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Enhancing plasmid transformation efficiency and enabling CRISPR‐Cas9/Cpf1‐based genome editing in Clostridium tyrobutyricum
Author(s) -
Zhang Jie,
Hong Wei,
Guo Liang,
Wang Yifen,
Wang Yi
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27435
Subject(s) - genome editing , plasmid , crispr , cas9 , transformation (genetics) , transformation efficiency , computational biology , biology , chemistry , gene , genetics , agrobacterium
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 × 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 Δ RMI Δ 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 Δ RMI Δ 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 Δ RMI Δ NP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.