L-threonine high-yield genetic engineering strain and application thereof
A technology of genetically engineered bacteria and threonine, applied in the field of genetic engineering, can solve the problems of affecting the expression of exogenous genes, adverse effects on cell metabolism, and reducing the synthesis of threonine
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Embodiment 1
[0050] Example 1 Construction of L-threonine high-yield genetically engineered bacteria
[0051] (1) Knock out the pka gene of E.coli THRD by Red homologous recombination technology
[0052] a. After transforming the plasmid pKD46 into E. coli THRD competent cells, spread it on an ampicillin-resistant LB plate and culture overnight at 30°C.
[0053] b. The single colony on the resistance plate was verified by PCR with the upstream and downstream primers (pKD46-up and pKD46-down) of the plasmid pKD46, and positive recombinants were screened. PCR reaction system (15 μL): sterile water 6.7 μL, green enzyme 7.5 μL, upstream primer 0.4 μL, downstream primer 0.4 μL.
[0054] c. Cultivate positive recombinants containing pKD46 plasmid to OD 600= 0.1-0.2, add 1M L-arabinose (to make the final concentration 10-50mM), continue to culture to OD 600 =0.6-0.8 to prepare competent cells.
[0055] d. Overlap PCR
[0056] ① Using pka-1 and pka-2 as primers and genomic DNA as a template t...
Embodiment 2
[0074] Embodiment 2 adopts the method for preparing threonine by E.coli JLTHRGQQ
[0075] (1) Activated strain culture
[0076] ①The slant strains of E.coli JLTHRGQQ were inserted into the activated slant medium for activation, and the primary strains were obtained after static culture at a temperature of 37°C for 14-16 hours;
[0077] ②The primary strains were activated for the second time, and cultured statically at a temperature of 37°C for a time period of 8 hours. The activated strains were obtained after the microscopic examination of the healthy and free bacteria.
[0078] (2) Preparation of high-yield threonine fermentation broth
[0079] ① Seed liquid culture: wash the four activated slopes with sterile distilled water into a 5L fermenter containing 3L seed medium, the culture conditions are 37°C, pH 7.0, and dissolved oxygen is controlled at 20%-40%. The temperature can be automatically adjusted by the circulating heating device, the pH can be adjusted by ammonia w...
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