CBP策略合成木糖醇的米曲霉工程菌构建、途径递进强化和代谢调控机制研究

项目名称： CBP策略合成木糖醇的米曲霉工程菌构建、途径递进强化和代谢调控机制研究

项目编号： No.21476093

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 数理科学和化学

项目作者： 陈宏文

作者单位： 华侨大学

项目金额： 80万元

中文摘要： 以构建米曲霉工程菌实现半纤维素直接转化为木糖醇的一体化生物加工（Consolidated Bioprocessing，CBP）为目标，无痕敲除米曲霉染色体上ku基因，提高米曲霉基因打靶效率；无痕敲除木糖醇脱氢酶基因，阻断木糖醇分解途径；通过辅酶工程手段，增强PPP途径代谢流，维持基因缺失后辅酶氧化还原平衡；过量表达木聚糖降解酶转录因子AoXlnR基因，激活木聚糖酶转录表达。通过多轮递进式途径改造，探明米曲霉木糖代谢调控机理、辅因子对碳物质代谢和能量代谢的作用机制、胞外酶和微生物协同作用机制，从木糖醇发酵和木聚糖降解两方面强化米曲霉性能，实现木聚糖酶生产、半纤维素胞外酶解和木糖胞内发酵，只用一个菌株、一步发酵完成的木糖醇CBP加工模式。本研究结果开辟了一条生物法高效生产木糖醇的新途径，可以有效提高生态和资源效益，并将丰富CBP途径工程基本理论，为促进化学品生物制造替代的工程应用奠定科学基础。

中文关键词： 米曲霉；一体化生物加工；木糖醇；途径工程

英文摘要： The aim of this research is direct xylitol conversion from hemicelluloses by consolidated bioprocessing (CBP) using constructed engineering Aspergillus oryzae. Firstly, a highly efficient gene targeting system in A. oryzae is developed based on Ku gene marker-free deletion. Then, enhanced xylitol production mutants are constructed by xylitol dehydrogenase gene marker-free deletion. Furthermore, the metabolic flux of PPP pathway is increased by cofactor engineering, keeping coenzyme redox equilibrium after gene deletion. Finally, xylanolytic activities in the culture supernatant are elevated by overexpression of the transcriptional activator, XlnR, which controls the expression of genes encoding xylanolytic enzymes in A. oryzae. After multi-rounds of chromosome modification, we hope to understand regulation mechanism of xylose metabolism, interplay between the operation of cofactors and carbon metabolism or energy metabolism, and cooperation of enzyme and microbe. This is helpful for realizing CBP mode by integrations of hemicellulase production, hemicellulase hydrolysis, and product fermentation into one step and one strain. This study provides a new idea for high efficient bioproduction of xylitol, can improve the ecological and resource efficiency, enrich the basic theory of CBP pathway engineering and lay a scientific foundation for large-scale biomass-based chemicals production.

英文关键词： Aspergillus oryzae;consolidated bioprocessing (CBP);xylitol;metabolic engineering