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Modified Escherichia coli engineering bacterium and method for producing citramalic acid by using modified Escherichia coli engineering bacterium

A technology of Escherichia coli and engineering bacteria, applied in the direction of microorganism-based methods, biochemical equipment and methods, genetic engineering, etc., can solve the problem of low conversion rate of citric acid

Active Publication Date: 2022-07-29
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aiming at solving the technical problem that the conversion rate of citramalic acid synthesized by microbial method is not high in the prior art

Method used

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  • Modified Escherichia coli engineering bacterium and method for producing citramalic acid by using modified Escherichia coli engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Escherichia coli engineering bacteria inserted with T7 RNA polymerase gene

[0036] The lactate dehydrogenase gene locus of Escherichia coli S17-3 was replaced and inserted into the T7 RNA polymerase gene by gene recombination, and then the kanamycin resistance gene as a selection marker was eliminated to obtain recombinant Escherichia coli Helicobacter sp. Escherichia coli S17-3-T7. The specific construction method includes the following steps:

[0037]The first step is to amplify the upstream and downstream long homology arm sequences, resistance fragments and T7 RNA polymerase fragments of the lactate dehydrogenase gene (ldhA). Homologous primers were designed to amplify the 300bp upstream and downstream DNA sequences of the Escherichia coli S17-3 ldhA gene by PCR respectively, and the pKD4 plasmid (commercial product) was used as a template for PCR amplification to obtain FRT sites at both ends. The gene fragment of kanamycin resistance is carried in th...

Embodiment 2

[0041] Example 2 Loss of function of the co-response factor RcsA

[0042] Using the method of gene recombination to replace and insert the resistance gene fragment at the auxiliary response factor gene locus of Escherichia coli S17-3-T7, and then eliminate the kanamycin resistance gene as a selection marker to obtain recombinant Escherichia coli Herba Escherichia coli S17-3-T7ΔrcsA, the construction process is as follows:

[0043] First, the upstream and downstream long homology arm sequences and the resistance gene fragments of the auxiliary response factor gene (rcsA) are amplified. The DNA sequences of 300bp upstream and downstream of the rcsA gene of Escherichia coli S17-3-T7 were amplified by PCR respectively. Then, PCR amplification was carried out using the pKD4 plasmid as a template to obtain a gene fragment with kanamycin resistance in the middle of the FRT site at both ends. Then use fusion PCR method to connect the above three fragments to form a recombinant gene ...

Embodiment 3

[0044] Example 3 Loss of function of the co-regulatory factor RcsB

[0045] Using the method of gene recombination to replace and insert the resistance gene fragment at the auxiliary response factor gene locus of Escherichia coli S17-3-T7, and then eliminate the kanamycin resistance gene as a selection marker to obtain recombinant Escherichia coli Helicobacter sp. Escherichia coli S17-3-T7ΔrcsB, the construction process is as follows:

[0046] First, the upstream and downstream long homology arm sequences and resistance gene fragments of the auxiliary response regulatory gene (rcsB) were amplified. The DNA sequences of 300bp upstream and downstream of the rcsB gene of Escherichia coli S17-3-T7 were amplified by PCR respectively. Then, PCR amplification was carried out using the pKD4 plasmid as a template to obtain a fragment containing the FRT site at both ends with a kanamycin resistance gene in the middle. Then use fusion PCR method to connect the above three fragments to ...

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Abstract

The invention discloses a modified Escherichia coli engineering bacterium and a method for producing citramalic acid by using the modified Escherichia coli engineering bacterium. According to the method, Escherichia coli S17-3 is taken as an original strain, and a T7 RNA polymerase gene for identifying a T7 promoter is inserted into a genome of the original strain, so that the modified escherichia coli engineering strain is obtained. The modified escherichia coli engineering bacteria further highly express citramalic acid synthase genes and one or more functional proteins of an inactivation auxiliary response factor RcsA, a response regulation factor RcsB, transmembrane sensing kinase RcsC, phosphate transporter RcsD and outer membrane lipoprotein RcsF. The invention provides novel industrial escherichia coli and a fermentation culture method, the strain is easy to culture, citramalic acid can be efficiently biosynthesized by using a cheap carbon source, and the production efficiency is higher than that of the existing biosynthesis method.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering bacteria, in particular to a modified Escherichia coli engineering bacteria and a method for producing citramalic acid. Background technique [0002] Citral malic acid, a commodity chemical, can be used as a synthetic precursor of methacrylic acid in the resin production industry, as an acidulant for soft drinks in the food industry, and as a skin care product with anti-wrinkle effects. In the field of medicine, the substance also has clinical effects on skin diseases such as ichthyosis. Citral malic acid is a five-carbon compound with a carboxyl group at the 1st and 4th positions, a hydroxyl group and a methyl group at the 2nd position, and its configuration includes (R)-citramalic acid and (S) -Citramalic acid. The production route of citramalic acid mainly includes chemical synthesis method and microbial transformation method. In the chemical production process, citramalic acid...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/60C12N15/31C12N15/70C12P7/44C12R1/19
CPCC12N9/1247C12Y207/07006C12Y401/03022C07K14/245C12N9/12C12N9/88C12N15/70C12P7/44Y02A50/30
Inventor 孙俊松陈艾谢雨康史吉平
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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