Recombinant escherichia coli for synthesizing glutathione and application of recombinant escherichia coli

A technology of recombinant Escherichia coli and glutathione, applied in the field of bioengineering, can solve the problems of high price of acetyl phosphate and low glutathione yield

Active Publication Date: 2021-09-14
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patent CN201310538982 overexpresses bifunctional glutathione synthase (STH) and acetate kinase (ack) in Escherichia coli. Although this method can achieve the purpose of ATP regeneration, the price of acetyl phosphate is relatively high
Zhang Xing et al. (J.Biotechnol.2017; (241): 163-169) combined recombinant GshF with polyphosphate kinase to form an ATP regeneration system in vitro to produce GSH, and reacted at 45°C for 5 hours The accumulation of GSH reached 28.5mM, and the yield based on cysteine ​​reached 81.4%. Although the yield based on cysteine ​​was high, the yield of glutathione was relatively low

Method used

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  • Recombinant escherichia coli for synthesizing glutathione and application of recombinant escherichia coli
  • Recombinant escherichia coli for synthesizing glutathione and application of recombinant escherichia coli
  • Recombinant escherichia coli for synthesizing glutathione and application of recombinant escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1. Connecting SpyCatcher / SpyTag to the N-terminus of GshF and the C-terminus of PPKJT to construct different recombinant expression plasmids and engineering bacteria

[0031] Select the PPKJT (amino acid sequence as shown in SEQ ID NO.4) screened by the laboratory and the GshF (amino acid sequence as shown in SEQ ID NO.2) of directed evolution, and respectively pass flexible linker(GGGGS) 2(nucleotide sequence as shown in SEQ ID NO.17: ggtggcggtg gctcgggcgg tggtgggtcg) connect SpyCatcher (amino acid sequence as shown in SEQ ID NO.1) and SpyTag (amino acid sequence as shown in SEQ ID NO.3) (SpyCatcher and SpyTag And the flexible linker was synthesized by Shanghai Bioengineering Co., Ltd.), CM3 was connected to the pET28a vector by homologous recombination, and PPKJT was synthesized by Shanghai Bioengineering Co., Ltd. Each gene has a separate promoter or RBS regulation, and the plasmid Transfer to BL21(DE3) respectively. The specific primer sequences are shown ...

Embodiment 2

[0036] Example 2. Construction of recombinant expression plasmids optimized for assembly protein expression and engineering bacteria

[0037] Insert different gene copy numbers into the same plasmid and regulate the expression of assembled protein through promoter or RBS, construct five different plasmids by homologous recombination, and transfer the plasmids into BL21(DE3) respectively. The specific primer sequences are shown in Table 1, and the sequences were synthesized by Shanghai Bioengineering Co., Ltd.

[0038] Preparation of linearized vector: PCR amplification was performed with pET28a as template and pET28a-F and pET28a-R as upstream and downstream primers respectively. The reaction system is: template 2 μl; PrimerSTAR Max 25 μl; forward and reverse primers 20 mM each; add sterile water to a total volume of 50 μl. The reaction conditions were: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s; annealing at 60°C for 30 s; extension at 72°C for 4 min, ...

Embodiment 3

[0041] Example 3. Verification of CM3 and JTT intracellular self-assembly and ATP regeneration

[0042] ATP is an essential cofactor in the production of GSH, so the regeneration of ATP is an important factor to reduce the cost of GSH production. Polyphosphate kinase PPK can use polyphosphate (PolyP) and ADP as substrates to achieve efficient regeneration of ATP. Compared with single-enzyme catalysis, the efficiency of intermediate transfer and cofactor regeneration can be improved by aggregating several enzymes involved in metabolic pathways in organisms to form a multi-enzyme complex, and at the same time, it can also reduce the feedback inhibition of products. GshF is intracellularly coupled to PPK to solve the problem of ATP regeneration during whole-cell catalyzed synthesis of GSH. In this study, it is hoped that SpyCatcher and SpyTag can realize dual-enzyme intracellular self-assembly and be used for ATP regeneration. The result is as figure 1 As shown in A and B, a l...

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Abstract

The invention provides recombinant escherichia coli for synthesizing glutathione and application of the recombinant escherichia coli. The N end of bifunctional glutathione synthetase GshF and the C end of polyphosphoric acid kinase PPK are connected with SpyCatcher and SpyTag respectively, two modified gene segments are integrated to the same plasmid vector at the same time, and the recombinant escherichia coli is obtained through conversion. After the obtained recombinant escherichia coli is subjected to high-density culture and optimization of the inducible expression condition, the maximum expression of a multi-enzyme compound is obtained, escherichia coli thalli are collected for efficient catalysis of whole cells or assembly of the multi-enzyme compound, centrifugation or membrane separation is directly conducted after the reaction is finished, and the cells or the multi-enzyme compound can be repeatedly used after being simply recycled. According to the process, the production cost of glutathione is effectively reduced, the utilization efficiency of a catalytic system is improved through efficient repeated utilization, the recombinant escherichia coli is simple to operate, and the application prospect is good.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant Escherichia coli for glutathione synthesis and an application thereof. Background technique [0002] Glutathione (GSH) is a biologically active tripeptide compound formed by condensation of L-glutamic acid, L-cysteine ​​and glycine through peptide bonds. In organisms, glutathione can participate in the oxidation-reduction process in the body, combine peroxides and free radicals to resist the damage of sulfhydryl groups by antioxidants, and at the same time resist the damage of free radicals to important organs. In addition, glutathione improves human immunity and anti-aging, and its effect on the retarded cells of the elderly is greater than that of young people. Glutathione also has a very significant effect on the treatment of leukopenia caused by radiation and radiopharmaceuticals. Effect. [0003] Enzymatic production of glutathione has the advantages o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P21/02C12R1/19
CPCC12N9/93C12Y603/02003C12N9/1229C12N15/70C12P21/02C12Y207/04001
Inventor 李志敏崔向伟
Owner EAST CHINA UNIV OF SCI & TECH
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