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Construction method and application of Pseudomonas jy-q fructose metabolism gene deletion strain

A technology of JY-Q and Pseudomonas, applied in the field of environmental microbial metabolic engineering, can solve problems such as degradation of nicotine

Active Publication Date: 2021-03-30
ZHEJIANG UNIV OF TECH
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  • Description
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Problems solved by technology

So far, there have been no reports at home and abroad on the degradation of nicotine and other pollutants by related strains for the deletion of the first step of fructose metabolism genes

Method used

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  • Construction method and application of Pseudomonas jy-q fructose metabolism gene deletion strain
  • Construction method and application of Pseudomonas jy-q fructose metabolism gene deletion strain
  • Construction method and application of Pseudomonas jy-q fructose metabolism gene deletion strain

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Embodiment Construction

[0028] (1) Based on the genome-wide information of Pseudomonas sp.JY-Q, this experiment analyzed the pathways related to fructose metabolism through the existing gene annotation information. We found that there may be two fructose metabolism initiation enzyme genes in the first step of fructose metabolism, PTS1 and PTS2, respectively, are isoenzymes, both annotated as fructose phosphotransferase.

[0029](2) Gene knockout: the present invention adopts the double exchange homologous recombination method to realize seamless gene knockout. First, construct a knockout vector for the gene of interest. Design primers, add an EcoR Ⅰ restriction site at the 5' end of the forward primer of the upstream homology arm of the target gene, and add a BamH Ⅰ restriction site at the 5' end of the reverse primer of the downstream homology arm of the target gene to fuse PCR After the upstream and downstream homology arms (about 1000bp) of the target gene are amplified, they are recombined with ...

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Abstract

The invention discloses a construction method and application of pseudomonas JY-Q fructose metabolism gene deleted strain. JY-Q / 5Delta is used as an original strain, the following two isozyme target genes expressing fructose phosphotransferase are determined through genome information analysis of the strain; a knockout vector of a target gene is constructed; the knockout vector is transformed intoan escherichia coli deleted strain WM3064 by heat transfer, which is a donor strain, and pseudomonas JY-Q is used as a receptor bacterium for carrying out parent conjugation; the recombinants exchanged for the first time are screened by utilizing the Kanamycin resistance gene on the vector; and after the obtained strain is subjected to overnight culture expansion, a reverse screening marker SacBgene carried on a carrier is utilized to carry out second exchange of recombinants to obtain the selectively degraded pseudomonas JY-Q fructose metabolic deleted strain. The pseudomonas JY-Q fructosemetabolism deleted strain is applied in nicotine degradation of waste tobacco leaf water extract (TWE).

Description

technical field [0001] The invention belongs to the technical field of environmental microbial metabolism engineering. The enzyme gene that catalyzes the initial step of fructose metabolism in Pseudomonas sp. It is applied to degrade nicotine more efficiently in the environment where nicotine and fructose coexist (such as waste tobacco leaf water extract, TWE). [0002] technical background [0003] Nicotine, also known as nicotine, is composed of three elements C, H and N. It is a nitrogen-containing heterocyclic organic compound with a molecular weight of 162.23Da and a molecular formula of C 10 h 14 N 2 , chemically named as 1-methyl-2-(3-pyridyl)pyrrolidine, is a well-known toxic substance, which will have varying toxic and side effects on different human bodies and environments, so it is necessary to treat nicotine and its related Treatment of sources of pollution. At present, there are countless examples of microbial degradation of various organic pollutants in the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/78C12N15/90C12N15/54A62D3/02A62D101/26
Inventor 钟卫鸿章柳泽王艳颖何子良胡祯怡金维华
Owner ZHEJIANG UNIV OF TECH
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