The application of the transfer carrier gene to improve the production efficiency of l-tryptophan in Escherichia coli
A technology of Escherichia coli and tryptophan, applied in the fields of metabolic engineering and biology, can solve the problems of large differences in affinity, poor transport effect, and difficulty in meeting industrial production, and achieve the effect of improving yield and broad application prospects.
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Embodiment 1
[0047] Example 1: E.coli TRP 05 strain construction
[0048] 1. Methods of gene editing
[0049] 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 The two plasmid maps used are in the appendix 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.
[0050] The concrete steps of this method are as follows:
[0051] 1.1 pGRB plasmid construction
[0052] The purpose of constructing...
Embodiment 2
[0110] Example 2: Production of L-tryptophan by Escherichia coli genetic engineering bacteria shake flask fermentation
[0111] A kind of concrete operation that utilizes Escherichia coli genetically engineered bacteria to carry out shake flask fermentation to produce L-tryptophan is as follows:
[0112] Slant culture: Streak inoculation of -80°C preserved strains on the activated slant, culture at 37°C for 12 hours, and passage once;
[0113] 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 8-10h;
[0114] Shake flask fermentation culture: Inoculate 10-15% (v / v) inoculum into a 500mL Erlenmeyer flask with fermentation medium (final volume is 30mL), seal with nine layers of gauze, culture at 37°C, 200r / min shaking, During the fermentation process, maintain the pH at 7.0-7.2 by adding ammonia water; add 6...
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