Zhang Bo, Liu Zhi-Qiang, Liu Chang, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic of China.
Biotechnol Lett. 2016 Dec;38(12):2153-2161. doi: 10.1007/s10529-016-2207-z. Epub 2016 Sep 13.
To construct, test and exploit the CRISPRi system for enhancement of shikimic acid production with Corynebacterium glutamicum.
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.
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.
构建、测试和利用CRISPRi系统来提高谷氨酸棒杆菌莽草酸的产量。
CRISPRi系统用于在转录水平调控谷氨酸棒杆菌的基因表达。将Hfq蛋白介导的小调控RNA系统与CRISPRi系统进行了比较。采用效率更高的CRISPRi系统来调整涉及莽草酸（SA）合成途径的代谢通量。在包括转录调节因子在内的11个候选基因中，有3个靶点对提高SA产量有效。通过过表达ncgl1512并下调ncgl2008、ncgl2809、ncgl1856的表达，在250 ml摇瓶中SA产量提高了115%，达到7.76 g/l，在5 l发酵罐中达到23.8 g/l，这是报道的谷氨酸棒杆菌最高的莽草酸产量。
构建了CRISPRi系统，它是一种用于操纵谷氨酸棒杆菌中多个基因以生产莽草酸的高性能且省时的方法。此外，CRISPRi系统在调节转录调节因子的表达方面也很有效。
