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A kind of genetically engineered bacterial strain producing L-citrulline and its application

A technology of genetically engineered strains and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of limited bacterial growth, long production cycle, and easy fluctuation of production, and achieve the effects of stable fermentation process, high production intensity, and short fermentation cycle

Active Publication Date: 2022-04-08
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the deficiencies of the prior art, the present invention provides an Escherichia coli genetically engineered bacterium and its construction method for producing L-citrulline by microbial fermentation, which solves the problems of low production intensity, long production cycle, limited bacterial growth and production The problem of easy fluctuation, and provide corresponding fermentation process control scheme, in order to apply to the efficient industrial production of L-citrulline

Method used

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  • A kind of genetically engineered bacterial strain producing L-citrulline and its application
  • A kind of genetically engineered bacterial strain producing L-citrulline and its application
  • A kind of genetically engineered bacterial strain producing L-citrulline and its application

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Experimental program
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Effect test

Embodiment 1

[0041] Construction of genetically engineered bacteria CIT 4:

[0042] 1 Methods of gene editing

[0043] 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 See Figure 1 for the two plasmid maps used. 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.

[0044] The concrete steps of this method are as follows:

[0045] 1.1 pGRB plasmid construction

[0046] The purpose of constructing the ...

Embodiment 2

[0103] Comparison of shake flask fermentation results between genetically engineered bacteria CIT 1 and E.coli MG1655 Figure 7 To verify the effect of knocking out the host's own argG on the accumulation of L-citrulline.

[0104] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0105] Shake flask seed culture: Scrape a ring of slanted 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 8h;

[0106] Shake flask fermentation culture: Inoculate 10% 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 the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation pe...

Embodiment 3

[0112] Comparison of shake flask fermentation results of genetically engineered bacteria CIT 2, CIT 3, and CIT 4, such as Figure 8 shown.

[0113] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;

[0114] Shake flask seed culture: Scrape a ring of slanted 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 8h;

[0115] Shake flask fermentation culture: Inoculate 10% 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 the pH at 7.0-7.2 by adding ammonia water; add 60% (m / v) glucose solution to maintain the fermentation; the fermentation period is 28h;

[0116] The composition of the slant medium is: glucose 1g / L, pepton...

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Abstract

The present invention provides a genetically engineered strain CIT 4 for efficient and stable production of L-citrulline. The strain uses Escherichia coli as a host. First, the host lacks the activity of argininosuccinate synthase to block the degradation of L-citrulline. It is arginine succinate; the gene argG encoding Escherichia coli argininosuccinate synthase is also integrated on the host genome, and is controlled by the tryptophan promoter P trp Regulate expression; also make the host lack of acetylornithine deacetylase activity; also integrate the gene argJ of Corynebacterium glutamicum encoding glutamate acetyltransferase on the host genome, so as to strengthen the synthesis of glutamate Metabolic flux in the process of acetylglutamate; also integrated on the host genome the genes pyrAA and pyrAB encoding the two subunits of carbamoyl phosphate synthase in the Bacillus subtilis mutant strain A260 to release arginine for carbamoyl phosphate Feedback inhibition of synthetases increases the supply of the precursor carbamoyl phosphate.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering strain for producing L-citrulline and its construction method and application. Background technique [0002] As a non-protein α-amino acid, L-citrulline has important biochemical and physiological functions. At present, L-citrulline has been widely used in medicine, industry, food, cosmetics, animal husbandry and other fields, and has important economic and social value. The production methods of L-citrulline mainly include chemical synthesis, enzyme catalysis and microbial fermentation. The chemical synthesis method is mainly to hydrolyze L-arginine under alkaline conditions to directly obtain L-citrulline. This method has low yield, high environmental pollution, and easy to produce D-type optical enantiomer in the production process, resulting in high cost of separation and purification of later products, so chemical synthesis met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/71C12P13/10C12R1/19
CPCC12N9/1029C12N9/80C12N9/93C12Y603/04005C12Y305/01016C12Y203/01035C12Y603/04016C12N15/52C12N15/71C12P13/10
Inventor 谢希贤文晨辉蒋帅吴鹤云秦臻朱彦凯王加初朱永铎李镠
Owner TIANJIN UNIV OF SCI & TECH
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