Yang Zhiheng, Li Zilong, Li Bixiao, Bu Ruihong, Tan Gao-Yi, Wang Zhengduo, Yan Hao, Xin Zhenguo, Zhang Guojian, Li Ming, Xiang Hua, Zhang Lixin, Wang Weishan
State Key Laboratory of Bioreactor Engineering, and School of Biotechnology, East China University of Science and Technology (ECUST), 200237, Shanghai, China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China.
Nat Commun. 2023 Oct 4;14(1):6193. doi: 10.1038/s41467-023-41973-5.
Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 10 CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.
嗜热细胞工厂在工业应用方面具有显著广泛的潜力，但受到缺乏基因操作工具和转化难的限制。在此，我们从嗜热栖热糖芽孢杆菌中鉴定出一种嗜热I-B型CRISPR-Cas系统，并发现其表现出高效的转录抑制或DNA切割活性，分别通过将crRNA长度调整为小于或大于26 bp来切换，且不影响Cas3核酸酶活性。然后，我们利用该I-B型系统在嗜热菌和嗜温菌宿主中开发了一种用于基因组编辑和转录抑制的正交工具。借助这一工具，我们设计了一种策略来筛选参与转化效率的基因组规模靶点，并通过时间多路复用抑制建立了高效（~10 CFU/μg DNA）的动态可控超感受态嗜热栖热糖芽孢杆菌细胞。我们还通过基因组规模鉴定和多个靶点的组合操作，展示了在高温发酵中构建迄今最高效价的嗜热核黄素细胞工厂。这项工作实现了嗜热栖热糖芽孢杆菌中多种高效基因操作，并促进了嗜热细胞工厂的工程化。
