米曲霉Sedolisin家族基因相关联的外源蛋白表达分泌机制的研究

It has been shown that the bottleneck in heterologous proteins production by Aspergillus oryzae is possibly caused by proteolytic degradation and posttranslational processes in the secretory pathway. In this study, we selected the sedolisin family which consist of four proteases (AoSedA, AoSedB, AoSedC, AoSedD) as the candidate potentially degrading the heterologous protein, and perform localization analysis of the fusion protein by each of sedolisins and EGFP to characterise the sedolisins, and then each sedolisins gene disruptant will be constructed to investigate the effect on the production level of heterologous proteins and protease activity. Afterwards, we focus on post-Golgi apparatus secretory pathway and select a vacuolar protein sorting receptor AoVps10 as a candidate potentially which probably influence the secretory pathway of heterologous protein. The localization analysis of heterologous proteins (chymosin) will be performed in wild type strain and Aovps10 gene disruptant, together with the localization analysis AoCpyA, a known cargo of AoVps10, we try to reveal the interaction of heterologous proteins and AoVps10, and the distinct sorting of heterologous proteins to the vacuoles with that of AoCpyA. Furthermore, the localization analysis of each sedolisins and EGFP in wild type strain and Aovps10 gene disruptant will be performed, together with the localization analysis AoCpyA, we try to reveal the interaction of sedolisins and AoVps10, and the distinct sorting of sedolisins to the vacuoles with that of AoCpyA. These results will demonstrate sedolisin family play two role （vacuolar protease and secretory protease）in proteolytic degradation, which will make them significantly effect on heterologous protein production. Finally, the disruption of the Aovps10 gene and four genes of sedolisin family will be constructed to enhance the heterologous protein production by A. oryzae.

米曲霉自身蛋白酶对外源蛋白的降解和外源蛋白在分泌路径中遇到的障碍极大影响了外源蛋白表达分泌效率。本课题基于申请人对米曲霉蛋白酶及蛋白分泌路径的前期研究，筛选出对米曲霉外源蛋白分泌有重要作用的Sedolisin家族四个蛋白酶基因作为研究对象；通过EGFP荧光融合蛋白胞内定位、和检测分别敲除该家族基因前后外源蛋白等一些列酶的活性变化, 解析该家族成员性质和功能。同时，着眼于高尔基体后运输路径，通过两种荧光蛋白共定位分析，解析该家族成员与液泡蛋白运输载体AoVps10的关系，以及其相互关系对外源蛋白分泌的影响，验证因为该家族中的三肽基肽酶同时具备液泡蛋白酶与分泌蛋白性质，所以对外源蛋白的分泌有重要影响的猜想。最终，连续敲除Aovps10和Sedolisin家族，通过改造外源蛋白高尔基体后分泌路径和减少胞内胞外蛋白酶对外源蛋白降解相结合的策略提高外源蛋白分泌效率，为米曲霉高效遗传育种开辟新的途径。

米曲霉自身蛋白酶对外源蛋白的降解和外源蛋白在分泌路径中遇到的障碍极大影响了外源蛋白表达分泌效率。本课题基于申请人对米曲霉蛋白酶及蛋白分泌路径的前期研究，筛选出对米曲霉外源蛋白分泌有重要作用的Sedolisin家族四个蛋白酶基因作为研究对象；通过EGFP荧光融合蛋白胞内定位、和检测分别敲除该家族基因前后外源蛋白等一些列酶的活性变化，解析该家族成员性质和功能。同时，着眼于高尔基体后运输路径，通过两种荧光蛋白共定位分析，解析该家族成员与液泡蛋白运输载体AoVps10的关系，以及其相互关系对外源蛋白分泌的影响，验证了该家族中的三肽基肽酶同时具备液泡蛋白酶与分泌蛋白性质。最终，连续敲除Aovps10和Sedolisin家族，通过减少分泌路径中外源蛋白由于过量生产而在液泡内的聚集和减少蛋白酶对外源蛋白的降解相结合的方法提高了米曲霉外源蛋白的胞外产量，使外源蛋白（小牛凝乳酶）的胞外分泌量提高了5倍，为米曲霉高效遗传育种开辟新的途径。