Method for improving yield of L-glutamic acid synthesized by corynebacterium glutamicum
A technology of Corynebacterium glutamicum and glutamic acid, applied in the field of bioengineering, can solve problems such as insufficient thoroughness and can not solve the production of L-glutamic acid well, and achieve the effect of promoting the accumulation amount
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Embodiment 1
[0029] Example 1: Knockout of transcriptional regulator Ncgl1124
[0030] Using the Corynebacterium glutamicum G01 genome as a template, the primer pair Ncgl1124-L F / Ncgl1124-L R and Ncgl1124-R F / Ncgl1124-R R were used to amplify the upstream and downstream 500bp of the gene Ncgl1124 respectively, and then purified The obtained upstream and downstream fragments were used as templates. Using the primer pair Ncgl1124-L F / Ncgl1124-R R, the upstream and downstream fragments were fused through overlap extension PCR and the integrated homology arm with a full length of 1000 bp was recovered. At the same time, the plasmid pK18 was used as a template, and the linearized vector pK18 was amplified by reverse PCR with the primer pair pK18 F / pK18 R. After recovery, the One-stephomologous recombination kit (purchased from Novizym, Nanjing) was used to combine the integrated homology arms Ligate at 30°C for 30 minutes, then transform the ligated system into Escherichia coli, then use colony...
Embodiment 2
[0037] Example 2: Overexpression of transcriptional regulator Ncgl1124
[0038]Using the genome of Corynebacterium glutamicum G01 as a template, the primer pair Ncgl1124 F / Ncgl1124 R was used to amplify the nucleotide sequence of the transcriptional regulator Ncgl1124, and the target fragment was recovered. And use the One-step homologous recombination kit (purchased from Novozyme, Nanjing) to connect the obtained target DNA fragment and the linearized vector pXMJ19 that has been digested and purified with EcoRI and BamHI at 30°C for 30 minutes, and transform into Escherichia coli Then, the positive transformant was identified by colony PCR, and the plasmid was extracted to obtain the successfully constructed recombinant plasmid pXMJ19-Ncgl1124. The successfully constructed recombinant knockout plasmid was electrotransformed into Corynebacterium glutamicum G01, and the recombinant strain G01 / pXMJ19-Ncgl1124 was obtained through colony PCR identification.
[0039] Ncgl1124 F: ...
Embodiment 3
[0041] Example 3: Transcription factor Ncgl1124 and promoter P GlnA Verification of the binding effect
[0042] Verification of Ncgl1124 protein and promoter P by EMSA assay GlnA combination.
[0043] In order to carry out the EMSA test, it is necessary to obtain the purified Ncgl1124 protein. First, the present invention uses Escherichia coli BL21 as a host to construct a recombinant bacterium E.coli / pET28a-Ncgl1124, which heterologously expresses the transcriptional regulatory factor Ncgl1124 derived from Corynebacterium glutamicum G01. Recombinant strain E.coli / pET28a-Ncgl1124 was cultured to OD at 37°C and 180rpm 600 At about 0.5-0.8, add 0.5mM isopropylβ-D-1-Thiogalactopyranoside (IPTG) and transfer to 16°C, induce protein expression under the condition of 180rpm. After induction of expression for 10-12 hours, the cells were collected by centrifugation, and the cells were resuspended in 5 mL Tris-HCl buffer (50 mM, pH 7.4), and then the resuspended cells were treated ...
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