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 × 10³ 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 ΔRMIΔNP. 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 ΔRMIΔNP 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 ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.

中文翻译：

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在酪丁酸梭菌中提高质粒转化效率并实现基于 CRISPR-Cas9/Cpf1 的基因组编辑。

Clostridium tyrobutyricum ATCC 25755 被认为是一种天然的超丁酸生产者，具有巨大的潜力，是一个极好的平台，可用于利用可再生资源进行有价值的生化生产。然而，有限的转化效率和缺乏基因操作工具阻碍了这种微生物的更广泛应用。在本研究中，通过基因缺失研究了 I 型限制性修饰系统和天然质粒对酪丁酸梭菌结合效率的影响。I 型限制性核酸内切酶的缺失导致结合效率提高了 3.7 倍，而天然质粒的额外消除进一步将结合效率提高到 6.05 ± 0.75 × 10 ³CFU/ml 供体，比野生型菌株高 15.3 倍。发酵结果表明，这两个遗传元件的缺失并未显着影响所得突变体 Δ RMI Δ NP的终产物生产。由于偶联效率的提高，以前无法在酪丁酸梭菌中实施的 CRISPR-Cas9/Cpf1 系统成功用于 Δ RMI Δ NP 中的基因组编辑，效率为 12.5-25%。总之，我们在此开发的方法为在非模型微生物中建立有效的 DNA 转化方法提供了宝贵的指导。Δ RMI Δ NP 突变体可以作为一个很好的底盘，用于各种有价值的生物燃料和生化生产。