A kind of genetically engineered bacteria producing l-leucine and its application
A technology of genetically engineered bacteria and leucine, applied in the field of metabolic engineering, can solve the problems of slow growth, unstable fermentation and nutritional deficiencies of L-leucine production strains, and achieve short fermentation cycle, fast growth and high conversion rate Effect
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Embodiment 1
[0057] Example 1: Isopropylmalate Synthase Encoding Gene leuA Relieving L-Leucine Feedback Inhibition M the acquisition
[0058] (1) Screening of mutants resistant to L-leucine structural analogues
[0059] ① Preparation of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 bacterial suspension
[0060] Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 was inoculated into LB liquid medium, cultured at 32°C, 200rpm for 12h, collected by centrifugation, washed with sterile saline for 3 times and then resuspended to make the OD 600 =0.6-0.8, take 10 μL of the bacterial suspension and spread it on the slide.
[0061] ② Plasma mutagenesis at atmospheric pressure and room temperature
[0062] The mutagenesis parameters were as follows: the slide was placed at 2mm from the airflow port, the power was 120W, the airflow was 10SLM, and the action time was 20s.
[0063] ③ Screening of mutant strains resistant to L-leucine structural analog α-aminobutyri...
Embodiment 2
[0085] Example 2 Release the acetohydroxyacid synthase coding gene ilvBN of L-isoleucine feedback inhibition M the acquisition
[0086] (1) Screening of mutants resistant to L-isoleucine structural analogues
[0087] ① Preparation of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 bacterial suspension
[0088] Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 was inoculated into LB liquid medium, cultured at 32°C, 200rpm for 12h, collected by centrifugation, washed with sterile saline for 3 times and then resuspended to make the OD 600 =0.6-0.8, take 10 μL of the bacterial suspension and spread it on the slide.
[0089] ② Plasma mutagenesis at atmospheric pressure and room temperature
[0090] The mutagenesis parameters were as follows: the slide was placed at 2mm from the airflow port, the power was 120W, the airflow was 10SLM, and the action time was 25s.
[0091] ③ Screening of mutant strains resistant to L-isoleucine structural analog ...
Embodiment 3
[0110] Embodiment 3: Construction of L-leucine producing bacteria TE03
[0111] (1) Recombinant fragment UHF-leuA M - Construction of DHF
[0112] synthetically containing leuA M The plasmid of the gene is used as a template, and LEUA-3 and LEUA-4 are used as primers for PCR amplification to obtain leuA M ;
[0113] Using the Escherichia coli W3110 genome as a template, using primers LEUA-1 and LEUA-2 and LEUA-5 and LEUA-6 to amplify the fragments UHF and DHF respectively, UHF and DHF are the upper and lower homology arms of the lacI gene respectively; UHF, DHF and leuA M As a template, use primers LEUA-1 and LEUA-6 for PCR amplification, and after recovery, it is the recombinant fragment UHF-leuA M -DHF.
[0114] (2) Recombinant fragment UHFA-ilvBN M - Construction of DHFB
[0115] Synthetic containing ilvBN M The plasmid of the gene is used as a template, and IlvB-3 and IlvB-4 are used as primers to perform PCR amplification to obtain ilvBN M ; Using the E. coli W3...
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