脂肪酸互营氧化过程电子载体甲酸的合成调控与高效跨膜转运的分子机制

As a critical step in the process of methane produced by anaerobic digestion, smooth oxidation of short-chain fatty acid is vital to maintain the efficiency and stability of an anaerobic system. The low metabolic rate of syntrophic fatty acid oxidizing bacteria (SFOB), one kind of functional microbes oxidating short-chain fatty acid, is a limiting step in the anaerobic process besides methanogenic phase. Therefore, enhancing the ecological function of SFOB is a vital breakthrough to further increase the efficiency and stability of an anaerobic system on the existing conditions. The interspecific transformation of formic acid is proved to be an important way for the conversion of short chain fatty acids and prone to be a dominant way on certain conditions. Nevertheless, the catabolism of VFAs through SFOB is strictly controlled by the concentration of products, back to its causes, thermodynamic repression and feedback inhibition caused by accumulation of formic acid are the essential reasons restricting the metabolic efficiency and stability of syntrophic oxidation dominated by formic metabolism. Hence, a novel idea is proposed to solve these bottleneck: first, at the genetic level, enhance the tolerance of related enzymes to formic acid, one of electronic carrier, improving the synthesis of formic acid in the cell; second, boost the transmembrane transport of formic acid to accelerate the electron carrier entering the cell and initiating interspecific electron transfer process. In order to realize these research ideas, it is necessary to make clear the molecular mechanism of "syntrophic metabolism of SFOB regulated by formic acid to produce acetic acid and formic acid.” Accordingly, synthesis technique of radioactive isotope tracer, proteomics, metabolomics, high-throughput sequencing, real-time quantitative PCR and the fluorescent protein markers are proposed to be adopted to carry out the following research from the molecular and project level: (1) the molecular mechanism of feedback regulation on metabolic enzymes in SFOB with accumulation of formic acid, (2) genetically removing thermodynamic inhibition of formic acid on metabolism of SFOB producing formic and acetic acid, (3) study on the engineering regulation on increasing the synthesis and transmembrane transport of intracellular formic acid. The expected results of this project will enrich the theory system of anaerobic digestion and provide technical support for engineering regulation with great scientific significance.

短链脂肪酸（VFA）互营氧化产甲酸产乙酸代谢是其降解的途径之一，其重要性已被证实。该过程为厌氧消化枢纽步骤是系统高效运行的关键，但主导该过程的脂肪酸互营氧化菌（SFOB）代谢水平低是制约提高厌氧消化效能的重要原因。SFOB代谢受控于产物浓度，溯其根源甲酸引起的热力学阻碍和反馈抑制是制约其代谢能力的本质。针对该瓶颈项目提出新的解决思路：从基因水平提升关键酶对甲酸的耐受力实现甲酸合成调控，提高反向电子传递效率；同时通过甲酸外排机制研究，加快甲酸进入胞外并快速启动种间电子传递过程。为实现此思路，需解决甲酸调控VFA互营代谢的分子机制的关键科学问题。拟以多组学为核心手段开展SFOB关键酶受反馈调节的分子机制、基因水平解除SFOB代谢热力学阻碍效应及强化SFOB胞内甲酸合成与跨膜转运能力的工程学方法等研究。预期成果可对现有厌氧消化理论体系进行丰富与完善并可为工程调控提供技术支持，具有重要的科学意义。

厌氧消化作为一种以微生物代谢为核心的生物技术，其特点为有机物的最终矿化成甲烷过程是在产酸发酵菌、SFOB 和产甲烷菌对底物梯级利用下协作完成的。对于缺乏代谢分支途径的SFOB 受产能限制而决定其增殖缓慢，代谢能力低，是厌氧消化过程除产甲烷阶段外的另一关键步骤，提高SFOB 代谢能力是进一步提升厌氧消化系统效能的突破口。而SFOB 的产甲酸产乙酸代谢途径是VFAs 互营转化过程的一个重要组成部分。对基于关键酶的SFOB调控快速启动种间电子传递过程是保证VFAs 互营产甲酸产乙酸代谢高效进行的关键，对其系统研究有助于提高VFAs 的互营转化能力，进而达到在现有水平进一步提升厌氧消化产甲烷系统效能的目的。.基于以上认识，本项目通过开展SFOB 关键代谢酶受反馈调节的分子机制、解除电子载体甲酸对SFOB 菌代谢热力学阻碍效应、优化SFOB 代谢能力的工程学方法研究等三方面的工作，解决了电子载体甲酸调控VFAs 互营产甲酸产乙酸代谢的分子机制是什么的关键科学问题，确定了电子载体甲酸的反馈调控位点、解析了甲酸累积下SFOB 的关键代谢酶受反馈调节的分子机制、揭示了电子载体甲酸的种间电子传递机制、建立了解除甲酸对SFOB 产甲酸产乙酸代谢热力学阻碍效应的方法、获得了VFAs 互营产甲酸产乙酸代谢的最优化生态条件、确立了工程水平SFOB 产甲酸产乙酸代谢的最佳控制参数及调控策略。项目在研周期内发表学术论文31篇，其中重要国际学术期刊论文28篇，申请人为通讯作者的SCI论文26篇，申请人作为通讯作者发表中文核心期刊论文3篇；投递过程中SCI论文6篇；申请国家发明专利8项，其中6项获得授权；申请实用新型专利2，其中2项获得授权。参加学术会议与交流10人次并均做大会报告；培养博士生 4 人，硕士生 3 人。4人职称获得晋升。本项目的成果可对现有厌氧消化理论进行丰富与完善并可为工程调控提供技术支持，具有重要的科学意义。