多尺度解析全细胞催化木糖制取木糖酸的关键性抑制物及响应机制

Xylonic acid is confirmed to be a new way for xylose bioconversion and utilization in high efficiency. The strain of Gluconobacter oxydans performs the most promising future in industrial production as it produces 400 g/L of xylonic acid from single xylose using whole-cell catalysis. However, the cell capacity and productivity will decrease markedly or loss completely because of inhibitory effects of toxic degradation products derived from lignocellulosic materials processing. There is no applicable method or technology afforded yet for effective removal of these inhibitors as the result of little basic research efforts on the reaction pathway and mechanism before. In this proposal, the outstanding strain of G. oxydans NL71 will be studied for xylonic acid production. The responsed genes will be founded by high throughout RNA-Seq and comparison analysis of transcript profiles between the control and typical inhibitory samples, and their encoding functional proteins and enzymes will be chosen out, and then the metabolic pathway is drafted by the correlation analysis. On this basis, we can identify the inhibited targets involving of the key genes, proteins, enzymes and metabolism joints by multi-scale analysis using combinations method of cell, metabolism and molecular biology. By detecting and analyzing of these targets' responses, the key inhibitors will be disclosed and identified which are derivated from lignocellulosic materials during processing. Focusing on these key inhibitors, we will investigate their effects on these targets, including the key genes, proteins, enzymes and metabolism joints.Ultimately,we will discovery the cell response and regulation mechansim in the sight of the multi-scale analysis. It will be helpful to the study on the controlling strategy and removal method of these key inhibitory factors in the production of xylonic acid from lignocellulosic xylose using whole-cell catalysis.

木糖酸是破解木糖高效生物转化的重要出路。氧化葡萄糖酸杆菌（G.oxydans）全细胞催化纯木糖制取木糖酸的产物浓度超过400g/L，极具工业应用前景。但在木质纤维水解液中受抑制物效应导致细胞催化活性及生产性能显著下降。由于缺乏对抑制物及机制的基础研究，目前尚未能建立经济有效的脱毒生产技术与工艺。本项目基于前期工作以G.oxydans NL71为研究材料，针对代表性抑制物，从细胞分子转录水平入手，利用高通量转录组测序技术RNA-Seq开展抑制条件下的转录谱比较研究，筛选出主要响应基因，再注释得到对应的功能蛋白和酶，并映射至代谢途径；然后研究它们对抑制物的响应规律，进而从细胞、代谢和分子多尺度解析得到关键的响应基因、功能蛋白、酶及调控节点。以此为检测目标,筛选并发掘出木质纤维原料炼制过程中的关键性抑制物，再解析细胞的响应机制和调控规律，最终为关键性抑制物的控制策略及消减措施的研究提供理论指导。

围绕葡萄糖酸杆菌（G.oxydans）全细胞催化木质纤维水解液木糖合成木糖酸的抑制效应及细胞响应与调控机制，基于细胞代谢动力学、基因组学、转录组学的代谢网络的多尺度解析，阐明了细胞响应的分子及生化机制，发掘出主要抑制物、关键性响应基因及代谢调控节点。研究提出了富氧环境全细胞耐抑制物催化反应理论理论，首次建立COS-SSTR全细胞催化技术，将木糖酸产品浓度提高至586.3g/L，体积产率达4.69g/L.h，同时成功实现木质纤维稀酸水解液不经脱毒原位直接全细胞催化合成木糖酸的生产目标，产品木糖酸浓度达143.9/L，体积产率达0.97g/L.h，为木糖生物转化领域最高技术水平。木糖酸（盐）产品应用于水泥减水剂的使用性能良好，为木质纤维原料生物炼制技术的研发提供了十分重要的理论指导。COS-SSTR技术被拓展应用于糠酸、二羟丙酮、乙醇酸等具有生物毒性的系列化多元醇（酮）酸产品生物合成，产品浓度及体积产率均为当前报道的最高水平；利用细胞控机制的理论指导，研究并发现了二价锌离子、过氧化氢对G.oxydans催化氧化葡萄糖合成不同葡萄糖（酮）酸的调控规律，实现了葡萄糖酸、2-酮基葡萄糖酸高效定向调控的合成效果。本研究成果为G.oxydans菌株应用于包括木糖酸在内的多元醇（酮）酸生物基化学品的生物制造技术研发及商业化拓展提供了重要的理论指导和技术支撑。