酵母表面展示多酶分子机器对纤维素高效水解的研究

High costs of cellulase is one of the most important obstacles to the commercialization of cellulosic ethanol, due to the high enzyme production cost, low specific activity, and large enzyme loading. Therefore, enhancing hydrolysis efficiency and decreasing cellulase loading have been important contents in biorefinery reasearch. In view of this background, in present project, we plan to assemble multi-enzyme machine of cellobiose phosphorylase (CBP) and alpha-glucan phosphorylase (PGP) on the cell surface of Saccharomyces cerevisiae, replacing beta-glucosidase (BG) in cellulase and forming cellulose-multi-enzyme-yeast cell complex for in vitro biotransformation of a fraction of cellulose to artificial starch. At the same time, yeast can convert the remaining glucose to bioethanol, achieving the coproduction of bioethanol and artificial starch. To improve the apparent activity and hydrolysis efficiency of cellulase, the structure and ratio of CBP and PGP will investigated, as a result of the cellulase loading reduction. This project will provide new directions for biorefineries: the production of BG-free or low BG content cellulase, immobilization of enzyme machines on the surface of yeast to reduce the cellulase loading. Artificial starch, as a new product joint produced in biorefinery way by utilization nonfood biomass will be a strategic technology reserve for addressing national food security in the future.

纤维素酶生产成本高、比酶活低以及用量大是阻碍纤维素乙醇工业化生产的重要障碍之一，所以提高纤维素酶水解效率、降低纤维素酶用量已成为生物炼制行业的重要研究内容。鉴于此，本项目将在酵母表面组装纤维二糖磷酸化酶（CBP）、淀粉磷酸化酶（PGP）多酶分子机器，取代纤维素酶中的β-葡聚糖苷酶（BG），形成纤维素-多酶分子机器-酵母细胞复合体，将纤维素中的一部分转化为人造淀粉，同时酿酒酵母利用剩余葡萄糖生产生物乙醇，实现乙醇及人造淀粉联产。通过考察不同CBP、PGP结构与当量比对底物穿梭效应的影响，实现提高纤维酶的表观比酶活及水解效率，减少纤维酶用量。本项目将为生物炼制中纤维素乙醇的工业化提供新方向：生产无或低BG的工业纤维素酶，利用酿酒酵母为载体固定化多酶分子机器减少纤维素酶用量。非食用的生物质通过生物炼制技术联产新产品—人造淀粉，这将为中国粮食安全提供新的战略技术储备。

通过支架蛋白将多酶分子机器固定于Saccharomyces cerevisiae的细胞表面，形成纤维素-酶-酵母细胞复合体，实现底物穿梭效应，提高了纤维素酶的水解效率。本项目利用合成生物学手段，组装多酶分子机器、获得低beta-葡萄糖苷酶的纤维素酶，实现纤维素转化生产人造淀粉联产生物乙醇。同时阐明了有机溶剂对人造淀粉聚合度的促进作用。通过X-射线、体积排阻色谱等方法，对人造淀粉的性质进行了分析。本研究为下一步性质可控的人造淀粉的生产，建立高效定制工业酶催化体系提供指导。