Method for improving tolerance of corynebacterium glutamicum to inhibitors

A Corynebacterium glutamicum, inhibitor technology, applied in the field of bioengineering, can solve problems such as bacterial growth stress

Active Publication Date: 2022-04-22
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Corynebacterium glutamicum has a certain ability to detoxify inhibitors such as fu

Method used

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  • Method for improving tolerance of corynebacterium glutamicum to inhibitors
  • Method for improving tolerance of corynebacterium glutamicum to inhibitors
  • Method for improving tolerance of corynebacterium glutamicum to inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Knockout purU Genetic construction of recombinant Corynebacterium glutamicum

[0036] (1) with C. glutamicum The genomic DNA of ATCC13032 is used as a template, and it is designed to be knocked out according to the sequence of SEQ ID NO.3 purU The left and right homologous arms of the gene (about 1kb each) and their primer sequences were amplified by PCR respectively, and the products were purified and recovered, and then combined with enzyme-digested pK18 mobsacB The plasmid fragments are connected by homologous recombination of three fragments, and the connection products are introduced by chemical transformation E. coli DH5α competent cells were spread onto LB solid plates added with Kan resistance, and cultured overnight in a 37°C incubator. After the monoclonal grows, pick the monoclonal and inoculate it into the LB liquid medium added with Kan resistance, extract the recombinant plasmid from the bacterial solution, and obtain the recombinant pla...

Embodiment 2

[0038] Example 2 inactivation purU Genetic construction of recombinant Corynebacterium glutamicum

[0039] (1) According to the sequence design of SEQ ID NO.3 for inactivation purU The sgRNA target sequence of the gene (20 bp), synthesize two corresponding sense strand and antisense strand primers (24 bp each) according to the target sequence, and the primer contains a gap sequence (4 bp) introduced at the 5' end and paired with the vector . The two primers are annealed to form double-stranded DNA, and then combined with pnCas9(D10A)-AID-gRNA- ccdB TS Carrier plasmid for Golden Gate connection, transformation E. coli DH5α competent cells were spread onto LB solid plates added with Cm resistance, and cultured overnight in a 37°C incubator. After the monoclonal grows, pick the monoclonal and inoculate it into the LB liquid medium added with Cm resistance, extract the recombinant plasmid from the bacterial solution, and obtain the recombinant plasmid pnCas9(D10A)-AID- g...

Embodiment 3

[0041] Example 3 contains serA Construction of recombinant Corynebacterium glutamicum with mutant gene

[0042] 1. Through the suicide plasmid pK18 mobsacB The method of double exchange is constructed, and the steps are as follows:

[0043] (1) with C. glutamicum Genomic DNA of ATCC13032 is used as a template, designed for mutation according to the sequence of SEQ ID NO.4 serA The left and right homology arms of the gene (about 1kb each) and their primer sequences, the mutation site (Q184K) sequence was introduced into the primers, the left and right homology arms were amplified by PCR, the products were purified and recovered, and then combined with enzyme-digested pK18 mobsacB The plasmid fragments are connected by homologous recombination of three fragments, and the connection products are introduced by chemical transformation E. coli DH5α competent cells were spread onto LB solid plates added with Kan resistance, and cultured overnight in a 37°C incubator. Aft...

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Abstract

The invention discloses a method for improving the tolerance of corynebacterium glutamicum to an inhibitor, which is characterized in that the activity of corynebacterium glutamicum is enhanced by reducing or eliminating the catalytic activity of endogenous formyltetrahydrofolate deformylase of the corynebacterium glutamicum or mutating endogenous D-3-phosphoglycerate dehydrogenase of the corynebacterium glutamicum, or the tolerance of the corynebacterium glutamicum to the inhibitor is improved by combining the two methods. Therefore, the recombinant corynebacterium glutamicum with improved tolerance to inhibitors is obtained. The strain constructed by the invention provides reference for construction of subsequent high-performance industrial fermentation chassis strains, and is beneficial to production of biofuels or other high-added-value chemicals by corynebacterium glutamicum by taking lignocellulose hydrolysate as a raw material.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for improving the tolerance of Corynebacterium glutamicum to inhibitors such as furfural. Background technique [0002] Lignocellulose is an important class of renewable biomass resources that widely exist in nature. The development of microbial cell factories using lignocellulose such as straw as raw materials to produce biofuels such as ethanol and butanol will effectively reduce the current dependence on traditional fossil fuels, so researchers have paid attention in recent years. Due to the dense structure of lignocellulosic raw materials, it must undergo a series of pretreatment processes, such as dilute acid treatment, to destroy its rigid structure before it can be used by microorganisms. During acid treatment, some toxic by-products will be released, including furan aldehydes, weak acids, phenols and other substances. Furfural and 5-hydroxyme...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/55C12N15/77C12R1/15
CPCC12N9/80C12N9/0006C12N15/77C12Y305/0101C12Y101/01095
Inventor 王猛刘叶
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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