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Construction method of escherichia coli with high yield of L-tryptophan and low yield of L-alanine

A technology of Escherichia coli and construction method, applied in the biological field, can solve the problems of carbon source waste, target product purification and recovery problems, bacterial growth and metabolism hazards, etc., and achieve the effects of increasing yield, reducing product purification pressure, and reducing yield

Inactive Publication Date: 2022-06-07
FUJIAN NORMAL UNIV
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The production of L-tryptophan by Escherichia coli is often accompanied by the generation of by-products. The accumulation of by-products will not only cause waste of carbon sources, but also may bring difficulties to the purification and recovery of the target product, and even damage the bacteria. The growth and metabolism of the potential hazards

Method used

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  • Construction method of escherichia coli with high yield of L-tryptophan and low yield of L-alanine
  • Construction method of escherichia coli with high yield of L-tryptophan and low yield of L-alanine
  • Construction method of escherichia coli with high yield of L-tryptophan and low yield of L-alanine

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Experimental program
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Effect test

Embodiment 1

[0031] A kind of Escherichia coli construction method of high-yield L-tryptophan and low-yield L-alanine (the knockout method of transaminase genes alaA and alaC), comprising the steps:

[0032] (1) According to the published sequence information of E. coli K-12 alanine aminotransferase genes (alaA and alaC) in GenBank, design knockout primer pairs alaA-F1 / alaA-R1 with homology arms respectively , alaC-F1 / alaC-R1 (Table 1). Using the plasmid pKD13 as the template and the knockout primer pairs of each gene of alanine aminotransferase as the primers, PCR amplification was carried out, and the gene fragments containing the kanamycin resistance gene and homologous to the alaA or alaC gene were amplified respectively. After the PCR reaction, Dpn I enzyme was added proportionally to the reaction product, treated at 37°C for 1 h, placed in a water bath at 80°C for 10 min to terminate the reaction, and the product fragments were recovered by the kit and sent for sequencing.

[0033] ...

Embodiment 2

[0042] It was verified by shaking flask fermentation whether the Escherichia coli construction method of Example 1 achieved the purpose of reducing the yield of L-alanine by-product by knocking out aminotransferase, thereby increasing the yield of L-tryptophan.

[0043] (1) Seed cultivation

[0044] A small amount of strains before and after transformation were taken in a cryovial and were respectively inoculated on the LB solid slant, and cultured at 37°C for about 15h. In a sterile environment, the colonies on the slope were washed with sterile saline and transferred to a conical flask containing 30 mL of seed culture solution, and cultured with shaking at 35°C and 250 r / min for 12 h.

[0045] (2) Shake flask fermentation culture

[0046] The inoculation amount of 2% seed liquid was inoculated into the fermentation medium containing 30 mL of sterilized bacteria, and corresponding antibiotics were added if necessary, wherein the final concentration of ampicillin was 100 μg / m...

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Abstract

The invention discloses a construction method of Escherichia coli with high yield of L-tryptophan and low yield of L-alanine, which comprises the following steps: respectively designing knockout primer pairs with homologous arms according to sequence information of alaA and alaC in E.coli K-12 alanine transaminase genes of the E.coli K-12 alanine transaminase genes, and knockout amino transferase genes alaA and alaC to obtain L-tryptophan with high yield and low yield of L-alanine. The recombinant escherichia coli can more fully utilize the carbon metabolic flow to synthesize the L-tryptophan, so that the yield of the L-tryptophan is increased, the yield of an L-alanine by-product in a metabolic pathway of the escherichia coli is reduced, and the pressure of product purification is relieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing Escherichia coli with high L-tryptophan production and low L-alanine production. Background technique [0002] As one of the 20 amino acids necessary for humans and animals, L-Tryptophan (L-Trp) cannot be synthesized in humans and animals, and can only be obtained from plants and microorganisms. L-tryptophan is widely used in medicine, food and feed industries. [0003] The production methods of L-tryptophan include protein hydrolysis, chemical synthesis, microbial transformation and microbial fermentation. At present, microbial fermentation is mainly used at home and abroad to produce L-tryptophan, among which the main microorganisms are Escherichia coli and Corynebacterium glutamicum and other strains; in the breeding process of strains, physical mutagenesis or chemical induction are usually used Variation and combination of metabolic enginee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/54C12P13/22C12N1/21C12R1/19
CPCC12N15/70C12N9/1096C12Y206/01002C12P13/227C12N1/20Y02P60/87
Inventor 张怀东刘峰孟帅帅臧珊珊
Owner FUJIAN NORMAL UNIV
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