Abstract:Malonyl-CoA is an important precursor for the synthesis of plenty of valuable chemicals such as food and health products. The synthesis of malonyl-CoA has been a potential bottleneck in the production of target metabolites in E. coli. To solve this problem, this research aimed to promote accumulation of malonyl-CoA in E.coli through CRISPR interference(CRISPRi). Firstly, a malonyl-CoA biosensor was constructed to achieve fast and visible detection of the intracellular malonyl-CoA. Secondly, a CRISPRi/ddCpf1 system was constructed to repress gene transcription via ddCpf1(inactive Cpf1). And we attempted the fusion of Gp2 and ddCpf1(CRISPRi/ddCpf1-Gp2) and RNA polymerase. The results showed that Gp2 contributed a certain transcriptional inhibition effect, however, it seriously affected the growth. Finally, the CRISPRi/ddCpf1 system was applied to inhibit with a dual crRNA array, targeting acetaldehyde dehydrogenase & 3-oxoacyl-acyl carrier protein synthase II genes and 3-oxoacyl-acyl carrier protein synthase I & succinyl-CoA synthetase genes, greatly enhanced malonyl-CoA content in E. coli.
