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L-lysine gene engineering producing bacterium

A technology of genetic engineering and lysine, which is applied in the field of L-lysine genetic engineering production bacteria, can solve the problems of difficulty in obtaining high-yielding strains, restricted production efficiency, etc., achieve wide industrial application prospects, improve yield and sugar-acid conversion rate, the effect of a wide range of industrial application value

Active Publication Date: 2015-09-02
SHANGHAI RES & DEV CENT OF INDAL BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the optimal growth temperature of Corynebacterium glutamicum and Brevibacterium is 30 degrees, the production efficiency has restricted the demand for large-scale fermentation and production of lysine to a certain extent. In addition, due to the limitations of traditional mutagenesis methods, it is difficult to obtain high-yield strains. very difficult

Method used

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  • L-lysine gene engineering producing bacterium
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  • L-lysine gene engineering producing bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 : Preparation of strain MG1655 (ΔcadA) with knockout of cadA gene

[0066] (1) PCR amplification: use cadA-F(S) / cadA-R(S) as primers and templates, PCR amplify the cadA(HR) fragment, about 100bp, and recover from the gel;

[0067] (2) Competent cell preparation: pCas plasmid (sourced from literature: Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, Jiang Y, Chen B, et al. Appl Environ Microbiol, 2015) was transformed into MG1655 (purchased at From CGSC (E.coli Genetic Stock Center, Yale University, New Haven, Connecticut, USA)) competent cells (transformation method and competent preparation method refer to "Molecular Cloning III", chapter 1, page 96), pick MG1655 / A single colony of pCas was cultured in a 4ml LB test tube containing kanamycin at 30°C and 220r / min. 600 Induce to OD by adding a final concentration of 10 mM arabinose one hour before 0.4 600 is 0.4;

[0068] (3) Electroporation: Electroporation of cadA (HR) fragmen...

Embodiment 2

[0070] Example 2 : Preparation of strain MG1655 (ΔcadA, ΔldcC) with ldcC gene knockout

[0071] (1) PCR amplification: using ldcC-F(S) / ldcC-R(S) as primers and templates, PCR amplifies the ldcC(HR) fragment, about 100bp;

[0072] (2) Competent cell preparation: transform the pCas plasmid into the MG1655 (ΔcadA) competent cells obtained in Example 1 (refer to page 96, Chapter 1 of Molecular Cloning III for the transformation method and competent preparation method), and pick MG1655(ΔcadA) / pCas single colonies were placed in 4ml LB test tubes containing kanamycin, and the preparation method was the same as in Example 1(2);

[0073] (3) ldcC-N20-Spec plasmid construction: using the pTargetF-cadA plasmid in Example 1 as a template, ldcC-N20-F / pTargetF-R as primers, PCR amplified ldcC-N20 fragment, about 2.2kb, digested with DpnI After the PCR fragments were self-ligated, the ldcC-N20-Spec plasmid was obtained;

[0074] (4) Electroporation: Electroporation of ldcC(HR) fragment ...

Embodiment 3

[0076] Example 3 : Preparation of bacterial strain MG1655 (ΔcadA, ΔldcC, Ptac-ybjE) overexpressing ybjE gene

[0077] (1) PCR amplification: using the MG1655 genome as a template and ybjEup / ybjE-R (Ptac) as a primer, PCR amplifies the Ptac-ybjE1 fragment, about 500bp;

[0078] (2) PCR amplification: using the Ptac-ybjE1 fragment as a template and ybjEup / ybjE-R (40) as a primer, PCR amplifies the Ptac-ybjE (HR) fragment, about 600bp;

[0079] (3) ybjE-N20-Spec plasmid construction: using the pTargetF-cadA plasmid in Example 1 as a template, ybjE-N20-F / pTargetF-R as primers, PCR amplified ybjE-N20 fragment, about 2.2kb, digested with DpnI After the PCR fragments were self-ligated, the ybjE-N20-Spec plasmid was obtained;

[0080] (3) Competent cell preparation: transform the pCas plasmid into the MG1655 (ΔcadA, ΔldcC) competent cells obtained in Example 2 (for both the transformation method and the competent preparation method, refer to Chapter 1, page 96 of Molecular Cloning II...

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Abstract

The invention provides a L-lysine gene engineering bacterium strain of which the collection number is CCTCC M 2015232. The L-lysine gene engineering bacterium provided by the invention can implement effective accumulation of L-lysine in the fermentation liquid in the fermentation process, thereby enhancing the yield of the L-lysine and the conversion rate of sugar acid, and having wide industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an L-lysine genetic engineering production bacterium. Background technique [0002] As one of the essential amino acids for humans and animals, L-lysine is widely used in feed additives, food fortifiers and pharmaceutical products, among which about 90% are used in the feed industry, and 10% are used in the food and pharmaceutical industries. At present, L-lysine is the second largest amino acid species in the world. [0003] There are four main production methods of L-lysine, which are extraction method, chemical synthesis method, enzymatic method and microbial fermentation method. The first three methods are difficult to achieve industrial production due to the disadvantages of high precursor cost and complicated process. Objective: The preparation of L-lysine by fermentation is the main production method at present. The microorganisms that produce L-l...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P13/08
Inventor 杨晟蒋宇孙兵兵陈飚杨俊杰
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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