Improved Glutamic Acid Production Capacity of <i>Corynebacterium glutamicum</i> by the ARTP Mutagenesis Method
Lingling Shangguan,
Huiyan Zhang,
Zixiong Liu,
Feiran An,
Qiao Yang,
Xiaoling Zhang,
Lan Yao,
Shihui Yang,
Jun Dai,
Xiong Chen
Affiliations
Lingling Shangguan
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Huiyan Zhang
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Zixiong Liu
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Feiran An
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Qiao Yang
ABI Group, Donghai Laboratory, College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China
Xiaoling Zhang
ABI Group, Donghai Laboratory, College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China
Lan Yao
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Shihui Yang
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China
Jun Dai
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Xiong Chen
Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), National “111” Center for Cellular Regulation and Molecular Pharmaceutics, School of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.
