酿酒酵母结合纤维小体与寡糖代谢单步转化纤维素乙醇的研究

Direct conversion of cellulose to bioethanol remains a challenge if using the traditional two-step approaches by cellulosome (single module)-engineered Saccharomyces cerevisiae. In this work, a single-step conversion via two-module-engineered yeast is proposed based on our successful experience on self-assembly of a two-scaffoldin-derived minicellulosome. This research includes the following key points: i) Detach the extracellular single-module cellulosome into a compact two-scaffoldin-derived cellulosome (extracellular) and a cellodextrin metabolic pathway (intracellular). This module modification will reduce the metabolic load of the host and increase the surface display level of the cellulosome, thus the ability of the engineered S. cerevisiae to directly saccharify the cellulose will be enhanced. ii) Integrate the celullosome assembly and the ethanol fermentation into a single step via applying cellulose and galactose as the co-carbon sources. It will simplify the whole process of the cellulosic ethanol production and get the efficient use of the galactose as both the expression inducer and the fermentable sugar, thus the total bioethanol productivity is able to be increased. This research will point out the principles of designing and constructing of the cellulosome module and the cellodextrin-pathway module in S. cerevisiae, and illustrate the interaction mechanism between these two modules, and also conclude their co-working strategy. In addition, we will study the fundamentals and the regulation methods to simultaneously realize the cellulosome assembly, the cellodextrin pathway constructing and the ethanol co-sugar fermentation within one single step. This work will increase the cellulosic ethanol titer of the genetically engineered S. cerevisiae , and promote the application of Consolidated Bioprocessing (CBP) in non-food-based bioethanol fuel production.

针对纤维小体单模块两步法的酿酒酵母纤维素乙醇转化技术的缺陷，并结合申请人在双支架微型纤维小体自组装方面的成功经验，本项目提出酿酒酵母双模块单步法的创新性研究方案。核心内容包括：（1）将胞外单模块纤维小体拆分为双支架微型纤维小体（胞外）和低聚寡糖代谢途径（胞内）两个关联模块，以此精简纤维小体结构，降低宿主代谢负担，提高纤维小体的跨膜展示效率，进而强化酿酒酵母的单细胞纤维素糖化能力；（2）采用双碳源（纤维素和半乳糖）单步法模式，整合模块组装与乙醇发酵过程，简化传统工艺流程，解决半乳糖（诱导剂）利用效率低的问题，从而实现乙醇产量的最大化。本项目拟探讨纤维小体与寡糖途径的设计、构建原理以及模块间的互作机制和适配策略，阐明纤维小体组装、寡糖代谢途径构建、双碳源乙醇发酵的同步实现机理和调控方法。该项目有望提高重组酿酒酵母对纤维素乙醇的直接转化效率，研究成果有助于CBP技术在非粮型燃料乙醇中的应用。

针对纤维小体单模块两步法的酿酒酵母纤维素乙醇转化技术的缺陷，并结合申请人在双支架微型纤维小体自组装方面的成功经验，本项目提出酿酒酵母双模块单步法的创新性研究方案。核心内容包括：（1）将胞外单模块纤维小体拆分为双支架微型纤维小体（胞外）和低聚寡糖代谢途径（胞内）两个关联模块，以此精简纤维小体结构，降低宿主代谢负担，提高纤维小体的跨膜展示效率，进而强化酿酒酵母的单细胞纤维素糖化能力；（2）采用双碳源（纤维素和半乳糖）单步法模式，整合模块组装与乙醇发酵过程，简化传统工艺流程，解决半乳糖（诱导剂）利用效率低的问题，从而实现乙醇产量的最大化。本项目探讨了纤维小体与寡糖途径的设计、构建原理以及模块间的互作机制和适配策略，阐明了纤维小体组装、寡糖代谢途径构建、双碳源乙醇发酵的同步实现机理和调控方法。该项目有望提高重组酿酒酵母对纤维素乙醇的直接转化效率，研究成果有助于CBP技术在非粮型燃料乙醇中的应用。