Application of CRISPRi in Corynebacterium glutamicum for shikimic acid production.
OBJECTIVE
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.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Bo
B
Zhang
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Zhi-Qiang
ZQ
Liu
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
Chang
C
Liu
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.
Yu-Guo
YG
Zheng
D001426:Bacterial Proteins / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D012765:Shikimic Acid / Q000378:metabolism*
D001426:Bacterial Proteins; D012765:Shikimic Acid
CRISPRi; Corynebacterium glutamicum; Shikimate pathway; Shikimic acid