Corynebacterium glutamicum for producing lysine, and construction method and application of corynebacterium glutamicum
A technology of Corynebacterium glutamicum and amino acid, applied in the field of genetic engineering, can solve the problems of low acid production rate, can not meet large-scale industrial production, poor fermentation performance, etc. The effect of improving amino acid ability
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Embodiment 1
[0026] Example 1 Construction of the transformation vector pK18-NCgl0267 comprising the NCgl0267 gene coding region of the point mutation T539C
[0027] According to the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were designed and synthesized to amplify the sequence of the coding region of NCgl0267 gene, and were used in the strain YP97158 (preservation number: CGMCCNo.12856, preservation date: 2016 On August 16, 2009, depository unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing) introduced a point mutation into the coding region of the NCgl0267 gene (SEQ ID NO: 1) in the background, corresponding to the encoded protein The amino acid sequence is sequence 3, and the 539th T in the nucleotide sequence of the NCgl0267 gene is changed to C (SEQ ID NO: 2: NCgl0267 T539C ), corresponding to the amino acid sequence of the encoded protein (SEQ ID NO:...
Embodiment 2
[0033] Example 2 Construction of NCgl0267 comprising a point mutation T539C engineered strain
[0034] Construction method: allelic replacement plasmid pK18-NCgl0267 T539C Transformed into the patented L-lysine producing strain YP97158 by electric shock (for its construction method, please refer to WO2014121669A1; it was confirmed by sequencing that the wild-type NCgl0267 gene coding region was retained on the chromosome of the strain), and the single colonies produced by culture were respectively passed primers P1 and universal primer M13F were used for identification, and the strain amplified with a band of 1400 bp was a positive strain. The positive strains were cultured on a medium containing 15% sucrose, and the single colony produced by the culture was cultured on a medium containing kanamycin and a medium not containing kanamycin, respectively, and on a medium not containing kanamycin The bacterial strain that does not grow on the culture medium containing kanamycin f...
Embodiment 3
[0038] Embodiment 3L-lysine fermentation experiment
[0039] The bacterial strain YPL-4-016 constructed in Example 2 and the original bacterial strain YP97158 were used in the fermenter of the BLBIO-5GC-4-H model (purchased from Shanghai Bailun Biotechnology Co., Ltd.) with the medium and table shown in Table 1. The control process shown in 2 was used for fermentation experiments. Each strain was repeated three times, and the results are shown in Table 3.
[0040] Table 1 Fermentation Medium Formula
[0041] Element formula starch hydrolyzed sugar 30g / L ammonium sulfate 12g / L magnesium sulfate 0.87g / L molasses 20g / L acidified corn steep liquor 3mL / L phosphoric acid 0.4mL / L potassium chloride 0.53g / L Defoamer (2% foam enemy) 4mL / L ferrous sulfate 120mg / L Manganese sulfate 120mg / L Nicotinamide 42mg / L thbrthdrexvbdr 6.3mg / L Vitamin B1 6.3mg / L Copper, zinc salt solution 0...
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