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Construction method of pseudomonas denitrificans lacking malonic semialdehyde bypass metabolic pathway

A technology of Pseudomonas denitrification and malonate semialdehyde, applied in the biological field, can solve the problems of low yield and achieve the effect of increasing yield

Pending Publication Date: 2022-04-12
XUZHOU NORMAL UNIVERSITY
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Problems solved by technology

However, the yield of the bacterium converting acetic acid into 3-HP is low

Method used

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  • Construction method of pseudomonas denitrificans lacking malonic semialdehyde bypass metabolic pathway

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Embodiment 1

[0026] 1. Knockout of malonate semialdehyde bypass metabolic pathway

[0027] The starting strain of the present invention is Pseudomonas denitrificans ZAP01 constructed in the previous stage. The mmsAI, mmsAII, mmsAIII and BAPAT genes were knocked out separately and in combination in the genomic DNA of the strain to obtain mutant strains ZAP01ΔmmsAI, ZAP01ΔmmsAII, ZAP01ΔmmsAIII, ZAP01ΔBAPAT, ZAP01ΔmmsAIΔmmsAIIΔmmsAIII and ZAP01ΔmmsAIΔmmsAIIΔmmsAIIIΔBAPAT.

[0028] In the present invention, the above-mentioned genes are knocked out by using the technique of a traceless gene knockout. The specific operation is as follows (taking the knockout of the gene mmsAI as an example):

[0029] For the knockout of the gene mmsAI, at first the DNA fragment mmsAI US (SEQ ID No.2) of about 700bp upstream sequence of the mmsAI gene (SEQ ID No.1) was cloned using the genomic DNA of Pseudomonas denitrophila ATCC13867 as a template, and mmsAI The DNA fragment mmsI DS (SEQ ID No.3) of about 700...

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Abstract

The construction method of the pseudomonas denitrificans lacking malonic semialdehyde bypass metabolic pathway comprises the following steps: (1) starting from the constructed pseudomonas denitrificans ZAP01, respectively knocking out mmsAI, mmsAII, mmsAIII and BAPAT genes in genomic DNA (Deoxyribonucleic Acid) of the strain independently and in a combined manner to obtain a plurality of mutant strains; (2) by constructing a plasmid pUCPK-AM, overexpressing a 3-HP synthesis path related enzyme to convert acetic acid to produce 3-HP; and (3) screening on an LB plate containing 30mg / L kanamycin to obtain a target mutant recombinant bacterium. According to the method disclosed by the invention, malonic semialdehyde dehydrogenase and beta-alanine-pyruvate transaminase are knocked out from pseudomonas denitrificans, so that a malonic semialdehyde branch metabolic pathway is completely blocked, and more malonic semialdehyde is used for synthesizing 3-HP, so that the yield of 3-HP synthesized by acetic acid is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a strain of Pseudomonas denitrifica deficient in malonate semialdehyde bypass metabolic pathway. Background technique [0002] The metabolic pathway of malonyl-CoA to 3-hydroxypropionate (3-HP) is currently the main route of biosynthesis of 3-HP with high prospects. Intracellular malonyl-CoA is synthesized into 3-HP by acetyl-CoA carboxylase (ACC) carboxylating acetyl-coenzyme semialdehyde dehydrogenase (MCRN)). In the early stage of the laboratory, a recombinant strain that metabolized acetate and biosynthesized 3-HP through malonyl-CoA pathway was constructed by overexpressing ACC, MCRC and MCRN in Pseudomonas denitrifica ZAP. However, the conversion of acetic acid to 3-HP by the bacteria was low. After the activation of acetic acid to generate the intermediate metabolite acetyl-CoA, the intermediate products malonyl-CoA and malonate semialdehyde are seq...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12N15/53C12N15/54C12P7/42C12R1/38
Inventor 周生芳李红雨章颖莉
Owner XUZHOU NORMAL UNIVERSITY
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