Application of CRISPRi in Corynebacterium glutamicum for shikimic acid production.

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.