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Genetic Engineering Bacteria Deleting 21 Coding Exopolysaccharide Synthesis Genes and Its Application

An extracellular polysaccharide, encoding gene technology, applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low PHB yield and the like

Active Publication Date: 2021-01-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In the article "Engineering Escherichia coli for an efficientaerobic fermentation platform" (published date: October 12, 2009), Kang, Z. et al. continuously knocked out the glucose transport system gene ptsG and heteroacid synthesis bypass in Escherichia coli MG1655 PHB synthesis increased from 14.91% to 28.92% after gene poxB, pta and iclR, but PHB production is still low and needs to be further improved

Method used

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  • Genetic Engineering Bacteria Deleting 21 Coding Exopolysaccharide Synthesis Genes and Its Application
  • Genetic Engineering Bacteria Deleting 21 Coding Exopolysaccharide Synthesis Genes and Its Application
  • Genetic Engineering Bacteria Deleting 21 Coding Exopolysaccharide Synthesis Genes and Its Application

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Embodiment 1

[0045] Construction of embodiment 1 knockout plasmid

[0046] Using the CRISPR / Cas9 knockout system to knock out exopolysaccharide synthesis and transport gene clusters requires the construction of a knockout plasmid pTargetF-galF-wza. The construction process of the plasmid is as follows:

[0047] (1) Through the analysis of the website http: / / crispr.mit.edu / , select a 20nt N that is complementary to the target sequence of the target gene 20 Sequence (see the underlined sequence in the primer pTargetF-galF-wza-F in Table 1).

[0048] (2) Using the plasmid pTargetF as a template, use the forward primer pTargetF-galF-wza-F (the 5' end is the N in step (1) 20 sequence) and reverse primer pTargetF-R, amplified to obtain the introduction of N 20 Sequence of the open-circle plasmid. The PCR amplification product was verified by electrophoresis and purified and recovered.

[0049] (3) Since the recovered product may contain the template plasmid pTargetF, which will affect subseq...

Embodiment 2

[0053] The construction of embodiment 2 genetically engineered bacteria WJW09

[0054] Using the CRISPR / Cas9 knockout system to knock out the exopolysaccharide synthesis and transport gene cluster to obtain WJW09, the gene cluster includes wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, wcaG, wcaH, wcaI, There are 21 genes including manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM, and galF. The NCBI accession numbers of these gene sequences are "BAE76576.1", "BAE76575.1", "BAA15913.1", "BAA15912. 1", "BAA15911.1", "BAE76574.1", "BAE76573.1", "BAE76572.1", "BAA15910.1", "BAA15909.1", "BAA15908.1", "BAA15907.1" , "BAA15906.1", "BAA15905.1", "BAA15901.1", "BAA15900.1", "BAA15899.1", "BAE76571.1", "BAA15898.1", "BAA15897.1", " BAA15896.1". Specific steps are as follows:

[0055] (1) Construction of homology arm knockout fragments

[0056] Extract the genome of Escherichia coli W3110, use the genome as a template, and use the homology arm primers galF-wza-U-F / galF-wza-U-R, galF...

Embodiment 3

[0065] The growth status of embodiment 3 bacterial strain WJW09

[0066] LB medium: yeast powder 5g / L, tryptone 10g / L and NaCl 10g / L.

[0067] WJW09 and W3110 were inoculated into LB medium respectively, cultured at 37°C, and their growth curves were measured.

[0068] The results show that: if image 3 As shown, the growth of strains WJW09 and W3110 is basically the same, indicating that knockout of 21 exopolysaccharide synthesis and transport-related genes does not affect cell growth and can be used in industrial production.

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Abstract

The invention discloses a genetically engineered bacterium with deficiency of 21 encoded exopolysaccharide synthetic genes and application thereof, and belongs to the field of gene engineering and fermentation engineering. A mutant bacterium WJW09 is obtained by knocking out 21 genes in charge of exopolysaccharide synthesis and transfer on an escherichia coli W3110 genome through a CRISPR / Cas9 knockout system, with the WJW09 strain as a host bacterium, a PHB synthesis gene cluster phaCAB is heterologously expressed through pBHR68, PHB synthesized through WJW09 / pBHR68 can reach 57.2% of the drycell weight, while PHB synthesized through W3110 / pBHR68 only accounts for 1.5% of the dry cell weight. The PHB volumetric production of the recombinant bacterium WJW09 / pBHR68 is 38 times that of thecontrast bacterium W3110 / pBHR68 (1.5%).

Description

technical field [0001] The invention relates to a genetically engineered bacterium lacking 21 coding exopolysaccharide synthesis genes and its application, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Extracellular polysaccharides (EPSs) refer to polysaccharide-containing structures secreted by microorganisms into the surrounding environment, and are polysaccharide components outside the cell membrane of Gram-negative bacteria. Genes related to exopolysaccharide synthesis exist in the genome in the form of gene clusters, encoding glycosyltransferases, polymerases and branching enzymes, and are responsible for adding modification components. In Escherichia coli, exopolysaccharides are mostly synthesized in the form of clarified acid, which is attached to the outer layer of the outer cell membrane and consists of multiple sugar units, each sugar unit contains 6 sugar molecules, and the synthesis of these sugar molec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N9/1029C12N9/18C12P7/625C12Y101/01036C12Y301/01075
Inventor 王小元王建莉马文渐王甜忆周晴汪晨卉李烨
Owner JIANGNAN UNIV
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