviated as UGT94B1 M0) constructed in the earlier stage. In one embodiment, the amino acid sequence of glycosyltransferase UGT94B1 M0 is shown in SEQ ID No. 3. In one embodiment, the mutation is any one of (1) to (5): (1) Mutating alanine at position 11 to leucine; (2) Mutating threonine at position 96 to methionine; (3) Mutating threonine at position 219 to lysine; (4) Mutating threonine at position 281 to asparagine; (5) Serine 327 is mutated to proline. In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.3, with alanine at position 11 mutated to leucine and threonine at position 219 mutated to lysine, designated UGT94B1 M1. In one embodiment, the mutant is one in which alanine at position 11 is mutated to leucine, threonine at position 96 is mutated to methionine, threonine at position 219 is mutated to lysine, and threonine at position 281 is mutated to asparagine based on the amino acid sequence shown in SEQ ID NO.3, designated UGT94B1 M2. In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.3, with alanine at position 11 mutated to leucine and threonine at position 96 mutated to methionine and threonine at position 219 mutated to lysine and threonine at position 281 mutated to asparagine and serine at position 327 mutated to proline, designated UGT94B1 M3. In one embodiment, the amino acid sequence of glycosyltransferase mutant UGT94B1 M3 is shown in SEQ ID No. 1. The invention also provides genes encoding the glycosyltransferase mutants. The invention also provides an expression vector carrying the gene. The invention also provides recombinant microbial cells expressing the glycosyltransferase mutants. In one embodiment, the recombinant microorganism hosts E.coli. In one embodiment, the recombinant microorganism expresses the glycosyltransferase mutant using E.coli BL21 (DE 3) as a host and pET-21b (+) as a vector. In one embodiment, the recombinant bacterium further co-expresses the glycosyltransferase mutant with a sucrose synthase. In one embodiment, the recombinant bacterium expresses the glycosyltransferase mutant with pET-21b (+) vector and expresses sucrose synthase with pACYCDuet-1 vector. In one embodiment, the amino acid sequence of the sucrose synthase is as shown in SEQ ID NO. 5. The invention also provides a biocatalyst comprising the glycosyltransferase mutant and sucrose synthase. In one embodiment, the biocatalyst comprises the glycosyltransferase mutant and an active enzyme protein of sucrose synthase, or comprises recombinant microbial cells expressing the glycosyltransferase mutant and sucrose synthase