Despite the long-term interest in solventogenic clostridia-based ABE (acetone-butanol-ethanol) fermentation, clostridial butanol tolerance and its underlying mechanism remain poorly understood, which is a major obstacle hindering further improvements of this important fermentative process. In this study, a two-component system (TCS), BtrK/BtrR, was identified and demonstrated to positively regulate butanol tolerance and ABE solvent formation in Clostridium acetobutylicum, a representative species of solventogenic clostridia. The transcriptomic analysis results showed that BtrK/BtrR has a pleiotropic regulatory function, affecting a large number of crucial genes and metabolic pathways. Of the differentially expressed genes, btrTM, encoding a putative ABC-type transporter (named BtrTM), was shown to be under the direct control of BtrR, the response regulator of the BtrK/BtrR TCS. Furthermore, BtrTM was shown to contribute to more butanol tolerance (46.5% increase) by overexpression, revealing a novel regulatory mechanism consisting of the BtrK/BtrR TCS and the BtrTM transporter in C. acetobutylicum. Based on these findings, we achieved faster growth and solvent production of C. acetobutylicum by overexpressing BtrK/BtrR or its direct target BtrTM, although no significant improvement in the final butanol titer and yield. These results further confirm the importance of BtrK/BtrR and BtrTM in this organism. Also, of significance, a specific number of btrR-btrT-btrM-btrK-like gene clusters were identified in other Clostridium species, including the pathogens Clostridium perfringens and Clostridium botulinum, indicating a broad role for this regulatory module in the class Clostridia.

中文翻译：

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由两组分系统和ABC型转运蛋白组成的新型调控途径有助于丙酮丁醇梭菌对丁醇的耐受性。

尽管长期以来对产溶剂的梭状芽胞杆菌ABE（丙酮-丁醇-乙醇）发酵感兴趣，但梭菌对丁醇的耐受性及其潜在机理仍知之甚少，这是阻碍这一重要发酵过程进一步改进的主要障碍。在这项研究中，确定了一种两组分系统（TCS）BtrK / BtrR，它可以正向调节丙酮丁醇梭菌中的丁醇耐受性和ABE溶剂的形成，这是一种产溶剂梭菌的代表种。转录组分析结果表明，BtrK / BtrR具有多效性调节功能，影响大量关键基因和代谢途径。在差异表达的基因中，编码推定的ABC型转运蛋白（称为BtrTM）的btrTM被显示为受BtrR的直接控制，BtrK / BtrR TCS的响应调节器。此外，BtrTM被证明通过过表达有助于提高丁醇耐受性（增加46.5％），从而揭示了由BtrK / BtrR TCS和丙酮丁醇梭菌中BtrTM转运蛋白组成的新型调节机制。基于这些发现，我们通过过量表达BtrK / BtrR或其直接靶标BtrTM实现了丙酮丁醇梭菌的更快生长和溶剂生产，尽管最终丁醇滴定度和收率没有明显改善。这些结果进一步证实了BtrK / BtrR和BtrTM在该生物中的重要性。同样重要的是，在其他梭菌属物种中也发现了特定数量的btrR-btrT-btrM-btrK样基因簇，包括病原体产气荚膜梭菌和肉毒梭菌，表明该调控模块在梭菌属中具有广泛的作用。