Identification of serine/threonine kinases that regulate metabolism and sporulation in Clostridium beijerinckii

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Delta cbei0666 and Delta cbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Delta cbei0566, Delta cbei1929, and Delta cbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2 g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains.