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Recombinant Escherichia coli engineering bacteria and its method for preparing s-adenosylmethionine

A technology for recombining Escherichia coli and adenosylmethionine, applied in the field of enzyme engineering, can solve the problems of high cost, easy inactivation, instability of S-adenosylmethionine synthase and the like

Active Publication Date: 2021-08-20
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method is mainly limited by the acquisition of highly catalytically active S-adenosylmethionine synthetase and the high cost of ATP (600-700 yuan / kg), and in the in vitro catalytic process, S-adenosylmethionine Acid synthase is very unstable and easy to inactivate, so the cost of enzymatic catalytic synthesis is much higher than that of microbial fermentation, and there is no report on the industrial application of this method

Method used

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  • Recombinant Escherichia coli engineering bacteria and its method for preparing s-adenosylmethionine
  • Recombinant Escherichia coli engineering bacteria and its method for preparing s-adenosylmethionine
  • Recombinant Escherichia coli engineering bacteria and its method for preparing s-adenosylmethionine

Examples

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Effect test

Embodiment 1

[0038] Example 1: Construction of S-adenosylmethionine synthetase expression strain

[0039] 1. Construction of expression strain BL21(DE3) / pET30a-SUMO-MATI

[0040] At the same time, download the amino acid sequence of S-adenosylmethionine synthase MATI derived from rat liver in GenBank (SEQ ID NO.1 in this article, corresponding to GenBank accession number: NP_036992.2) and the small molecule ubiquitin-like modified protein (SUMO ) (SEQ ID NO.12 herein, corresponding to GenBank accession number: AQS95516.1). Link the SUMO amino acid sequence to the N-terminal of the MATI protein amino acid sequence to form a fusion protein sequence, which is provided to Beijing Qingke Biotechnology Co., Ltd. for the whole gene synthesis of the encoding nucleic acid (using E. coli preferred codons). It was constructed into the prokaryotic expression vector pET30a to form a recombinant plasmid vector pET30a-SUMO-MATI. Transform the recombinant plasmid vector pET30a-SUMO-MATI into Escherichia...

Embodiment 2

[0059] Embodiment 2: Construction of ATP synthetase recombinant expression vector

[0060] The amino acid sequence of ATP synthesis-related enzymes (the amino acid sequence of adenosine kinase (EC 2.7.1.20, AK) is shown in SEQ ID NO.9; the amino acid sequence of adenosine kinase (EC 2.7.4.3, ADK) is shown in SEQ ID NO .10; the amino acid sequence of polyphosphate kinase (EC 2.7.4.1, PPK) is shown in SEQ ID NO.11) provided to Beijing Qingke Biotechnology Co., Ltd. for the whole gene synthesis of the encoding nucleic acid (using E. coli preferred codons). Constructed into the prokaryotic expression vector pCDFDuet-1 to form a three-gene co-expression recombinant plasmid vector pCDFDuet-1-AK-ADK-PPK (see the construction flow chart figure 2 ). In pCDFDuet-1-AK-ADK-PPK, the expression genes of adenosine kinase AK and adenylate kinase ADK were constructed into the same reading frame of pCDFDuet-1 to form a double-gene tandem expression, and the expression gene of polyphosphate ki...

Embodiment 3

[0061] Example 3: Construction of S-adenosylmethionine synthetase and ATP synthetase co-expression strain

[0062] Using BL21(DE3) / pET30a-SUMO-MATI as the starting strain, chemically competent cells were prepared, and the three-gene co-expression recombinant plasmid vector pCDFDuet-1-AK-ADK–PPK ​​was transformed into competent cells by using calcium chloride heat shock transformation method Cells BL21(DE3) / pET30a-SUMO-MATI were cultured at 37°C and 220r / min for 60 min, and the transformed cells were coated with kanamycin sulfate (50 μg / mL) and streptomycin ( 25 μg / mL) solid LB medium for positive clone screening, the colony grown on the plate is the co-expression strain BL21(DE3) / pET30a-SUMO of S-adenosylmethionine synthase MATI and ATP synthase -MATI / pCDFDuet-1-AK-ADK-PPK.

[0063] Build in the same way:

[0064] S-adenosylmethionine synthase MATI-1 and ATP synthase co-expression strain BL21(DE3) / pET30a-SUMO-MATI-1 / pCDFDuet-1-AK-ADK-PPK,

[0065] The co-expression strain B...

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Abstract

The invention belongs to the technical field of enzyme engineering, and relates to a recombinant Escherichia coli engineering bacterium and a method for preparing S-adenosylmethionine by using the same. The recombinant Escherichia coli engineering bacterium contains a recombinant expression vector, and the recombinant expression vector contains a gene expressing S-adenosylmethionine synthetase and a gene expressing ATP synthetase. Compared with the traditional fermentation method and in vitro enzyme-catalyzed synthesis method, the recombinant Escherichia coli engineering bacteria of the present invention and the method for preparing S-adenosylmethionine can be prepared with simpler, lower cost and high yield. ‑adenosylmethionine.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a recombinant Escherichia coli engineering bacterium and a method for preparing S-adenosylmethionine by using it. Background technique [0002] S-adenosyl-L-methionine (SAM) is an important intermediate product in the metabolic process of organisms. It exists in all living cells and has the functions of transmethylation, transsulfurization and transaminopropylation. . SAM is a pair of chiral substances, there are two isomers: (R, S)-SAM and (S, S)-SAM, only (S, S)-SAM has biological activity. Clinically, SAM has extremely high medicinal value. It is mainly used to treat intrahepatic cholestasis, and can also be used to treat viral hepatitis and alcoholic liver disease, improve liver function, and relieve depression symptoms in patients with depression (with antidepressant effect), etc. . SAM can also be used in combination with L-dopa to treat Parkinson's disease, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N9/10C12N15/70C12P13/12C12R1/19
CPCC12N9/1085C12N9/1229C12N15/70C12P13/12C12Y205/01006C12Y207/0102C12Y207/04001C12Y207/04003
Inventor 周晶辉许岗黄斌曾红宇刘洋田艳
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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