A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction-modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction-modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.

中文翻译：

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通过完整的甲基化组分析和规避天然限制性修饰系统，合理开发热纤梭菌ATCC 27405中的转化。

代谢工程和基础生物学研究的主要障碍是大多数微生物缺乏遗传工具。一个例子是热纤梭菌ATCC 27405T，那里没有遗传工具来帮助验证数十年的假说。DNA转化的一大障碍是限制性修饰系统，该系统可防御与宿主甲基化程度不同的外源DNA。为了确定该菌株中的活性限制性修饰系统，我们通过单分子实时测序进行了完整的甲基化组分析，以检测6-甲基腺嘌呤和4-甲基胞嘧啶，而很少使用的全基因组亚硫酸氢盐测序，以检测5-甲基胞嘧啶。鉴定出多个活性系统，并从大肠杆菌染色体表达相应的DNA甲基转移酶以模拟C。甲基纤维素。质粒甲基化已通过实验验证，并成功地电穿孔入热纤梭菌ATCC27405。这种组合方法能够对热纤梭菌型菌株进行遗传修饰，并充当转化其他非模式微生物的蓝图。