Department of Biosystems Engineering, Auburn University, Auburn, Alabama.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture and Rural Affairs, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
Biotechnol Bioeng. 2020 Sep;117(9):2911-2917. doi: 10.1002/bit.27435. Epub 2020 Jun 4.
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 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.
凝结芽孢杆菌 ATCC 25755 是一种天然的超高丁酸盐生产者，具有很大的潜力，可作为从可再生资源中进行有价值的生物化学生产的优秀平台进行工程设计。然而，有限的转化效率和缺乏遗传操作工具限制了该微生物的更广泛应用。在这项研究中，通过基因缺失研究了 I 型限制修饰系统和天然质粒对凝结芽孢杆菌接合效率的影响。I 型限制内切酶的缺失导致接合效率提高了 3.7 倍，而天然质粒的进一步消除进一步将接合效率提高到 6.05 ± 0.75×10 CFU/ml-供体，比野生型菌株提高了 15.3 倍。发酵结果表明，这两个遗传元件的缺失对所得突变体 ΔRMIΔNP 中的终产物生产没有显著影响。由于接合效率的提高，先前无法在凝结芽孢杆菌中实施的 CRISPR-Cas9/Cpf1 系统成功地用于 ΔRMIΔNP 的基因组编辑，效率为 12.5-25%。总之，我们在此开发的方法为建立非模型微生物中的有效 DNA 转化方法提供了有价值的指导。ΔRMIΔNP 突变体可作为用于多种有价值的生物燃料和生物化学生产的优秀底盘。
