菌群演替与温水沤麻系统关键酶产生菌代谢组学特征的耦合机制

Microbial-ecosystem is an ideal model to understand the symbiosis of species and the reciprocity with their environment. Metabolites are the final response of cells to the changes of the environment. Metabolomics combining with other system biological 'omics' technologies are helpful for us to get further information about the process and function between microbial-community and their ecosystem. The growth of degumming bacteria and the metabolism of key enzymes jonitly determine the production cycle and quantity of flax fibres. The basic research has been finished, including separation of degumming bacteria, optimization of enzyme-producing condition and investigation of microbial-community structure. This present work takes the flax-retting microbial-ecosystem as a research model and aims at the feature of metabolome and proteome of the microbial-community, as well as the variation of environmental substances. Series of 'omics' and molecular technologies are planned to apply on this research, such as metabolomic fingerprinting, metabolomic profiling, two-dimensional electrophoresis, real-time quantitative PCR and denaturing gel gradient electrophoresis. According to the contrastive analysis on the variation of microbial-community structure and substance transition between natural flax-retting ecosystem and bacterial flax-retting ecosystem, general rule of the community succession, metabolism of key enzymes and substance transition is about to be discovered. The final goal is to establish the mechanisms on (1) enzyme-producing metabolomics of degumming bacterial community; (2) symbiosis among different key enzyme-producing communities. This project is not only a challenging try to bring the conception of eco-metabolomics to flax-retting ecosystem research but also applicablly meaningful to improve the production process of flax fibre in near future.

微生态系统是研究物种共生及其与环境相互作用机制的理想模型。代谢物是细胞对环境变化的终端响应，代谢组学技术与其他系统生物学组学技术结合应用，有助于深入了解微生物菌群及所处生态系统的过程和功能。脱胶菌群的生长及关键酶代谢决定亚麻纤维生产周期及质量。本研究在脱胶菌株分离、产酶优化及沤麻系统菌群结构研究等前期工作基础上，以温水沤麻微生态系统为模型，采用代谢指纹分析、代谢轮廓分析等代谢组学技术，结合2D电泳等蛋白质组学技术和定量实时PCR及变性梯度凝胶电泳技术，研究菌群代谢物组、蛋白质组特征及环境基质动态变化，通过天然及加菌温水沤麻的对比试验，分析菌群结构、数量和物质转换的差异，探讨菌群演替、关键酶代谢及其与基质变化的一般规律，建立温水沤麻系统中脱胶菌群产酶代谢组学机制和不同关键酶产生菌群的共生机制。本研究是生态代谢组学概念及技术引入温水沤麻系统的有益尝试，对沤麻工艺改进具有实践指导意义。

温水沤麻体系是一个复杂的生态系统，其脱胶实质是利用亚麻原茎上微生物产生的脱胶酶，降解果胶及胶接韧皮纤维或纤维束的其它非纤维素类物质，将亚麻纤维从非纤维类物质中分离出来。 通过对比分析天然温水沤麻和加菌温水沤麻系统中的菌群结构和物质转换的差异，探讨微生物菌群演替、脱胶关键酶及其代谢产物变化的一般规律。.构建加菌B. cereus HDYM-02温水沤麻体系（BA），以天然温水沤麻体系（CK）为对照，利用高通量测序测序研究微生物物种丰度及多样性。在发酵前期，B.cereus HDYM-02能够显著改善微生物群落结构，尤其是两体系中芽孢杆菌科（Bacillaceae）与假单胞菌科（Pseudomonadaceae）的物种丰度的差异；在发酵中后期，BA体系中脱胶优势菌科为梭菌科（Clostridiaceae_1）和假单胞菌科（Pseudomonadaceae）；CK体系中脱胶优势菌科为梭菌科（Clostridiaceae_1）、假单胞菌科（Pseudomonadaceae）和肠杆菌科（Enterobacteriaceae）。B.cereus HDYM-02能够促进脱胶优势菌群的生长，降低非脱胶菌群的物种丰度，降低了沤麻体系生态系统中的物种多样性。.在菌群结构方面，供试菌株B.cereus HDYM-02提高了温水沤麻体系中芽孢杆菌科（Bacillaceae）、假单胞菌科（Pseudomonadaceae）、梭菌科（Clostridiaceae_1）的物种丰度，分别为天然温水沤麻体系的8.9、2.2及1.1倍，且降低了肠杆菌科（Enterobacteriaceae）的物种丰度，为天然温水沤麻体系的0.2倍。在代谢物方面，表现为脂蜡质及木质素（即烷烃及其衍生物、酯、有机酸及芳香族化合物，为亚麻纤维表面的胶质成分）丰度的差异。主要胶质成分代谢物丰度在72-96 h时达到最大值，表明亚麻纤维脱胶进程的结束。.供试菌株B.cereus HDYM-02的加入能够促进脱胶优势菌群的生长，降低非脱胶菌群的物种丰度，改善沤麻体系生态系统中的物种多样性，引起沤麻体系中代谢产物的差异。且这些差异的代谢产物可以作为沤麻体系中优势菌群物种丰度及多样性变化和亚麻纤维脱胶进程的基础生物指示剂。