利用 CRISPR-AsCpf1 系统在枯草芽孢杆菌中开发高效的迭代基因组编辑方法。
Development of an efficient iterative genome editing method in Bacillus subtilis using the CRISPR-AsCpf1 system.
机构信息
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
出版信息
J Basic Microbiol. 2022 Jul;62(7):824-832. doi: 10.1002/jobm.202200134. Epub 2022 Jun 2.
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.
枯草芽孢杆菌是合成生物学和代谢工程领域中用于化学生产的有用底盘。在这里，我们构建了基于 CRISPR-Cpf1 的表达质粒，带有温度敏感复制子，用于枯草芽孢杆菌的迭代基因组编辑。该方法允许进行基因插入和大片段基因组缺失，编辑效率高达 80%-100%，并且可以快速进行质粒消除，从而促进枯草芽孢杆菌 168 的迭代基因组编辑。使用定制的 CRISPR-Cpf1 系统，我们成功且高效地在枯草芽孢杆菌 168 中实现了相关基因编辑，用于透明质酸（HA）生物合成、HA 合成酶基因（hasA）插入、UDP-葡萄糖脱氢酶基因（tuaD）插入和 eps 基因簇（epsA-O）缺失。通过工程菌株实现了异源 HA 的生产，产量为 1.39g/L。这些结果支持了 CRISPR-Cpf1 系统在细菌基因组编辑中具有高效性的发现，并为使用 CRISPR-Cpf1 系统进行枯草芽孢杆菌基因组工程提供了有价值的指导和重要参考。
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