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Method for efficiently synthesizing PHBs by knocking out LPS synthesis gene cluster

A gene cluster and gene technology, applied in the fields of genetic engineering and fermentation engineering, can solve the problems that PHB cannot meet the needs of industrial production

Active Publication Date: 2019-10-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Method for efficiently synthesizing PHBs by knocking out LPS synthesis gene cluster
  • Method for efficiently synthesizing PHBs by knocking out LPS synthesis gene cluster
  • Method for efficiently synthesizing PHBs by knocking out LPS synthesis gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] The construction of embodiment 1 engineering bacteria WJW00

[0045] 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:

[0046] (1) Obtaining of gmhD (also known as rfaD) gene knockout fragment

[0047] 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.

[0048] (2) Prepara...

Embodiment 2

[0054] Example 2 Construction of LPS streamlined strain WJW01

[0055] 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. Among them, the core sugar synthesis gene cluster contains 14 genes, namely waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD (also known as rfaD), the NCBI of its sequence The accession numbers are "BAE77660.1", "BAE77661.1", "BAE77662.1", "BAE77663.1", "BAE77664.1", "BAE77665.1", "BAE77666.1", "BAE77667.1 ", "BAE77668.1", "BAE77669.1", "BAE77670.1", "BAE77671.1", "BAE77672.1", "BAE77673.1".

[0056] On the basis of the strain WJW00 obtained in Example 1, the remaining 13 genes in the core sugar gene cluster gmdD(rfaD)-waaFC-waaQGPSBORYZUL except the gmhD gene were knocked out.

[0057] (1) With reference to the knockout process of the gmhD gene in Example 1, the waaQ g...

Embodiment 3

[0071] Example 3 Construction of LPS streamlined strain WJW02

[0072] The O-antigen synthesis gene cluster rmlBDACX-glf-wbbHIJKL contains 11 genes, namely wbbL::IS5, wbbK, wbbJ, wbbI, wzy, glf, wzx, rmlC, rmlA, rmlD, rmlB, and their sequence accession numbers are " BAA15873.1", "BAA15874.1", "BAA15875.1", "BAA15876.1", "BAA15877.1", "BAA15878.1", "BAA15879.1", "BAA15880.1", "BAA15881. 1", "BAA15882.1", "BAA15883.1", "BAA15884.1". WJW02 was obtained after further knocking out the O-antigen synthesis gene cluster on the basis of WJW01. The specific operation is as follows:

[0073] (1) Construction of tool plasmid pDTW202-cat

[0074] 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:

[0075] 1) Using the plasmid pDTW109 (se...

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Abstract

The invention discloses a method for efficiently synthesizing PHBs by knocking out an LPS synthesis gene cluster, and belongs to the field of gene engineering and fermentation engineering. By knockingout 14 genes of a core polysaccharide gene cluster on an escherichia coli genome in escherichia coli, a mutation strain WJW01 is obtained, then 11 genes of an O-antigen gene cluster on the escherichia coli genome continue to be knocked out to obtain a mutation strain WJW02, afterwards, three genes synthesized by the PHBs are transformed into the simplified strains WJW01 and WJW02 respectively, and the obtained recombinant strains WJW01 / pBHR68 and WJW02 / pBHR68 can respectively synthesize the PHBs which reach up to 80.6% and 82.0% of cell dry weight, and are 53.7 times and 54.7 times that of awild-type contrast strain W3110 / pBHR68 (1.5%).

Description

technical field [0001] The invention relates to a method for efficiently synthesizing PHB by knocking out an LPS synthesis gene cluster, 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 from hard and brittle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N9/1029C12N9/18C12P7/625C12Y301/01075
Inventor 王小元王建莉马文渐王雨舟王雨倩李烨
Owner JIANGNAN UNIV
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