酿酒酵母高效分泌元件与异源蛋白兼容性的规模化研究

Saccharmyces cerevisiae, a microorganism generally recognized as safe (GRAS), has a wide range of application. It is an ideal engineered candidate for microbial manufacturing to produce the functional eukaryote proteins, because of the excellent performance of industrial production and complete secretory pathway for performing the post-translational modification. Although engineering of secretory pathway could improve heterologous protein secretion in Saccharomyces cerevisiae, the compatibility is different between secretory components and various proteins. According to the modular and standardizing design specifications of synthetic biology, efforts are underway to characterize and standardize the key components of five parts of secretory pathway in Saccharomyces cerevisiae, containing translocation, protein folding, glycosylation, vesicle trafficking, mis-sorting and proteolytic degradation, by the effect of various eukaryote proteins extracellular activity (large and small weight protein, glycoprotein and protein containing high disulfide bonds). Based on this, optimal components were combined to form efficient modules for specified protein production. Furthermore, the reason of the improved activities of target proteins and the impact of secretory pathway by engineering components was investigated to analyze the compatibility. Our results will further improve the functional annotation of various components and contribute to the development of synthetic biology in yeast.

酿酒酵母是广泛应用的食品级安全性微生物，其良好的产业化生产性能及完善的蛋白质分泌、翻译后加工修饰系统，已成为真核生物源功能性蛋白微生物制造的研究热点。研究表明改造酿酒酵母的分泌途径，可以提高异源蛋白的胞外活性，但各分泌元件与特定靶蛋白间的兼容性不同。本项目依据合成生物学中元件模块化、标准化理念，选定不同特性(大/小分子量蛋白、糖蛋白及高二硫键蛋白)的真核生物源靶蛋白，针对酿酒酵母分泌途径的5个环节，即新生肽链易位、内质网中折叠、糖基化修饰、膜泡运输及错误蛋白分拣降解，规模化研究各环节不同分泌元件对这些靶蛋白胞外活性的影响，进而对作用大的元件进行组合优化分析，以建立不同特性蛋白高效分泌元件的标准化模块。在此基础上，研究各元件提高靶蛋白胞外活性原因、引起的分泌途径变化，及两者的关联性等兼容性机制。项目结果将进一步完善这些分泌元件的功能注释，对推进以酿酒酵母为底盘细胞合成生物学的研究有积极意义。

中文摘要：酿酒酵母是广泛应用的食品级安全性微生物，其良好的产业化生产性能及完善的蛋白质分泌、翻译后加工修饰系统，已成为真核生物源功能性蛋白微生物制造的研究热点。改造酿酒酵母的分泌途径，可以提高异源蛋白的胞外活性，但各分泌元件与特定靶蛋白间的兼容性不同。本项目选用四种不同的异源蛋白包括β-葡萄糖苷酶、内切葡聚糖酶、外切葡聚糖酶以及α-淀粉酶作为靶蛋白，针对酿酒酵母分泌途径的各个环节，即新生肽链易位、内质网中折叠、糖基化修饰、膜泡运输，深入的研究了各环节不同的分泌元件对这些靶蛋白胞外活性的影响，并解析了不同特性的蛋白在分泌途径不同的限制节点。首次发现新生肽链易位对蛋白分泌具有促进作用，解析了糖基化修饰的改造提高蛋白分泌的具体机理，发现并不是由于影响了蛋白活性，而是由于造成了细胞壁完整性的改变和分泌途径多个元件的上调而导致蛋白分泌量的增加。项目结果对推进以酿酒酵母为底盘细胞生产各种异源蛋白具有积极的意义。