食气梭菌化能固碳的乙酰化修饰调控机理

Gas-fermenting Clostridia are a group of special anaerobic microorganisms capable of using one-carbon gas (such as CO and CO2) for synthesis of a series of important chemicals and fuels. In the previous studies, we observed that, besides transcriptional level, post-translational modification also plays an important role in metabolic regulation of gas-fermenting clostridia. Interestingly, a mutual restraint exists between the transcriptional and post-translational regulation, and then may be involved in multiple physiological and biochemical processes, e.g., carbon fixation and solvent synthesis. However, the underlying mechanism remains unclear. Here, we will make an investigation on acetylation modification in regulating an important trait, i.e., carbon fixation, of Clostridium ljungdahlii. Firstly, through bioinformatics analysis and molecular biology techniques, the major acetylation system (acetyltransferase/deacetylation) that is involved in enzymatic modification will be identified, enabling a further investigation of the acetylation targets in the carbon fixation pathway under the condition of one-carbon gas fermentation. Then, by combining biochemical and phenotypic analysis, the effect of acetylation modification on the function of key enzymes responsible for carbon fixation will be revealed. On this basis, the above acetylation sites will be mutated, aiming to further confirm their regulatory roles as well as optimize the regulation process. Finally, The rule and molecular mechanism of the mutual coordination and restriction between the post-translational and transcriptional regulation on carbon fixation will be studied. Taken together, the purpose of this work is to understand the carbon fixation process from the perspective of the “modification regulation”, aiming to find new regulation targets and mechanisms, then providing new ideas for the design and reconstruction of industrial gas-fermenting clostridia.

食气梭菌是一类能够利用一碳气体（如CO、CO2）发酵合成化学品及燃料的特殊厌氧微生物。我们在前期的研究中发现，该菌的代谢调控除转录水平外，翻译后修饰调控也扮演着十分重要的角色，并且二者存在着有趣的相互制约关系，涉及碳固定、溶剂合成等多种生理生化过程，但作用机理尚不清楚。本项目拟围绕食气梭菌的重要性状—化能固碳的乙酰化修饰调控开展研究。以代表性菌株扬氏梭菌为研究对象，通过生物信息学和分子生物学技术，首先鉴定出参与酶催化修饰的主效乙酰化系统，找到在一碳气体培养条件下固碳途径中的乙酰化修饰靶点；然后结合生化与表型分析，阐明乙酰化修饰对固碳代谢关键酶功能的影响，并通过修饰位点的突变进行验证和调控过程的优化；发现固碳途径翻译后修饰水平与转录水平调控之间的相互协调与制约的规律，揭示其分子机制。本研究从“修饰调控”的角度来认识化能固碳过程，旨在发现新机制和新靶点，为工业食气梭菌的设计改造提供依据。

产溶剂梭菌作为重要的工业微生物，不仅能够利用廉价的木质纤维素原料，还可吸收和代谢CO、CO2等一碳气体，发酵合成乙醇、丁醇和乙酸等多种化学品和生物燃料。在前期研究中，我们发现除转录水平的代谢调控外，产溶剂梭菌还普遍存在蛋白质赖氨酸乙酰化修饰现象，即受到翻译后修饰的调控，提示转录调控以外的其他类型代谢调控在产溶剂梭菌中也可能扮演重要角色，值得深入探究。.本项目以食气产溶剂梭菌——扬氏梭菌为研究对象，研究蛋白质乙酰化修饰在该菌中的分布与作用，尤其聚焦一碳气体利用的化能固碳过程，同时解析乙酰化修饰与转录调控之间的互作与协调机制。为此，我们首先通过蛋白质乙酰化组学分析，比较全面地揭示了扬氏梭菌中存在乙酰化修饰蛋白及位点。在此基础上，通过体外生化分析筛查潜在的乙酰化系统，成功鉴定了在扬氏梭菌中起主效作用的乙酰基转移酶At2和去乙酰化酶Dat1。进一步研究发现，乙酰化修饰系统At2/Dat1参与了化能固碳关键酶——甲酸脱氢酶（FDH1）和碳代谢相关全局性调控蛋白CcpA的乙酰化修饰和去乙酰化修饰反应。FDH1的关键位点K29经乙酰化修饰后导致酶活性显著下降，CcpA关键位点K56经乙酰化修饰后会影响其与靶DNA结合的能力。有趣的是，转录因子CcpA不仅能调控甲酸脱氢酶基因fdh1，还能直接调控主效乙酰基转移酶基因at2的表达，从而与at2形成了一种交互影响甲酸脱氢酶基因fdh1活性的调控模式。体内酶活力分析进一步表明，FDH1的活性受到转录调控和乙酰化修饰的双重影响，而且前者的影响更大。基于上述认识，我们通过同时解除乙酰基转移酶At2和转录因子CcpA对甲酸脱氢酶FDH1的抑制作用，显著提升了扬氏梭菌的细胞性能，单批发酵时菌株对CO2的利用量以及总溶剂产量比原始菌株分别提高了129%和124%。.这些研究揭示了自养细菌中一种多层次协同调控固碳过程的新机制，也为菌株的遗传改造提供了有价值的信息。项目发表论文3篇，培养研究生3名，按计划完成了任务。