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Glycosyl transferase mutant and method for catalytically synthesizing rebaudioside M by using glycosyl transferase mutant

A technology of glycosyltransferase and mutant, applied in the field of bioengineering, can solve the problems of poor water solubility and low efficiency, and achieve the effects of increasing yield, high catalytic efficiency and simple operation

Active Publication Date: 2021-10-01
XINGHUA GL STEVIA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large molecular weight and poor water solubility of the intermediate product rebaudioside D, the efficiency of the wild-type enzyme UGT76G1 in catalyzing the synthesis of rebaudioside M from rebaudioside E is low, so it is necessary to carry out molecular modification of the glycosyltransferase UGT76G1 to improve The catalytic efficiency of its enzyme

Method used

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  • Glycosyl transferase mutant and method for catalytically synthesizing rebaudioside M by using glycosyl transferase mutant
  • Glycosyl transferase mutant and method for catalytically synthesizing rebaudioside M by using glycosyl transferase mutant
  • Glycosyl transferase mutant and method for catalytically synthesizing rebaudioside M by using glycosyl transferase mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Glycosyltransferase UGT76G1 mutation site screening

[0060] Transformation by directed evolution theory method. A crystal structure was selected and docked to obtain a complex model. A single-point mutation was performed on the amino acid residues in the structure and around RebE. The distance (Distance) between the NE2 of the key residue of the active center His25 in the structure and the O atom of the C13 glycoside of the substrate RebE was determined. Sort according to the value of affinity (Affinity). Combined with Affinity and Distance, the screened mutants are G87D, S147D, S147N, S195Q, L200Y, T284S, S285W, and G378P, as shown in Table 1.

[0061] Table 1 Mutant affinity value data

[0062]

[0063] Note: Affinity stands for affinity; Distance stands for distance between atoms; vdw stands for van der Waals force.

Embodiment 2

[0064] The construction of embodiment 2 double enzyme expression system

[0065] Select the double-gene expression vector pRSFDuet-1, insert the stevia-derived glycosyltransferase UGT76G1 at the NdeI / XhoI site of the vector, the nucleotide sequence is shown in SEQ ID NO: 2; insert sucrose at the NcoI / EcoRI site respectively Synthase StSUS1 or sucrose synthase McSuSy, the nucleotide sequences of which are shown in SEQ ID NO: 11 and SEQ ID NO: 12 constitute two corresponding recombinant expression plasmids, and the recombinant plasmids are respectively introduced into Escherichia coli BL21 (DE3) Transformation is carried out to obtain two double-enzyme co-expression recombinant strains.

Embodiment 3

[0066] Example 3 Preparation of Glycosyltransferase UGT76G1 Mutant

[0067] Using PCR amplification technology, the wild-type UGT76G1-StSUS1 recombinant plasmid was used as template DNA to carry out base-directed mutation.

[0068] Primers for G87D site-directed mutagenesis:

[0069] Forward primer: 5'-CCGCTGGCG GAC ATGCGTATTCCGATTATCAACGAA-3' (the underline is the mutant base), as shown in SEQ ID NO:13.

[0070] Reverse primer: 5'-AATACGCAT GTC CGCCAGCGGGCCGTGGGTCGGCAG-3' (the underline is the mutated base), as shown in SEQ ID NO:14.

[0071] Primers for S147D site-directed mutagenesis:

[0072] Forward primer: 5'-CTGATGACC GAC AGCCTGTTCAATTTTCATGCCCAC-3' (the underline is the mutant base), as shown in SEQ ID NO:15.

[0073] Reverse primer: 5'-GAACAGGCT GTC GGTCATCAGGACCAGGCGACGCAG-3' (the underline is the mutant base), as shown in SEQ ID NO:16.

[0074] Primers for S147N site-directed mutagenesis:

[0075] Forward primer: 5'-CTGATGACC AAC AGCCTGTTCAATTTTCATGCC...

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Abstract

The invention discloses a glycosyl transferase mutant and a method for catalytically synthesizing rebaudioside M by using the glycosyl transferase mutant. The mutant is obtained by performing mutation on the basis of a glycosyl transferase amino acid sequence shown as SEQ ID NO: 1, performing induced expression on a mutant strain to obtain a mutant enzyme, and catalyzing 20g / L RebE to synthesize 12.8 g / L RebM by using the mutant enzyme as a catalyst and the enzymic method. The kinetic parameters of the mutant S195Q on rebaudioside E and rebaudioside D and the Michaelis constant of the mutant are 56.34 + / -2.02 mu M and 214.48 + / -14.54 mu M respectively, and are 1 / 3 and 2 / 5 of those of a wild type. The glycosyl transferase mutant is coupled with sucrose synthase to realize efficient catalytic synthesis of rebaudioside M. According to the present invention, the recombinant strain of the glycosyl transferase UGT76G1 or the mutant thereof and the sucrose synthase is constructed so as to achieve the efficient catalytic synthesis of the rebaudioside M; the method has the optimal yield in the current enzymatic catalytic synthesis experiment of rebaudioside M, and is green, environment-friendly and pollution-free.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for catalyzing and synthesizing rebaudioside M by a glycosyltransferase mutant. Background technique [0002] Steviol glycoside is a diterpenoid compound with diterpenoid steviol as the basic skeleton. Steviol glycosides mainly exist in stevia leaves, which contain stevioside, rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, rebaudioside M and other natural Steviol glycosides, of which rebaudioside M has very little content in dry leaves, but its sweetness and taste are good, is currently recognized as the most ideal steviol glycoside product. As a new type of natural sweetener, rebaudioside M is derived from stevia rebaudiana, which has the advantages of high sweetness and low calorie, but there are few studies on the synthesis of rebaudioside M. [0003] Glycosyltransferases mainly exist in plants, which can connect sugar groups to d...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/56C12R1/19
CPCC12N9/1048C12N9/1062C12N15/70C12P19/56C12Y204/01013Y02P20/584C12N9/1051C12P19/18
Inventor 贾红华李艳陶叶慧余杰林磊马蕊琪
Owner XINGHUA GL STEVIA CO LTD
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