Bo Zhang1, Zhi-Qiang Liu1, Chang Liu2, Yu-Guo Zheng3. 1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. 2. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China. 3. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.
Abstract
OBJECTIVES: To construct, test and exploit the CRISPRi system for enhancement of shikimic acid production with Corynebacterium glutamicum. RESULTS: The CRISPRi system was used to regulate C. glutamicum gene expression at the transcriptional level. Hfq protein-mediated small regulatory RNAs system was compared with CRISPRi system. The more efficient CRISPRi system was used to adjust the metabolic flux involving the shikimic acid (SA) synthetic pathway. In 11 candidate genes, including transcription regulator, three targets were effective for increasing SA production. Through over-expression of ncgl1512 and down-regulating the expression of ncgl2008, ncgl2809, ncgl1856, the titers of SA increased 115 % to 7.76 g/l in 250 ml flasks and 23.8 g/l in 5 l fermentor, which is the highest shikimic acid yield reported for C. glutamicum. CONCLUSIONS: CRISPRi system was constructed and is a high-performance and time-saving method to manipulate multiple genes in C. glutamicum for shikimic acid production. Moreover, CRISPRi-system was also effective in regulating the expression of a transcription regulator.
OBJECTIVES: To construct, test and exploit the CRISPRi system for enhancement of shikimic acid production with Corynebacterium glutamicum. RESULTS: The CRISPRi system was used to regulate C. glutamicum gene expression at the transcriptional level. Hfq protein-mediated small regulatory RNAs system was compared with CRISPRi system. The more efficient CRISPRi system was used to adjust the metabolic flux involving the shikimic acid (SA) synthetic pathway. In 11 candidate genes, including transcription regulator, three targets were effective for increasing SA production. Through over-expression of ncgl1512 and down-regulating the expression of ncgl2008, ncgl2809, ncgl1856, the titers of SA increased 115 % to 7.76 g/l in 250 ml flasks and 23.8 g/l in 5 l fermentor, which is the highest shikimic acid yield reported for C. glutamicum. CONCLUSIONS: CRISPRi system was constructed and is a high-performance and time-saving method to manipulate multiple genes in C. glutamicum for shikimic acid production. Moreover, CRISPRi-system was also effective in regulating the expression of a transcription regulator.
Authors: Christopher Nosala; Kari D Hagen; Nicholas Hilton; Tiffany M Chase; Kelci Jones; Rita Loudermilk; Kristofer Nguyen; Scott C Dawson Journal: J Cell Sci Date: 2020-08-27 Impact factor: 5.285
Authors: S G McInally; K D Hagen; C Nosala; J Williams; K Nguyen; J Booker; K Jones; Scott C Dawson Journal: Mol Biol Cell Date: 2018-10-31 Impact factor: 4.138