Escherichia coli engineering bacteria and fermentation production method of n-acetylglucosamine
A technology of acetamido and Escherichia coli, applied in the field of agricultural biology, can solve the problems of energy waste, low conversion efficiency and the like
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Embodiment 1
[0037] Example 1 Construction of Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf Chassis cells
[0038] Mutant Escherichia coli chassis cells are Escherichia coli hosts in which key genes in the glycolytic pathway and pentose phosphate pathway are knocked out, and the key gene in the glycolytic pathway that is knocked out is ATP-dependent 6-phosphofructokinase isoenzyme 1 coding gene wxya , ATP-dependent 6-phosphofructokinase isozyme 2 coding gene wxya and the coding gene of glucose-6-phosphate dehydrogenase, a key gene of pentose phosphate pathway zwf .
[0039] 1. Construction of Escherichia coli MG1655-∆ wxya strain
[0040] The pRed_cas9_recA_poxb300 plasmid and wxya Gene Targeted Knockout Vector p ∆pfkB -T3 was introduced into the E. coli MG1655 host. Using arabinose to induce the expression of Cas9 protein, cutting the genome sequence of Escherichia coli MG1655 under the guidance of gRNA, and obtaining wxya Knockout Escherichia coli MG1655-∆ wxya strain. The s...
Embodiment 2
[0066] Example 2 Construction of N-acetylglucosamine-producing E. coli MG1655- ∆pfkA∆pfkB∆zwf -pGGY engineering strain
[0067] 1. Construction of expression plasmid pGGY-1 containing genes related to N-acetylglucosamine synthesis
[0068] Will Escherichia coli Gene encoding glutamine fructose-6 phosphate transaminase glms mutant gene and Caenorhabditis legans glucosamine 6-phosphate N-acetyltransferase encoding gene gna -1 mutant gene, gene encoding fructose-1-phosphatase YqaB wxya respectively placed in P PGI Expression under the control of the promoter. The expression cassettes of each gene were assembled on the expression plasmid, and the plasmid was named pGGY-1; the specific construction method is as follows:
[0069] Amplify the nucleotide sequence of about 1800bp such as the glutamine fructose-6 phosphate transaminase gene of SEQ ID NO: 4 glms the mutant gene; Escherichia coli The MG1655 genome was used as a template for PCR to obtain a 360bp P PGI Pr...
Embodiment 3
[0076] Example 3 Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf -pGGY engineering strain fermented to produce N-acetylglucosamine
[0077] N-acetylglucosamine-producing Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf-The pGGY engineering strain was inoculated in 50 mL of LB liquid medium (ampicillin 50 μg / mL), and rejuvenated at 37°C and 200 rpm for 16 hours.
[0078] Preparation of mixed carbon source fermentation medium: Accurately weigh 6.78 g of disodium hydrogen phosphate, 3 g of potassium dihydrogen phosphate, 1.5 g of sodium chloride, 1 g of ammonium chloride, 10 g of glucose, 0.011 g of calcium chloride, and sulfuric acid heptahydrate Magnesium 0.493 g, glycerol 5 g, yeast extract 0.5 g, peptone 1 g, water as a solvent to make up to 1 L, 115 ℃ autoclaving for 30 minutes to obtain a mixed carbon source fermentation medium.
[0079] Escherichia coli MG1655- ∆pfkA∆pfkB∆zwf -The pGGY engineering strain was transferred to 200mL mixed fermentation medium (ampicillin 50 μg / mL) accordi...
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