基于过氧化物酶体吞噬机制提高毕赤酵母表达外源蛋白的研究

Pichia pastoris is a widely used microbial producer of heterogenous protein. However, the delimma of Pichia pastoris high cell density fermentation is low methanol metabolic rate, high cell death ratio at the presence of methanol, and pexophagy and repress of alcohol oxidase promoter in the presence of glucose with the result of low heterogenous protein productivity. This proposal analyzes Pichia pastoris metabolism at different pexophagy type and growth condition to obtain the relationship between cell energy state, the phosphorylation of regulate protein(Atg30) during pexophagy and the heterogenous protein production. With this results, Pichia pastoris growth and induction condition can be controlled at the maximum energy state with high alcohol oxidase promoter activity, and higher heterogenous production. This proposal will clarify the intracellular mechanism of Pichia pastoris alcohol oxidase promoter acitvition and repression based on the protein cascaded regulation. Furthermore, atg30 gene will be knocked out to block pexophagy at the high energy charge. Finally, a continuous culture system will be established to express hydrophbin (RodA) from Aspergillus niger by Pichia pastoris efficiently at the condition of high energy charge without pexophagy to confirm our hypothesis. This work will make a way to higher heterogenous protein at high cell energy charge, which will have significant influence on microbial process control and heterogenous protein production.

毕赤酵母是成功表达多种外源蛋白质的细胞工厂。本项目为了阐明毕赤酵母培养过程中细胞生理状态、过氧化物酶体吞噬和外源蛋白表达的关系，解决毕赤酵母高密度发酵过程中甲醇代谢慢、细胞死亡率高，添加葡萄糖导致过氧化物酶体吞噬，损失蛋白质表达量的关键问题。通过分析毕赤酵母启动过氧化物酶体吞噬的临界生理状态，以过氧化物酶体吞噬过程的调控蛋白磷酸化水平为指标，阐明细胞能荷、调控蛋白和过氧化物酶体吞噬（微吞噬和宏吞噬）之间级联反应的量化关系。探索细胞能荷和过氧化物酶体吞噬的规律，力图达到保证醇氧化酶启动子活力，且不发生过氧化物酶体吞噬的最大碳源代谢量，提高外源蛋白在毕赤酵母中的表达。最后以表达黑曲霉疏水蛋白（RodA）的重组毕赤酵母为示范，建立连续、高效的高密度培养系统。本研究可以阐明过氧化物酶体吞噬的启动和控制因素，对解决毕赤酵母高密度发酵过程中的关键问题有重要意义。

毕赤酵母表达是外源蛋白的一个重要工业微生物。本研究探究毕赤酵母启动过氧化物酶体2种类型吞噬的临界非甲醇碳源浓度为0.05%-0.1%葡萄糖、2.0%-3.0%乙醇，能荷临界值为0.75。还通过敲除过氧化物酶体吞噬的启动蛋白基因atg30，考察敲除菌株的生理生化指标。进而以黑曲霉木聚糖酶基因和芽孢杆菌环糊精糖基转移酶基因为外源基因，通过控制过氧化酶体吞噬的条件提高木聚糖酶和环糊精糖基转移酶的表达量，建立了高效的表达体系。