CRISPRi/ddCpf1促进大肠杆菌中丙二酰辅酶A的积累

丙二酰辅酶A是多种有价值化合物（包括食品、保健品等）合成的重要前体物质,其合成已成为大肠杆菌中生产目标代谢物的潜在瓶颈。为解决其生物合成的瓶颈问题,作者通过CRISPR干扰（CRISPR interference,CRISPRi） 促进丙二酰辅酶A的积累。首先,构建了丙二酰辅酶A的生物传感器,实现了其快速、可视化的胞内测量。随后,构建了CRISPRi/ddCpf1系统,实现了基因转录的抑制,并尝试用ddCpf1（催化失活的Cpf1）和RNA聚合酶抑制蛋白Gp2融合表达（CRISPRi/ddCpf1-Gp2）。结果表明,Gp2虽然有一定的转录抑制效果,但是严重影响了生长。最后,利用CRISPRi/ddCpf1系统进行两组双靶点基因抑制,分别靶向乙醛脱氢酶和3-氧代酰基-酰基载体蛋白合酶II基因,以及3-氧代酰基-酰基载体蛋白合酶I和琥珀酰辅酶A合成酶基因,均实现了丙二酰辅酶A浓度的提高。

Accumulation of Malonyl-CoA in E. coli Promoted by CRISPRi/ddCpf1

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.