丙丁梭菌孤立组氨酸激酶的多效调控机理及丁醇发酵协同解毒策略研究

High cell density culture could enhance the stress tolerance of Clostridium acetobutylicum and decrease the specific butanol production rate, but the mechanism underlying these phenomena is not clear. In our previous work, the specific butanol production rate could be increased by a histidine kinase inactivation, but be inhibited by the addition of signal molecule oligopeptide in the medium, simultaneously with the influence of sporulation, which may be involved in quorum sensing with the histidine kinase. In this project, through the inactivation and complementation of one or multiple histidine kinases by ClosTron Group II intron retargeting technology, we will investigate the pleiotropic functions of orphan histidine kinases as well as their differences and collaboration in Clostridium acetobutylicum with high cell density. The effect of histidine kinases on sporulation and cell proliferation, signal transduction, stress tolerance, carbon metabolism and global gene expression will be discussed with/without butanol stress conditions by the method of transcriptome and metabolic flux analysis as well as integrated product removal of inhibitory butanol. This project is expected to solve the scientific problem of the decrease of specific butanol production rate under high cell density condition, and establish the synergetic strategy of detoxification with better compatibility for high efficiency of butanol fermentation. Finally, the achievements not only contribute to the improvement of butanol conversion in the fermentation with high cell density, but also provide new idea for the regulation of secondary metabolism by other bacteria.

高密度细胞培养可以提高丙丁梭菌细胞的胁迫耐性，但单位细胞的产丁醇能力显著降低，其机理尚不明确。前期研究发现，丙丁梭菌的产丁醇能力在孤立组氨酸激酶失活时显著增加，但受寡肽类信号物质抑制，孢子发育同时受到影响，这可能与由组氨酸激酶参与信号转导的群体效应有关。本项目拟利用二类内含子基因失活技术，通过对单个或多个孤立组氨酸激酶同时失活，研究组氨酸激酶在高密度细胞培养体系中对丙丁梭菌的多效调控以及它们功能的特异性和协同作用。从全局基因表达和代谢分析入手，探讨组氨酸激酶对孢子和细胞发育、信号感应和转导、胁迫耐性、碳源代谢以及全局基因表达的多效调控机制，建立发酵分离耦合解除丁醇抑制调控与孤立组氨酸激酶调控下的细胞发育和代谢规律相匹配的协同解毒策略，解决高密度培养体系中单位细胞产丁醇能力降低的科学问题。研究成果不仅有助于提高丙丁梭菌高密度发酵的丁醇转化效率，还为其它菌株生产次级代谢产物的调控提供新思路。

丁醇具有能量密度高等优点，被认为是比燃料乙醇更优质的新一代生物能源。丁醇对丙丁梭菌细胞的抑制作用，导致发酵终点的丁醇浓度较低，造成分离纯化的能耗成本较高。组氨酸激酶不仅与群体感应和信号转导密切相关，还对孢子发育和溶剂化有重要影响，其机理尚不明确。本项目利用基因工程技术，构建了孤立组氨酸激酶（CAC3319,CAC0323,CAC0437,CAC0903,CAC2730）单个或多个失活的组合文库，改变单个或多个孤立组氨酸激酶在丙丁梭菌中表达水平的差异，分别解析了梭菌中对产溶剂和细胞生长的必需组氨酸激酶基因。发现了不同组氨酸激酶对梭菌产溶剂调控的功能差异性，如CAC3319和CAC0323负责磷酸化，单个或同时失活促进梭菌产溶剂；而CAC0437负责脱磷酸化，单个失活抑制梭菌产溶剂。它们在梭菌中表达强度的变化影响孢子周期的重要调控因子，进而影响孢子形成，调控细胞的产溶剂能力。CAC3319和CAC0323失活，可以降低梭菌中一些与孢子周期相关蛋白的磷酸化水平（如Spo0A）,进而阻断孢子形成。梭菌不产生孢子，有利于梭菌长期维持在营养细胞状态（vegetative cells）和产溶剂期，减缓细胞自溶。因此，利用CAC3319和CAC0323同时失活的基因工程菌进行高密度发酵，丁醇浓度可达20.7 g/L （总溶剂33.6 g/L），比野生菌株提高~70%。在此基础上，我们制备的两端开口碳纳米管阵列PDMS复合膜对发酵液中的丁醇进行分离纯化，冷凝液中的分离丁醇浓度达到441.9 g/L,分离能耗降低到2.4 MJ/kg。因此，当发酵液中的丁醇浓度提高到2%，分离纯化能耗会显著降低。上述研究成果发表SCI论文14篇，申请中国发明专利2项，获省部级以上科技奖励2项。