组氨酸/丝氨酸/苏氨酸激酶协同调控丙丁梭菌合成生物丁醇的分子机制

Currently, strain development of Clostridium acetobutylicum is limited in the study on metabolic pathway genes and some of transcription factors (Spo0A、σ. etc), which leads to the bottleneck of butanol titer and low efficiency of target genes. In our previous work, butanol production could be increased by histidine kinases inactivation, which indicates that kinase phosphorylation plays a key role in metabolism regulation. Till now, there is less report on histidine/serine/threonine protein kinases that are responsible for post-translational modification of protein. In this project, the combinatorial mutants with 40 kinases inserted inactivation will be constructed using multi-plasmid co-transformation method, which will be used to investigate the pleiotropic and synergistic functions of histidine/serine/threonine protein kinases in Clostridium acetobutylicum. The effect of three kinds of kinases on sporulation and cell proliferation, signal transduction, stress tolerance, carbon metabolism and global gene expression will be investigated by the method of transcriptome and metabolic flux analysis. Furthermore, the interaction of histidine/serine/threonine protein kinases with transcriptional factor and protein kinases will be elucidated by phosphoproteome and in vitro phosphate transfer analysis. The difference, complementarity and coordination of these protein kinases will be studied, which is for strain construction with excellent ability of butanol production. Finally, the achievements not only contribute to the improvement of butanol conversion in the fermentation, but also provide new idea for the regulation of other products metabolism by various bacteria.

丙丁梭菌的改造局限于对代谢途径基因和转录因子（Spo0A、σ因子等）研究，存在产丁醇浓度低和靶点基因效果不明显等问题。我们研究发现，组氨酸激酶同时失活使梭菌产丁醇显著增加，说明激酶对菌株代谢有调控作用。目前对丙丁梭菌激酶的研究鲜有报道，其协同调控功能研究难点在于多激酶突变菌株的高效筛选。本项目利用多质粒共转化法，建立40个激酶（组氨酸/丝氨酸/苏氨酸激酶）的不同组合突变库，研究它们对梭菌生理代谢的全局协同调控功能。从全局基因表达和代谢分析入手，探讨三类激酶对孢子和细胞发育、胁迫耐性、碳源代谢以及全局基因表达的多效调控机制。在此基础上，通过磷酸化蛋白质组学和体外磷酸转移活性分析，揭示激酶之间以及与转录因子和蛋白酶的磷酸转移作用关系，阐明不同激酶功能特异性、互补性和协同作用，构建激酶全局调控的产丁醇能力优良菌株。研究成果既可以提高丙丁梭菌的丁醇转化效率，还可以丰富相关菌株生理代谢的理论认知。

梭菌中的激酶可参与信号转导，影响梭菌产丁醇能力，但梭菌内组氨酸/丝氨酸/苏氨酸激酶靶点有待挖掘，其对代谢调控和细胞发育的作用机制尚不明确。本项目创建了快速的梭菌多基因编辑方法，五个激酶基因组合突变菌株库构建时间由242天缩短至11天，激酶改造菌株选育效率大幅提高。揭示了不同激酶影响孢子形成/发育、细胞生长和产醇代谢的差异性与协同性调控作用，构建的多激酶改造菌株丁醇发酵浓度达20.6 g/L，较原始菌株提高70%，是目前报道最高值。结合细胞形态、转录组和磷酸化蛋白组分析，组氨酸激酶改造菌株可增强丁醇合成基因表达，阻断孢子形成，调节丝氨酸/苏氨酸激酶表达及其磷酸化模式。通过丁醇发酵与碳纳米管阵列复合膜分离耦合技术，发酵分离纯化丁醇浓度达441.9 g/L，分离能耗显著降低。.本项目在国际权威学术期刊发表SCI论文15篇，申请中国发明专利4项，培养博士后1人，研究生8人，获日内瓦国际发明展金奖、中国商业联合会全国商业科技进步奖特等奖、辽宁省科学技术进步奖一等奖、中国可再生能源学会科学技术奖一等奖、辽宁省自然科学学术成果奖一等奖，入选“科创中国”新能源技术应用案例库，完成成果技术转移2项，为构建优良菌株提供了重要的理论基础和技术支撑。