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A method for improving the 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: 2022-06-24
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 furfural, the growth of the bacteria is still under the stress of inhibitors such as furfural.

Method used

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  • A method for improving the tolerance of Corynebacterium glutamicum to inhibitors
  • A method for improving the tolerance of Corynebacterium glutamicum to inhibitors
  • A method for improving the tolerance of Corynebacterium glutamicum to inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 knockout purU Construction of Gene Recombinant Corynebacterium glutamicum

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

Embodiment 2

[0038] Example 2 Inactivation purU Construction of Gene Recombinant Corynebacterium glutamicum

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

Embodiment 3

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

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

[0043] (1) with C.glutamicum The genomic DNA of ATCC13032 was used as a template, and was 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, and the left and right homology arms were PCR amplified respectively. pK18 mobsacB The plasmid fragment is connected by three-fragment homologous recombination, and the ligation product is introduced by chemical transformation E. coli DH5α competent cells were plated on LB solid plates supplemented with Kan resistance, and cultured overnight in a 37°C incubator. After the single clone grows, pick the single clone and inoculate...

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Abstract

The invention discloses a method for improving the tolerance of Corynebacterium glutamicum to inhibitors, by reducing or eliminating the catalytic activity of endogenous formyltetrahydrofolate deformylase of Corynebacterium glutamicum, or by mutating glutamic acid Acid Corynebacterium endogenous D-3-phosphoglycerate dehydrogenase to enhance its activity, or the above two methods combined to obtain recombinant Corynebacterium glutamicum with increased tolerance to inhibitors. The strain constructed by the invention provides a reference for the construction of subsequent high-performance industrial fermentation chassis strains, and is beneficial to the production of biofuels or other high value-added chemicals by Corynebacterium glutamicum using lignocellulose hydrolyzate 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 a kind of important renewable biomass resource, which widely exists 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 it has attracted much attention from researchers in recent years. Due to the dense structure of lignocellulosic raw materials, a series of pretreatment processes, such as dilute acid treatment, are required to destroy its rigid structure before it can be used by microorganisms. During acid treatment, some toxic by-products are released, including furan aldehydes, weak acids, phenols and other substances. Furfural and 5...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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