基于蛋白质-代谢物调控网络的梅林青霉降解木质素的机制研究

The biodegradation of lignin has been a worldwide research focus and difficult problem. Clarifying the biodegradation mechanism of lignin is not only the primary key scientific issue which needs to be elucidated, but also the urgent theoretical basis for solving the practical application problems. This study intends to integrate and compare differential protein and differential metabolite expressed at different stages through analyzing differential proteome expression and its sequential metabolic profile of Penicillium melinii under different culture conditions using the technology of proteomics and metabonomics. The aim is to expound corresponding complicated and consecutive cooperative change of metabolic regulatory pathway during the degradation of lignin, thereby determining the key enzymes corresponding to the key resilient nodal points of the entire metabolic network and its regulatory mechanism. On that basis, the key enzyme is isolated and purified, and the study also will be carried out on its physical and chemical properties, molecular structure, biological function and the effect and mechanism displayed in the process of degradation or disaggregation of natural lignocellulose substrate. Meanwhile, through validating and analyzing key protein and metabolites selected using metabolic flux analysis, the proper adjusting and controlling method of metabolism of microbial fermentation process will be designed for improving fermentation process control. Further achieving the purpose of clarifying the regulation mechanism of lignin's enzyme production and degradation with improving the enzyme production and degradation ability of strain. This will lay the foundation for future applying the efficient strain to biopulping, biobleaching and bioconversion of lignocellulosic wastes etc.

木质素的生物降解一直是世界性的研究热点和难题，全面弄清木质素的生物降解机理是需首要阐明的关键科学问题，也是解决实际应用问题迫切需要的理论基础。本研究拟采用蛋白质组学和代谢组学技术，通过对梅林青霉在不同培养条件下蛋白质组表达差异及其相继的代谢轮廓分析，将不同阶段表达的差异蛋白质与差异代谢物进行整合比对，阐述菌株木质素降解过程中相应的代谢调控通路复杂、连续的协同变化，确定整个代谢网络的关键弹性节点对应的关键酶及其调控机制。进而分离纯化关键酶组分并对其理化性质、分子结构、生物学功能及在天然木质纤维素底物降解或者解聚过程中发挥的作用和机理进行研究。并通过代谢通量分析对甄选的关键蛋白和代谢物进行验证和解析，设计适当的微生物发酵过程代谢调控方法，改善发酵过程控制。达到弄清其产酶和降解的调控机理，进而提高菌株的产酶和降解能力的目的，为高效菌株应用于生物制浆、生物漂白和木质纤维废料的生物转化等奠定基础。

全面弄清木质素的生物降解机理是生物制浆、生物漂白和木质纤维废料的生物转化等实践应用首要解决的关键科学问题。. 本研究利用iTRAQ技术对Aspergillus Fumigatus G-13分别以木质素模型化合物和天然木质纤维素为底物，对培养过程中表达的全部蛋白质进行蛋白质组学分析。结果表明，以模型化合物为底物时，共有3862个蛋白质点发生明显变化，与木质素降解相关的差异蛋白主要富集于过氧化酶体相关蛋白质、纤维二糖/醌还原酶系及芳香环开裂酶系和阿魏酸酯酶相关蛋白质，菌株利用这些酶作用于羧酸酯键、连结木质素结构单元之间的醚键和酯键和电子转移链中的末端，差异蛋白质主要富集于碳源和能量、甘油酯代谢、核苷酸代谢、单胺菌素代谢相关途径。降解天然木质素底物差异蛋白质主要富集于碳源和能量、核苷酸代谢相关途径。综合来看，在A. Fumigatus G-13降解木质素过程中，阿魏酸酯酶作为诱导酶对木质素的降解起关键作用，差异蛋白质主要富集于碳源和能量、核苷酸代谢相关途径。. 利用基于LC/MS的非靶向代谢组学技术对菌株在降解木质素模型化合物各个阶段的中间代谢产物进行检测。结果表明，正负离子模式分别获得了8832和4223个代谢物，其中4173和1596个被注释。代谢物主要为氨基酸、脂质、碳水化合物、有机酸及其衍生物、苯环型化合物及有机杂环化合物。A. fumigatus G-13存在若干与木质素的降解相关的途径，其中淀粉和蔗糖代谢、磷酸戊糖途径、谷胱甘肽代谢及二羟基化芳环的邻位裂解途径与木质素降解紧密联系。通过Label-free蛋白质组学分析，降解木质素模型化合物的关键蛋白为儿茶酚双加氧酶、谷胱甘肽还原酶、葡聚糖酶、异戊醇氧化酶、甘油醛-3-磷酸脱氢酶、超氧化物歧化酶。经蛋白质组学和代谢组学整合比对分析，发现菌株降解木质素主要通过断裂木质素结构中Cα-Cβ化学键、氧化侧链和催化苯环裂解，将木质素解聚生成二芳基丙烷和芳基醚等低聚体，然后双加氧酶作用于苯环促使其裂解，再进入到β-酮己二酸途径。. 项目研究为揭示微生物木质素降解机理及产酶调控机理，提高微生物产酶能力奠定了理论基础。