A strain of Escherichia coli with reduced membrane wall and its high-production phb application

A technology of Escherichia coli and recombinant bacteria, applied in the fields of genetic engineering and fermentation engineering, can solve the problems such as the inability of PHB to meet the needs of industrial production

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods make the improvement of PHB output still unable to meet the needs of industrial production

Method used

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  • A strain of Escherichia coli with reduced membrane wall and its high-production phb application
  • A strain of Escherichia coli with reduced membrane wall and its high-production phb application
  • A strain of Escherichia coli with reduced membrane wall and its high-production phb application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of Engineering Bacteria WJW00

[0055] The engineering bacteria WJW00 has been published in the SCI paper "Construction and Characterization of an Escherichia coli Mutant Producing Kdo 2 -Lipid A" (disclosure date: March 13, 2014). The specific construction process is as follows:

[0056] (1) Obtaining the gmhD gene knockout fragment

[0057] The gmhD gene knockout fragment is obtained by chemical total synthesis or PCR step-by-step amplification, and its two ends are the upstream and downstream homology arms of the gmhD gene, and the middle is a kan fragment with an FRT site. The nucleotide sequence of the gmhD gene knockout fragment is shown in SEQ ID NO.2. The gmhD gene knockout fragment was cloned into the plasmid pBlueScriptIISK(+) to obtain the recombinant plasmid pBlueScript IISK(+)-gmhDU-Fkan-gmhDD. Using the plasmid as a template, the knockout fragment gmhDU-Fkan-gmhDD can be amplified.

[0058] (2) Preparation and electrotransformatio...

Embodiment 2

[0064] Example 2 Construction of LPS streamlined strain WJW01

[0065] Lipopolysaccharide (LPS) includes three parts: lipid A, core sugar and O-antigen, among which lipid A is a highly conserved region, while core sugar and O-antigen are composed of different sugar molecules. The core sugar synthesis gene cluster contains 14 genes, namely waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD. On the basis of the strain WJW00 obtained in Example 1, the remaining 13 genes in the core sugar gene cluster gmdD(gmhD)-waaFC-waaQGPSBORYZUL except the gmhD gene were knocked out.

[0066] (1) With reference to the knockout process of the gmhD gene in Example 1, the waaQ gene is knocked out

[0067] 1) Obtaining the waaQ gene knockout fragment

[0068] The waaQ gene knockout fragment is obtained by chemical total synthesis or PCR step-by-step amplification, and its two ends are the upstream and downstream homology arms of the waaQ gene, and the middle is a ...

Embodiment 3

[0080] Example 3 Construction of LPS streamlined strain WJW02

[0081] The O-antigen synthesis gene cluster rmlBrmlBDACX-glf-wbbHIJKL contains 11 genes, namely wbbL::IS5, wb bK, wbbJ, wbbI, wzy, glf, wzx, rmlC, rmlA, rmlD, rmlB. WJW02 was obtained after further knocking out the O-antigen synthesis gene cluster on the basis of WJW01. The specific operation is as follows:

[0082] (1) Construction of tool plasmid pDTW202-cat

[0083] The knockout of O-antigen gene cluster wbbL-rmlB adopts lox LR site-specific recombination method. Therefore, when constructing the Escherichia coli genome streamlining system, it is necessary to construct a tool plasmid pDTW202-cat. The construction process of the plasmid is specifically as follows:

[0084] 1) Using the plasmid pDTW109 (see Section 3.2.1 of Tan Yanzhen's master's degree thesis "Construction of the Corynebacterium glutamicum Gene Knockout System", publication date: 2012.03.27) as a template, using primers Tac-M-cat-F and Tac-M...

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Abstract

The invention discloses an Escherichia coli membrane wall-reduced chassis strain and the application of the high-yielding PHB strain, belonging to the fields of genetic engineering and fermentation engineering. The present invention knocks out the core sugar gene cluster gmhD-waaQ and the O-antigen gene cluster wbbL-rmlB of the lipopolysaccharide on the wild-type Escherichia coli W3110 genome, the extracellular polysaccharide gene cluster galF-wza, and the type 4 capsular polysaccharide synthesis gene cluster yccC- ymcD, three flagellar gene clusters flhE-motA, fliY-fliR, flgN-flgL, and a pilus gene cluster fimB-fimH to obtain strain WJW08, and then transform the genes related to PHB synthesis into the strain to obtain recombinant strain WJW08 / pBHR68, Under normal fermentation conditions, the recombinant bacterium can synthesize 81.9% PHB of dry cell weight, which is 54.6 times that of the wild control bacterium W3110 / pBHR68 (1.5%).

Description

technical field [0001] The invention relates to an Escherichia coli membrane wall-reduced chassis strain and the application of the high-yielding PHB strain, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Polyhydroxyalkanoates (PHA) are a class of renewable and degradable polymers with multiple material properties synthesized by microorganisms, and have broad application prospects in the fields of medicine, materials and environmental protection. Polyhydroxyalkanoate is widely present in microbial cells, mainly as a carbon source and energy storage carrier. The higher the ratio of C:N in the growth environment, the more favorable the synthesis of PHA. PHA exists in the form of hydrophobic particles in cells, and its content can exceed 90% of the dry weight of cells under certain conditions. According to different types of monomers and polymerization methods, PHA has a series of diverse material properties ranging ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N9/88C12N15/52C12P7/625
Inventor 王小元王建莉马文渐王甜忆梁浩杨惠婷丁玲李烨
Owner JIANGNAN UNIV
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