Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum

文献类型： 外文期刊

第一作者： Zhang, Jie

作者： Zhang, Jie;Hong, Wei;Wang, Yifen;Wang, Yi;Zhang, Jie;Hong, Wei;Guo, Liang;Wang, Yifen;Wang, Yi

作者机构：

关键词： Clostridium tyrobutyricum; conjugation; CRISPR-Cas9; Cpf1; genome engineering; restriction-modification system

期刊名称：BIOTECHNOLOGY AND BIOENGINEERING （ 影响因子：4.53； 五年影响因子：4.63 ）

ISSN： 0006-3592

年卷期： 2020 年 117 卷 9 期

页码：

收录情况： SCI

摘要： Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 +/- 0.75 x 10(3) CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant Delta RMI Delta NP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in Delta RMI Delta NP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The Delta RMI Delta NP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.

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