Genetically engineered bacterium for producing L-arginine and construction method and application thereof

A technology of genetically engineered strains and construction methods, applied in the field of stable and efficient production of L-arginine, genetically engineered bacteria and its construction, can solve the problems of arginine accumulation and production intensity that cannot meet industrial production

Active Publication Date: 2020-04-07
NINGXIA EPPEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the fermentation period of this strain is significantly shortened, its arginin...

Method used

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  • Genetically engineered bacterium for producing L-arginine and construction method and application thereof
  • Genetically engineered bacterium for producing L-arginine and construction method and application thereof
  • Genetically engineered bacterium for producing L-arginine and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0066] Construction of genetic engineering bacteria E.coli W3110 ARG10:

[0067] 1 Methods of gene editing

[0068] The gene editing method used in the present invention is carried out with reference to the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 mediated genome editing. Metabolic engineering, 2015, 31:13-21.), the method For the two plasmid maps used see figure 1 . Among them, pREDCas9 carries gRNA expression plasmid pGRB elimination system, bacteriophage λ Red recombination system and Cas9 protein expression system, spectinomycin resistance (working concentration: 100mg / L), cultured at 32°C; pGRB uses pUC18 as the backbone, including the promoter J23100, gRNA-Cas9 binding region sequence and terminator sequence, ampicillin resistance (working concentration: 100mg / L), cultured at 37°C.

[0069] The concrete steps of this method are as follows:

[0070] 1.1 pGRB plasmid construction

[0071] The purpose of const...

Embodiment 2

[0146] The method for producing arginine by fermenting genetically engineered bacteria E.coli W3110 ARG10 is as follows:

[0147] (1) Shake flask fermentation:

[0148] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0149] Shake flask seed culture: Scrape a ring of slant seeds with an inoculation loop and inoculate in a 500mL Erlenmeyer flask containing 30mL of seed medium, seal with nine layers of gauze, and incubate at 37°C and 200rpm for 7-10h;

[0150] Shake flask fermentation culture: Inoculate 15% of the seed culture solution volume into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, 37°C, 200r / min shaking culture, fermentation process Maintain pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain fermentation; fermentation cycle 26-30h;

[0151] The composition of the slant medium is: glucose 1g / ...

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Abstract

The invention discloses a genetically engineered bacterium for producing L-arginine and a construction method and application thereof. According to the invention, a gene for encoding carbamyl phosphate synthetase and a gene for encoding L-arginine biosynthetic pathway enzyme are integrated in escherichia coli; a synthetic route of arginine in escherichia coli and metabolic flux related to argininein a whole amino acid metabolic network are analyzed and reconstructed to obtain genetically engineered bacteria which are clear in genetic background, do not carry plasmids, are not mutated and canstably and efficiently produce L-arginine; and the genetically engineered bacteria have good L-arginine production capacity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium for stably and efficiently producing L-arginine, a construction method and application thereof. Background technique [0002] L-Arginine is a semi-essential basic amino acid or a conditionally essential amino acid in humans and animals, and has important biochemical and physiological functions. At present, L-arginine has been widely used in medicine, industry, food, cosmetics, animal husbandry and other fields, and has important economic and social value. [0003] The production methods of L-arginine mainly include protein hydrolysis and microbial fermentation. Compared with protein hydrolysis and extraction, microbial fermentation has the advantages of relatively simple production process, relatively small impact on the environment, and high product purity, which is suitable for large-scale production. industrial produc...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/10C12R1/19
CPCC12N9/93C12N9/88C12N9/1029C12N9/80C12P13/10C12Y401/01019C12Y203/01109C12Y305/01016C12N15/70C12N2310/20C12Y603/05005C12Y603/04016C12N15/52C12R2001/19C12R2001/15C12N15/77C12N1/205C12N2800/101
Inventor 谢希贤蒋帅文晨辉吴鹤云刘益宁李旋田道光熊博
Owner NINGXIA EPPEN BIOTECH
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