枯草芽孢杆菌
清脆的
基因组编辑
基因组工程
质粒
生物
基因组
代谢工程
Cas9
基因
合成生物学
计算生物学
反式激活crRNA
遗传学
细菌
作者
Xingcong Zhao,Xi Chen,Yali Xue,Xuedong Wang
标识
DOI:10.1002/jobm.202200134
摘要
Bacillus subtilis is a useful chassis in the fields of synthetic biology and metabolic engineering for chemical production. Here, we constructed CRISPR-AsCpf1-based expression plasmids with the temperature-sensitive replicon for iterative genome editing in B. subtilis. This method allowed gene insertion and large genomic deletion with an editing efficiency of up 80%-100% and rapid plasmid curing to facilitate the iterative genome editing in B. subtilis 168. Using the customized CRISPR-AsCpf1 system, we successfully and efficiently implemented the related gene editing in B. subtilis 168 for hyaluronic acid (HA) biosynthesis, HA synthase gene (hasA) insertion, UDP-glucose-dehydrogenase gene (tuaD) insertion, and eps gene cluster (epsA-O) deletion. The heterologous production of HA was realized by the engineered strain with a yield of 1.39 g/L. These results support the finding that the CRISPR-AsCpf1 system is highly efficient in bacteria genome editing and provide valuable guidance and essential references for genome engineering in B. subtilis using the CRISPR-AsCpf1 system.
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