肠杆菌降解木质素新途径的分子机制

With the dwindling supplies of oil based fuels and real situations in China, it is very urgent and crucial to develop the second generation biofuels and bioproducts from renewable biomass. Lignin, closely associated with cellulose filaments,is the most abundant and renewable source of aromatic biopolymer in nature. There are consideration interests in the utilization of lignin for production of bioenergy and renewable chemicals.Compared with lignin degradation by chemical and physical methods, biodegradation of lignin by microorganisms shows significant advantages of friendly environment and strong specificity. Nowadays, it is generally considered that lignin biodegradation is completed through either dissimilatory or assimilatory actions by microorganisms in nature.However, in our previous works, it has been shown that Enterobacter sp. can breakdown lignin via both dissimilatory and assimilatory actions through 4-hydroxyphenylacetate degradation pathway, which is significantly different with those reported in literature. But the molecular mechanism of this original lignin biodegradation pathway has not been reported so far.Therefore, the main purpose of this project is to shed new lights on the molecular mechanisms of this novel pathway of lignin biodegradation in Enterobacter sp. YL1301, a lignin degradation bacteria, based on proteomics,transcriptomics and metabolomics technologies. First of all, we would like to elucidate the specific secrete enzymes and their proteomics profilings of this strain growing under the anaerobic conditions and lignin as sole carbon source. Then, we also address the functional transcriptome gene monitoring the "core enzymes" and "accessory enzymes" of this strain involved in lignin biodegradation via transcriptomics sequencing technology. Finally, we further detect the compositions and contents of intermediate products as well as electronic transport chain during lignin bio-breakdown pathway through NMR-NOESY and HPLC methods based on metabolomics analysis.The primary objective of this project is to provide insights on lignin biodegradation in microorganisms through a multi-omics approach, as well as to provide the scientific theory basis and technique supports for efficient bioconversion to biofuels and chemicals from lignin.

随着石化资源日益枯竭，生物能源和生物基化学品研发十分迫切，清洁高效降解丰富的木质素资源亟待解决。生物降解木质素因专一性强和环境友好等特点最具发展优势。普遍认为，纯种微生物只能通过同化或异化作用降解木质素。然而，研究发现，肠杆菌能同时通过同化和异化作用降解木质素，开辟生物降解木质素新途径。但是，这种新途径的分子机制仍不清楚。针对上述科学问题，本项目以自主筛选的木质素降解菌-肠杆菌YL1301为对象，围绕酶蛋白及其转录因子和降解产物三方面，多维度诠释肠杆菌降解木质素新途径的分子机制。首先，利用酶蛋白组学和质谱技术，阐明肠杆菌降解木质素"核心酶"和"附属酶"等特异性分泌酶蛋白的动力学变化规律和指纹谱；然后，通过转录组学和生物信息分析，揭示 "核心酶"和"附属酶"的功能转录因子；最后，耦合NMR和HPLC技术，探明该菌降解木质素代谢产物和电子传递链的变化,为木质素的高效利用奠定理论基础和技术支撑。

随着石化资源日益枯竭，第二代生物质能源研发十分迫切，清洁高效降解丰富的木质素资源亟待解决。生物降解木质素因专一性强和环境友好等特点最具发展优势。普遍认为，纯种微生物只能通过同化或异化作用降解木质素。然而，研究发现，肠杆菌能同时通过同化和异化作用降解木质素，开辟生物降解木质素新途径。但是，这种新途径的分子机制仍不清楚。.针对上述科学问题，本项目以自主筛选的木质素降解菌—肠杆菌YL1301为对象，围绕酶蛋白及其转录因子和降解产物三方面，多维度诠释肠杆菌降解木质素新途径的分子机制。首先，利用酶蛋白组学和质谱技术，阐明肠杆菌降解木质素“核心酶”和“附属酶”等特异性分泌酶蛋白的动力学变化规律和指纹谱；然后，通过转录组学和生物信息分析，揭示 “核心酶”和“附属酶”的功能转录因子；最后，耦合NMR和HPLC技术，探明该菌降解木质素代谢产物和电子传递链的变化，为木质素的高效利用奠定理论基础和技术支撑。.据此，我们采用生化和分子生物学鉴定了该木质素降解细菌，命名为Enterobacter sp. YL1301，初步研究了该菌发酵特性和降解产物组成，具有较好的降解木质素效果，降解率为58%。该菌降解木质素途径可能为 4-羟基-苯乙酸酯降解新途径，完全不同于文献报道细菌降解木质素的原儿茶酸降解途径和3-O-没食子酸甲酯降解途径。实验中我们还对纤维素、半纤维素和木质素的结构和应用进行了探索。.项目按照任务书内容和要求，有计划地开展研究工作，4年来，基本完成了任务书规定的工作内容，总体上达到了预期研究目标和考核指标。然而，由于前期肠杆菌培养出了点小问题，致使微生物蛋白组和转录组学方面的工作仍在进行中，预计2019年测序完成。截止目前，标注该项目已发表SCI论文10篇，中文核心1篇；授权专利1项。培养博士研究生1名和硕士研究生3名。.项目经费使用按照《国家自然科学基金财务管理办法》执行，支出合规合理，共支出841,814.41元，结余58,185.59元。结余经费将于2019年用于肠杆菌降解木质素蛋白质测序和转录组学分析。