基于氮源/多价离子介导的微生物絮凝剂合成及分子调控机制

Microbial flocculant is a kind of biodegradable macromolecular flocculant secreted by microorganisms. Compared with the traditional chemical flocculants, it has received more scientific and biotechnological attention in recent years with their high efficiency, harmlessness and free of secondary pollution and biodegradability. However, the concerning problems such as low flocculant-producing capacity, low producing rate and higher production cost has limited severely the applications of microbial flocculant These problems might be solved efficiently by means of using nitrogen sources/multi-valence ions to mediate the synthesis of microbial flocculants. Two high-efficient flocculant-producing strains A-9 and BS-5 have been obtained in previous study. They will be used for cloning and functional localization of flocculation polysaccharide synthesis genes after regeneration and flocculant-producing culture. In different culture stage，the chemical constituents and molecular structure of flocculating active metabolic intermediates will be analyzed, and the activity of key enzymes will be detected for exploring the biosynthetic pathway and constructing optimized metabolism network of microbial flocculants, after then, the methods and mechanism of metabolism remolding will be built. By mediated with nitrogen sources and multivalence ions, the molecular mechanism of microbial flocculant synthesis will be explored. The new, efficient, low production cost and environment-friendly synthesis technology of microbial flocculant will be obtained. The theoretical system about microbial flocculant can be enriched and developed, the sphere of metabolomics will be broaden and the technical support for the further industrialization of microbial flocculant is also be provided by above research results.

微生物絮凝剂不但具有传统化学絮凝剂的特性，而且还具有易生物降解、无二次污染、对有机胶体絮凝效率高等独特优点。但微生物絮凝剂产生菌的产絮能力低、絮凝剂产量少及生产成本高等问题严重地限制其实际应用。研究"基于氮源/多价离子介导的微生物絮凝剂合成及分子调控机制"，可有效解决上述问题。本项目以课题组已筛选、保存的A-9、BS-5两株高效产絮菌为研究对象，通过复壮和产絮培养，进行产絮菌絮凝多糖合成基因的克隆与功能定位；对不同培养阶段的絮凝活性代谢中间产物进行分析、检测关键酶的活性，探明微生物絮凝剂合成途径并构建优化的微生物絮凝剂合成代谢网络，研究其改造机制；通过氮源与多价离子的介导，探索微生物絮凝剂合成的分子调控机制，以期获得高效、低成本的环境友好型微生物絮凝剂制备新技术。研究成果既可丰富和发展微生物絮凝剂的理论体系，又可拓展代谢组学的研究领域，并为新型高效微生物絮凝剂的产业化放大生产提供技术支持。

为了揭示高效微生物絮凝剂（MBF）合成代谢过程与调控机制、探求提高MBF 产量的途径与方法，本研究通过定向富集筛选分离了高效MBF产生菌，应用系统生物学多相分类手段确定了高效絮凝菌A9的分类归属与新菌种地位；开展了A9利用葡萄糖代谢合成絮凝多糖参数优化及动力学研究，建立了A9和模式菌在发酵过程中的菌体生长、产物合成及底物消耗的动力学方程，各个方程拟合度均大于97%；利用等离子体诱变技术对A9进行诱变选育，与出发菌A9 相比，突变株发酵液的絮凝率和多糖产量分别有所提高；探究了氮源与金属离子对A9生长和MBFA9合成的影响，发现通过减少氮源或增加碳源的投加、添加适量Mg2+、Mn2+可实现对MBFA9产量的调控；利用茚三酮显色、Molish反应、FTIR、SEM及HPLC，确定MBFA9主要成分为非均一多糖，其分子量约为9.66×106Da；MBFA9 对水中Pb2+ 具有较高去除作用， MBFA9 吸附质量浓度为56.20 mg/L的Pb2+，25 min 后去除率最高可达92. 73%， 其捕集Pb2+ 的理论最大值为196.08 mg/g；结合红外光谱检测、场发射扫描电镜和能谱分析，确定了MBFA9 去除Pb2+的机理；此外，利用分子生物学手段系统研究了高效絮凝菌A9的基因组学、转录组学、蛋白组学及代谢组学特征，完成了A9胞外多糖合成蛋白的克隆和异源表达，通过软件分析，确定了菌株A9胞外多糖合成蛋白为跨膜蛋白。以A9全基因组测序结果与KEGG数据库比对得到的葡萄糖代谢路径为基础，基于Petri网理论构建A9的葡萄糖代谢网络。利用着色Petri网对应软件CPN Tools对葡萄糖代谢网络进行定性分析，混合函数Petri网对应软件Cell Illustrator对A9的葡萄糖代谢网络进行定量分析，阐释了A9在利用葡萄糖合成多糖代谢过程中起主要作用的4种中间产物；利用味精废水代替发酵培养基中的碳源和氮源，在最佳条件下发酵培养36h时，MBFA9 的多糖产量高达6.73g/L，絮凝率最高可达94.71%。上述研究对解析絮凝多糖MBFA9合成途径及调控机制、降低微生物絮凝剂生产成本，实现产业化推广与应用提供重要的理论依据。