阻断糖基化修饰造成酵母分泌途径增强的机制解析及应用

Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production in industrial production. However, there are several restrictions in secretory pathway, which often result in low efficiency of protein secretion. Herein, we have found that secretory pathway was strengthened through selectively up-regulated expression of various secretory elements by N-glycosylation disruption. The strengthened secretory pathway significantly increased the production of recombinant proteins with different properties; whereas the engineered strains showed severely growth defects which limit their industrial applications. Thus, whether engineering mechanism of secretory pathway regulated by N-glycosylation disruption in the wild type could markedly improve protein secretion and also avoid cell growth defects. In this study, proteomics coupled with CRISPR technology will be used to construct high-throughput libraries for screening key regulatory proteins, and then the regulation mechanism of secretory pathway controlled by N-glycosylation disruption will be unfolded. Based on this, multiple regulatory proteins are modified and optimized simultaneously in the wild type for enhancing recombinant protein secretion. This study affords a new strategy and a new view for engineering secretory pathway and has valuable industrial applications.

酿酒酵母分泌重组蛋白被广泛的应用于工业生产中。但分泌途径存在多个限制节点，往往导致重组蛋白产量较低。前期实验结果发现阻断酿酒酵母糖基化修饰，能通过相关调控机制有选择性的协调多个分泌元件表达，从而增强分泌途径，实现不同特性靶蛋白高效分泌；但其严重影响细胞的正常生长，这限制了改造菌株在工业生产中的应用。假设在野生型菌株中改造使分泌途径增强的相关调控机制，能否避开阻断糖基化修饰造成的细胞生长问题，提高重组蛋白的胞外分泌量？本项目拟采用蛋白组学技术结合CRISPR技术建立高通量筛选体系，揭示阻断糖基化修饰造成分泌途径增强的调控机制，挖掘可以大幅度提高重组蛋白分泌的调控因子。在此基础上，在野生型菌株中组合改造、优化多个调控因子，构建高效分泌重组蛋白的健康酿酒酵母。本项目的完成将为促进重组蛋白分泌提供新方法和新视野，还可能产生较大的工业应用价值。

酿酒酵母分泌重组蛋白被广泛的应用于工业生产中。但分泌途径存在多个限制节点，往往导致重组蛋白产量较低。前期实验结果发现阻断酿酒酵母糖基化修饰，能通过相关调控机制有选择性的协调多个分泌元件表达，从而增强分泌途径，实现不同特性靶蛋白高效分泌；但其严重影响细胞的正常生长，这限制了改造菌株在工业生产中的应用。因此，本研究围绕阻断酿酒酵母糖基化修饰造成细胞壁损伤和分泌途径上调两方面展开。首先建立基因敲除和过表达库，从参与细胞壁合成和囊泡运输过程的基因中，筛选获得能够提高重组蛋白在酿酒酵母中表达活性的关键基因。再通过组合改造这些关键基因，进一步提高重组蛋白的在酿酒酵母中的表达活性。进而，验证该策略对不同重组蛋白的通用性。此外，分析了重组菌株的细胞生长、细胞壁完整性等生理指标，验证了重组菌株的健康情况。最终，通过蛋白组学结合反向代谢工程手段，揭示了重组菌株高效合成重组蛋白的机制。本项目完成为促进重组蛋白分泌提供新方法和新视野，还可能产生较大的工业应用价值。