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Glycosyltransferase mutant and application thereof

A technology of glycosyltransferases and mutants, applied in the biological field, can solve the problems of non-specificity of substrates and weak catalytic activity

Active Publication Date: 2020-12-15
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since the glycosyltransferase UGT76G1 participates in the multi-step glycosylation reaction in the synthesis of steviol glycosides, there are problems such as nonspecific substrate specificity and weak catalytic activity.

Method used

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  • Glycosyltransferase mutant and application thereof
  • Glycosyltransferase mutant and application thereof
  • Glycosyltransferase mutant and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0118] The above-mentioned method for preparing rebaudioside M can be carried out intracellularly or extracellularly. As a preferred mode of the present invention, a method for intracellular production of rebaudioside M is provided: the mutant glycosyltransferase UGT76G1 corresponding to the 284th position of SEQ ID NO: 1 mutated to Ser and the aforementioned "to Enzymes that convert rebaudioside A to rebaudioside D", "Enzymes that convert stevioside to rebaudioside A", "enzymes that catalyze the aglycone steviol to stevioside or rebaudioside A" And / or the gene encoding "enzyme converting rebaudioside A or stevioside into rebaudioside D" is transformed into host cells, and the cells are cultured to produce rebaudioside M.

[0119] In the present invention, a series of mutants that weaken the catalytic activity of the glycosyltransferase UGT76G1 are also provided, and the mutation occurs at position 147, 155, 146 or 380 corresponding to the sequence of SEQ ID NO: 1, etc., for e...

Embodiment 1

[0126] Example 1, UGT76G1 protein expression, purification, crystallization and structural analysis

[0127] 1. Construction process of wild-type UGT76G1 expression vector pQZ11

[0128]Using the codon-optimized UGT76G1 gene cloning vector as a template, specific primer pairs (Table 1) were used to amplify the target gene. The PCR product was cloned into the BamHI / HindIII site of the vector pETDuet1, and the obtained expression vector pQZ11 was verified by sequencing.

[0129] Table 1. Primers used in the construction of wild-type UGT76G1 expression vector

[0130]

[0131] 2. Protein expression and purification

[0132] Transfer the overnight cultured E. coli BL21(DE3) carrying the wild-type UGT76G1 expression vector pQZ11 to 1L LB at 1% v / v, and cultivate to OD at 37°C and 200rpm 600 ≈1.0. Induced by IPTG with a final concentration of 0.1 mM, the cells were collected after culturing overnight at 16°C for 18 hours. Use resuspension buffer to resuspend cells, add 1mM P...

Embodiment 2

[0139] Embodiment 2, mutant protein construction and expression

[0140] According to UGT76G1-substrate rebaudioside B ( Figure 4 ) and UDP complex structure and repeated verification, the inventors are located in the substrate binding pocket, and have determined several key amino acids located in the substrate binding pocket ( Figure 5 ), which interact with the glycosyl donor, glycosyl acceptor or aglycon core, respectively. According to the function of amino acids involved in the glycosylation process, the inventors divided them into 4 categories (Table 2), carried out single-point or multi-point mutations on these amino acids, and determined the role of mutant proteins in participating in the glycosylation process through in vitro enzymatic tests. Changes in catalytic activity and substrate recognition specificity.

[0141] Table 2. Amino acid mutation sites

[0142]

[0143] 1. Mutant construction

[0144] Using primers containing point mutation sites (Table 3), ...

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Abstract

The invention relates to a glycosyltransferase mutant and application thereof. According to the mutant glycosyltransferase UGT76G1 disclosed by the invention, the catalytic activity, substrate specificity and / or substrate specificity of the mutant glycosyltransferase UGT76G1 are / is changed, and the mutation at a specific site can obviously promote the catalytic activity of 1, 3 glycosylation of asubstrate containing 1, 2-diglucosyl (sophorosyl); so that catalytic activity of 1, 3 glycosylation on the basis of a glucose monosaccharide substrate is obviously weakened. Meanwhile, the invention also discloses a series of mutants for weakening the catalytic activity of glycosyltransferase UGT76G1, and the accumulation of specific stevioside intermediates can be increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a glycosyltransferase mutant and application thereof. Background technique [0002] Glycosylation is one of the most widespread modifications in natural product synthesis. In plants, glycosylation modification changes the solubility, stability, toxicity and physiological activity of natural products, and has the functions of detoxifying metabolites, preventing biological invasion, and changing the distribution interval of substances. The glycosylation of many plant-derived natural products is catalyzed by UDP-dependent glycosyltransferases (UGTs), which use UDP-activated sugars as glycosyl donors to specifically transfer sugar molecules to the glycosylation sites of acceptor molecules superior. At present, more than 2300 plant-derived UGTs have been discovered or annotated, but only about 20 UGTs have been analyzed for their protein structures. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N15/70C12P19/56
CPCC12N9/1048C12N15/63C12N15/70C12P19/56C12N9/10C12Q1/686
Inventor 王勇刘志凤孙雨伟吕华军张鹏李建戌刘海利李建华陈卓
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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