Corynebacterium glutamicum recombinant bacterium capable of effectively using pyruvic acid and construction method and application of corynebacterium glutamicum recombinant bacterium

A technology for encoding Corynebacterium glutamicum and alanine aminotransferase, applied in the field of genetic engineering

Active Publication Date: 2020-04-10
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether these genes promote or inhibit the synthesis of pyruvate is not known according to the literature

Method used

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  • Corynebacterium glutamicum recombinant bacterium capable of effectively using pyruvic acid and construction method and application of corynebacterium glutamicum recombinant bacterium
  • Corynebacterium glutamicum recombinant bacterium capable of effectively using pyruvic acid and construction method and application of corynebacterium glutamicum recombinant bacterium
  • Corynebacterium glutamicum recombinant bacterium capable of effectively using pyruvic acid and construction method and application of corynebacterium glutamicum recombinant bacterium

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Experimental program
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Effect test

Embodiment 1

[0033] The construction of embodiment 1 recombinant plasmid

[0034] Using the genome of Corynebacterium glutamicum ATCC XQ-9ΔltbR as a template, and using pyc-U-F / pyc-U-R and pyc-D-F / pyc-D-R as primers respectively (Table 1), pyc-U and pyc were obtained after PCR amplification -D PCR product, the obtained pyc-U and pyc-D fragments were subjected to fusion PCR to obtain the homology arm fragment Δpyc. The above PCR product was enzyme-ligated with the linearized plasmid pK18mobsacB after double digestion with XbaI and HindIII to construct the plasmid pK18mobsacB-Δpyc.

[0035] Using the genome of Corynebacterium glutamicum ATCCXQ-9ΔltbR as a template, using avtA-U-F / avtA-U-R and avtA-D-F / avtA-D-R as primers respectively (Table 1), avtA-U and avtA-U and avtA- Fragment D, the obtained avtA-U and avtA-D fragments were seamlessly cloned with the linearized plasmid pK18mobsacB cut with SmaI and HindIII to construct the plasmid pK18mobsacB-ΔavtA.

[0036] According to the construct...

Embodiment 2

[0045] Construction of embodiment 2 recombinant strain WL-1

[0046] Using Corynebacterium glutamicum ATCC XQ-9ΔltbR (named ΔLtbR) as the starting strain, the correct plasmid pK18mobsacB-Δpyc verified in the above steps was electroporated to transform C. glutamicumXQ-9ΔltbR competent, and the target recombinant strain C. glutamicum XQ-9ΔltbRΔpyc, which was named WL-1. The steps for screening the target recombinant strain are as follows: the first homologous recombination transformant was obtained based on 30° C. cultivation and screening on LBG solid culture containing 50 μg / mL kanamycin. Then, insert the recombined transformant into LBGS liquid culture containing 100g / L sucrose and cultivate it at 30°C. The sucrose in the medium will cause the linearized integrated gene fragment containing sacB gene to undergo a second reaction with the target gene in genomic DNA. Homologous recombination, LBGS cultured bacteria were separated by streaking on LBG plate, the colony grown on t...

Embodiment 3

[0047] Construction of embodiment 3 recombinant strain WL-2

[0048] Using Corynebacterium glutamicum ATCC XQ-9ΔltbR (named ΔLtbR) as the starting strain, the correct plasmid pK18mobsacB-Δppc verified in the above steps was electroporated to transform C. glutamicumXQ-9ΔltbR competent, and the target recombinant strain C. glutamicum XQ-9ΔltbRΔppc, which was named WL-2. The method for screening the target recombinant strain is the same as in Example 2, except that the upstream and downstream primers ppc-U-F / ppc-D-R of the target gene ppc are used to perform PCR respectively, and the PCR products are sequenced and identified, and PCR is performed respectively.

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Abstract

The invention relates to a corynebacterium glutamicum recombinant bacterium for enhancing effective utilization of pyruvic acid and improving the yield of leucine and a construction method of the corynebacterium glutamicum recombinant bacterium, and belongs to the field of gene engineering. According to the invention, a genetic engineering method is applied to knock out a pyruvate carboxylase genepyc participating in an intracellular oxaloacetic acid supplementing pathway and an alanine transaminase gene avtA participating in alanine synthesis in corynebacterium glutamicum; meanwhile, in order to weaken synthesis capacity of alanine in a competing branch, a T3 terminator is added in front of an alaT gene to weaken the expression level of the alaT gene, so that effective utilization of recombinant bacterial intracellular pyruvic acid is realized, and the recombinant corynebacterium glutamicum with an improved L-leucine yield is obtained. The leucine yield of the recombinant corynebacterium glutamicum is increased by 15.3% compared with an original strain. According to the invention, utilization of pyruvic acid is successfully strengthened, the synthetic flux of pyruvic acid to L-leucine is improved, and a new idea is provided for breeding pyruvic acid amino acid high-yield bacteria.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant Corynebacterium glutamicum capable of effectively utilizing pyruvate and its construction and application. Background technique [0002] L-leucine is one of the eight essential amino acids required by humans and animals. It is collectively referred to as branched-chain amino acids because it has the same branched methyl side chain structure as L-valine and L-isoleucine. L-leucine has a variety of physiological functions and is widely used in food industry, feed industry and pharmaceutical industries. At the same time, the amount of L-leucine in amino acid intravenous infusion is increasing day by day. It is one of the indispensable raw materials used in clinical amino acid compound infusion, and it plays an active role in maintaining the nutritional needs of critically ill patients and saving their lives. Corynebacterium glutamicum is currently the main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/15
CPCC12N9/93C12N9/1096C12Y604/01001C12Y206/01002C12N15/77C12P13/06
Inventor 张伟国王颖妤史可徐建中郝宇宸朱晗
Owner JIANGNAN UNIV
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