Dickeya dadantii DCE-01菌株苎麻脱胶的酶催化机制研究

The ramie industry is one of characteristic industry in China. Bio-degumming is the development direction of ramie degumming. We have selected a highly effective degumming strains DCE-01, which can finish ramie degumming within 6 to 8 h. Its whole genome sequences have been obtained, and the bio-degumming technology has been popularized and used in 13 companies. However, the strain is not stable, and the degumming is not completely. The enzymatic catalytic mechanism is not clear, which hinders the construction of high-stable and high-efficiency degumming strain, and the further development of bio-degumming technology. In this project, the key degumming protein components will be revealed by proteomics research; the key degumming genes will be revealed by whole genome and transcriptome method; by cloning, identifying and knockouting the key enzyme genes of ramie degumming, the gene regulation and protein expression mechanism will be revealed; and the mode of action between key enzyme and non-cellulose will be revealed by study on the site of the substrate, function order and catalytic function. The project has important theoretical value and practical significance to promote the sustainable development of ramie industry.

苎麻产业是我国的特色产业，生物脱胶是苎麻脱胶的发展方向。项目组筛选到一株高效脱胶菌株Dickeya dadantii DCE-01，该菌株能在6-8h内完成苎麻的非纤维素降解，已完成该菌株的全基因组测序草图，并已在13家企业获得推广应用，但该菌仍存在稳定性不足以及脱胶残胶高等问题。因苎麻生物脱胶的酶催化机制不清楚，阻碍了高稳定、高效能脱胶菌株的构建，阻碍了生物脱胶技术的进一步发展。本项目拟通过蛋白组学研究，揭示关键脱胶酶组分与比例；通过全基因组重测序及转录组学研究，揭示关键脱胶酶编码基因；进一步克隆、敲除、鉴定苎麻脱胶关键酶基因，揭示基因表达及调控机制；继而追踪整个脱胶过程中的关键酶对底物的切入位点、作用顺序及催化功能，揭示关键酶催化苎麻胶质的协同作用模式。项目的实施对促进苎麻产业的可持续发展具有重要理论意义和实用价值。

苎麻生物脱胶的酶催化机制不清楚，阻碍了高稳定、高效能脱胶菌株的构建，阻碍了生物脱胶技术的进一步发展。项目以本课题组选育到的苎麻生物脱胶高效菌株D. dadantii DCE-01为研究对象，利用蛋白质组学分析技术，开展了苎麻生物脱胶过程中的关键酶种类及表达的动态变化研究；利用基因组重测序技术，分析了苎麻生物脱胶过程中的关键酶基因类别；通过克隆、敲除、表达等手段，明确了关键酶基因的功能及协同表达调控机制；通过脱胶酶基因分子改造，构建了基因工程菌株。. 通过实施本项目，形成了菌株DCE-01全基因组完成图，注释到的编码基因为4361个，非编码RNA条目为246个，筛选到21个关键脱胶酶基因；鉴定到胞外蛋白总数为1474个，肽段数量8038条，获得二级图谱总数242275张；通过差异蛋白表达分析发现与苎麻脱胶相关的关键脱胶酶有14个；克隆到四个特质果胶酶基因pel207、pel225、pelG32、pel027，构建了四个重组菌株；采用理性设计对果胶裂解酶进行分子改造，从PelG403突变酶和Pel419突变酶库中各筛选到1个正向突变酶（PelG403-A129V和Pel419-V52A）；对果胶裂解酶PelG403和Pel419进行同源建模获得三维结构；采用插入法构建了一株纤维素酶缺陷基因eglS失活菌株；首次表明CitT可能正调控苎麻纤维的生物脱胶过程，是果胶降解的重要调控因子；探究了双组分系统CitST中应答调节蛋白CitT对苎麻脱胶效果的影响。. 本项目进一步揭示了苎麻生物脱胶的酶催化机制，有利于高稳定、高效能脱胶菌株的构建，项目的实施对促进苎麻产业的可持续发展具有重要理论意义和实用价值。