Method for quickly and efficiently screening L-arginine-producing strain

A technology for high-yielding strains and arginine, applied in the direction of microbial-based methods, biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of high screening work intensity, high cost, and high price, and achieve improved screening Work efficiency and cost saving effects

Active Publication Date: 2012-03-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large number of structural analogues of L-arginine and the high price, the screening work is intensive, inefficient and expensive

Method used

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  • Method for quickly and efficiently screening L-arginine-producing strain
  • Method for quickly and efficiently screening L-arginine-producing strain
  • Method for quickly and efficiently screening L-arginine-producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: The kanamycin resistance gene (Kan) was inserted into the arginine succinylase gene in the Escherichia coli BL21L-arginine synthesis pathway to achieve the knockout purpose and obtain the defective strain:

[0025] Amplification of argBH and construction of plasmid pMD-18T-argBH::Kan: According to the argBH sequence published in GeneBank, PCR was designed to obtain the primer P15'-CGC of argBH GAATTC ATGAATCCATTAATTATCAAACTGG-3'(EcoR I)P25'CGC GTC GAC TTACCCTAAACCGAGCCTGC-3' (Sal I). Using E.coli BL21 genomic DNA as a template, the argBH gene was amplified with primers P1 and P2. Amplification cycle conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 40s, annealing at 56°C for 90s, extension at 72°C for 90s, 35 cycles, Amplified products were detected by 0.8% agarose gel electrophoresis, such as image 3As shown in a, it can be seen that there is a bright band at 2.2kb, which is consistent with the expected size of the argBH gene...

Embodiment 2

[0027] 10 5 Cells per mL were mixed with E.coli BL21 L-arginine-deficient strain to make a screening plate, and 4 strains of Corynebacterium blunt tooth with different L-arginine yields preserved in our laboratory were planted on the screening plate with the same amount , incubate at 30°C for 5-7 days, spray sterile 1 μg / mL TTC solution (membrane filter sterilization) evenly on the ultra-clean bench, place at 37°C for 1-2h, and observe the color development on the plate. Such as Figure 5 As shown, the experimental results show that the size of the red circle is directly proportional to the L-arginine production of the strain, indicating that this method is effective and feasible for screening high-production L-arginine mutants.

Embodiment 3

[0028] Example 3: Application of plate color method for rapid screening of high-yield L-arginine strains

[0029] 10 5 Cells per mL were mixed with E.coli BL21L-arginine-deficient strains to make a screening plate, and the strains to be screened were made into a certain concentration of bacterial suspension, 10-fold serial dilution was spread on the plate, and cultured at 30°C On day 5-7, spray sterile 1 μg / mL TTC solution (membrane filtration sterilization) evenly on the ultra-clean workbench, place at 37°C for 1-2 hours, and observe the growth of the strain.

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Abstract

The invention discloses a method for quickly and efficiently screening an L-arginine-producing strain, belonging to the technical field of industrial microbial breeding. The traditional method for screening an L-arginine-producing strain is characterized by screening a strain containing anti-L-arginine structural analogs. In the invention, an Escherichia coli L-arginine defective strain is constructed as an L-arginine secretion indicator bacterium; and TTC (2,3,5-triphenyltetrazolium chloride) is a viable cell indicator and can be reduced into red by reducing hydrogen produced by viable cells. By using the L-arginine defective strain as the L-arginine secretion indicator bacterium and using the TTC as the developer through an interactive co-culture method to realize the qualitative screening of the L-arginine-producing strain based on the concentration of the extracellular L-arginine, a strain having high L-arginine synthesis capability or good secretion performance can be preliminarily screened on a flat plate. The method greatly improves the screening efficiency in strain breeding and saves the cost.

Description

technical field [0001] The invention relates to a method for rapidly and efficiently screening L-arginine high-yield strains based on L-arginine-deficient strains, and belongs to the technical field of industrial microorganism breeding. Background technique [0002] L-arginine is a semi-essential basic amino acid in humans and animals, an important raw material for the synthesis of protein creatine, and an important intermediate metabolite of the urea cycle in organisms, so it is widely used in the pharmaceutical and food industries. [0003] Fermentative production of L-arginine is an effective and economical method for commercial production at present, and excellent L-arginine high-yielding strains are the key to fermentative production of L-arginine. At present, the breeding of high-yield strains of L-arginine is mainly obtained through traditional mutation breeding and modern genetic engineering breeding. Among them, genetic engineering breeding is favored by researchers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04C12N15/70C12R1/19
Inventor 饶志明许正宏杨娟徐美娟窦文芳
Owner JIANGNAN UNIV
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