Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same

A technology of glycosyltransferase and mutants, which is applied in the field of bioengineering, can solve the problems of low soluble expression, limited efficient application of glycosyltransferase, etc., and achieves high catalytic efficiency, high yield, and improved enzymatic activity.

Active Publication Date: 2021-02-19
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The soluble expression level of stevia-derived UGT76G1 in Escherichia coli is very low, which limits the efficient application of glycosyltransferase

Method used

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  • Glycosyltransferase mutant and method for catalytically synthesizing rebaudioside A by using same

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Experimental program
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Effect test

Embodiment 1

[0042] The preparation of embodiment 1 glycosyltransferase mutant

[0043] PCR amplification of the coding gene of the site-directed mutant: using PCR amplification technology, the unmutated strain pRSFDUet-UGT76G1-AtSUS1 was used as the template DNA for rapid mutation:

[0044] The primers for site-directed mutagenesis of Q72E are:

[0045] Forward primer: 5'-AACGACCCG GAA GATGAACGCATCTCTAATCTGCCG-3' (the underline is the mutant base) as shown in SEQ ID NO:11

[0046] Reverse primer: 5'-GCGTTCATC TTC CGGGTCGTTATCCAGAATAAAACG-3' (the underline is the mutant base) as shown in SEQ ID NO:12

[0047] The primers for N196D site-directed mutation are:

[0048] Forward primer: 5'-GCGTACTCG GAC TGGCAGATTCTGAAAGAAATCCTG-3' (the underline is the mutant base) as shown in SEQ ID NO:13

[0049] Reverse primer: 5'-AATCTGCCA GTC CGAGTACGCTGACTTAATATCCTT-3' (the underline is the mutant base) as shown in SEQ ID NO:14

[0050] The primers for T319E site-directed mutation are:

[00...

Embodiment 2

[0062] The construction of embodiment 2 double enzyme expression system

[0063] Select the double gene expression vector pRSFDuet-1, in the vector Nde I / xho The nucleotide sequence of glycosyltransferase UGT76G1 (Accession: AY345974) or its mutant nucleotide sequence (Accession: AY345974) derived from Stevia rebaudiana is inserted at the I site. Nco I / Eco RI were respectively inserted into the nucleotide sequence of sucrose synthase AtSUS3 (Accession: AY051001) derived from Arabidopsis thaliana to constitute the corresponding recombinant expression plasmid, which was constructed on the pRSFDuet-1 vector. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) to form a double-enzyme co-expression recombinant bacterium.

Embodiment 3

[0064]Fermentation induction of embodiment 3 mutant enzyme

[0065] Spread the recombinant bacteria constructed by single-point mutants Q72E, N196D, T319E and multi-point mutants Q72E-N196D, N196D-T319E, Q72E-N196D-T319E of glycosyltransferase UGT76G1 (UniProt ID: Q6VAB4) on 50 µg / L kanamycin LB solid plate (NaCl 10 g / L, yeast powder 5 g / L, peptone 10 g / L, agar 20 g / L), placed in a 37°C incubator for overnight constant temperature cultivation. On the next day, pick a single colony from the plate to a shake tube filled with 5 mL of LB liquid medium (containing 50 μg / L kanamycin), and culture overnight at 37°C in a shaker at 200 rpm as a seed solution. The seed solution was transferred to 100 mL TB medium (containing 50 μg / L kanamycin) according to the inoculum volume of 1% (v:v). Place in a shaker at 37°C, 200 rpm, and shake for culture. After 2 h, adjust the temperature to 25°C and continue to cultivate for 20-22 h.

[0066] The fermentation broth was collected and refriger...

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Abstract

The invention discloses a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside A by using the same. The amino acid sequence of original glycosyltransferase is SEQ ID NO:1. According to the experiment, Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160 and Thr319 are screened on the basis of prediction obtained by starting from surface residues Q, N and T ofUGT76G1 and combining data analysis results of solvent accessible surface area, B-factor and the like, and finally, a UGT-SuSy system of the better mutant strain 76G1_Q72E-N196D-T319E is screened outby virtue of single-point or multi-point iterative mutation in Thr81, so as to realize efficient catalytic synthesis of the rebaudioside A. The glycosyltransferase UGT76G1 or a mutant gene thereof isconnected with a sucrose synthase gene to obtain a recombinant plasmid, and a double-enzyme co-expressed recombinant strain is constructed. By constructing a double-enzyme system, regeneration of in-vivo UDPG is realized, the problem of sources of expensive glycosyl donors is effectively solved, the cost is reduced, and application of biotechnology industry is promoted. The mutant is simple to prepare, the rebaudioside A is synthesized through efficient catalysis in a short time, and the biological enzyme catalysis method is green, environmentally friendly and pollution-free and is more suitable for current green industrial processing and production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a glycosyltransferase mutant and a method for catalyzing and synthesizing rebaudioside A. Background technique [0002] Steviol glycosides belong to tetracyclic diterpenoids, which are easily soluble in water and dioxane, and soluble in some organic reagents. Good heat resistance, not easy to decompose when exposed to light. The basic skeleton of steviol glycosides is tetracyclic diterpene glycosides, and the diversity of steviol glycosides is formed due to the type of glycosyl, the position of addition (C13, C19) and the number of glycosyls. Mainly include steviol, stevioside, rebaudioside A, B, C, D, E, M, etc. Among them, stevioside (Stevioside, St glycoside) has the highest content in Stevia rebaudiana, followed by rebaudioside A (Rebaudioside A, RA glycoside), accounting for about 3.8% of the dry weight of leaves. [0003] Rebaudioside A (Rebaudioside A,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/56C12P19/18C12R1/19
CPCC12N9/1051C12N15/70C12P19/56C12P19/18Y02P20/584
Inventor 李艳贾红华余杰孙萍齐雪莲严明
Owner NANJING UNIV OF TECH
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