Genetically engineered strain for producing L-citrulline and application of genetically engineered strain
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, low production intensity, and easily fluctuating production, and achieve the effects of stable fermentation process, high production intensity and short fermentation cycle.
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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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