Engineering Research Center of Application and Transformation for Synthetic Biology, School of Bioengineering, Dalian University of Technology, Dalian, 116024, China.
Ningbo Institute of Dalian University of Technology, Ningbo, 315016, China.
Appl Microbiol Biotechnol. 2022 Nov;106(22):7563-7575. doi: 10.1007/s00253-022-12234-0. Epub 2022 Oct 26.
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 Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 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. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation.
丝氨酸/苏氨酸蛋白激酶（STKs）在信号转导中起着重要作用，参与多种生理过程，包括细菌的细胞生长、中心代谢和孢子形成。然而，STKs 在产溶剂梭菌中的作用仍不清楚。本研究在拜氏梭菌中鉴定并全面研究了六个 STK 候选物。通过遗传组织分析、蛋白结构域预测和多重序列比对，将这些 STK 分为四个具有不同特征的组。Cbei0566 是 PrkA 家族的成员，与枯草芽孢杆菌的 PrkA 具有 41%的同一性，而 Cbei0666 和 Cbei0813 是双组分样 STK。Cbei1151 和 Cbei1929 属于 Hanks 家族 STKs，由细胞质催化结构域、跨膜区和细胞外传感器结构域组成。cbei0566、cbei0666、cbei1929 和 cbei2661 的框内缺失突变体与野生型相比具有相似的细胞生长。Δcbei0666 和 Δcbei2661 分别提高了丙酮丁醇乙醇（ABE）产量 14.3%（19.2g/L 比 16.8g/L），Δcbei0566、Δcbei1929 和 Δcbei2661 的孢子形成频率分别显著下降至 35.5%、55.1%和 44.8%。遗传互补后的恢复表型表明它们与 STKs 缺失直接相关。值得注意的是，cbei0566 的过表达导致孢子形成增加了 41.5%，cbei1929 的过表达将 ABE 产量从 19.3 提高到 24.2g/L，同时酸减少了 25%。这些结果表明 Cbei0566 和 Cbei1929 具有显著的调控功能。本研究扩展了 STKs 在原核生物中存在和功能的现有知识，并强调了 STK 介导的信号网络在开发优良菌株中的重要性。
