Hongmei Sun1, Zhiming Rao2, Xiupeng Li2, Meijuan Xu2, Xian Zhang2, Zhenghong Xu3. 1. Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi 214122, China. sun_hongmei0321@sina.com 2. Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi 214122, China. 3. Lab of Pharmaceutical Engineering, School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China.
Abstract
OBJECTIVE: We constructed an integrative recombinant Corynebacterium crenatum that could directly convert glucose in gamma-aminobutyric acid (GABA). METHODS: Using overlap PCR, we obtained delta argB fragment that lacked 491 bp of N-acetylglutamate kinase (NAGK) gene. The glutamate decarboxylase (GAD) gene attached tac promoter was amplified and inserted into delta argB fragment. Using the upstream and downstream of delta argB as homologous arms, we constructed pK18-delta argB::tacgad which was used for the integration of tacgad gene onto the C. crenatum genome. Through two homologous recombinations, we got an argB blocked strain C. crenatum delta argB::tacgad which could successfully express GAD. We also fermented the recombinant strain with glucose as substrate and the production of GABA was detected. RESULTS: In C. crenatum delta argB::tacgad, NAGK was totally inactivated and no L-arginine was detected though L-glutamic acid was accumulated. As a result of the efficient expression of GAD, part of L-glutamic acid was transformed into GABA, and the final yield of GABA was about 8.28 g/L. CONCLUSION: We successfully constructed a recombinant strain that could efficiently produce GABA by one-step fermentation from glucose. This research provided a new approach for GABA production.
OBJECTIVE: We constructed an integrative recombinant Corynebacterium crenatum that could directly convert glucose in gamma-aminobutyric acid (GABA). METHODS: Using overlap PCR, we obtained delta argB fragment that lacked 491 bp of N-acetylglutamate kinase (NAGK) gene. The glutamate decarboxylase (GAD) gene attached tac promoter was amplified and inserted into delta argB fragment. Using the upstream and downstream of delta argB as homologous arms, we constructed pK18-delta argB::tacgad which was used for the integration of tacgad gene onto the C. crenatum genome. Through two homologous recombinations, we got an argB blocked strain C. crenatum delta argB::tacgad which could successfully express GAD. We also fermented the recombinant strain with glucose as substrate and the production of GABA was detected. RESULTS: In C. crenatum delta argB::tacgad, NAGK was totally inactivated and no L-arginine was detected though L-glutamic acid was accumulated. As a result of the efficient expression of GAD, part of L-glutamic acid was transformed into GABA, and the final yield of GABA was about 8.28 g/L. CONCLUSION: We successfully constructed a recombinant strain that could efficiently produce GABA by one-step fermentation from glucose. This research provided a new approach for GABA production.