草酸青霉液泡蛋白分选受体在外源蛋白分泌过程中的作用机制

Filamentous fungi have served as an interesting alternative to other fungal secretory expression systems that are used to produce heterologous proteins and lignocellulolytic enzymes because of their high secretion capability and eukaryotic posttranslational modi?cations. However, a bottleneck in posttranscriptional processing in the secretory pathway limits protein yields. The vacuolar protein sorting receptors are responsible for the recognition and delivery of vacuolar hydrolases to the vacuole. Although several of them can also target and deliver recombinant and aberrant proteins to vacuolar for degradation, there is limited knowledge of the effect of VPS receptor disruption on heterologous protein production in filamentous fungi. In this study, cDNAs encoding vacuolar protein sorting (VPS) receptors from the ?lamentous fungus Penicillium oxalicum HP7-1 will be cloned and functional analysis. To determine the localization of VPS receptors in P. oxalicum, the transformant expressing VPS fused with green ?uorescent protein (GFP) will be constructed and observed by confocal laser microscopy. Moreover, the effect of the vps genes disruption in P. oxalicum on heterologous protein production and intracellular protein trafficking to vacuoles by VPS receptors will be investigated. This proposed project is of great theoretical significance in understanding the molecular mechanism which mediates protein traf?cking between the Golgi apparatus and vacuoles. Meanwhile, it will provide solid basis for directed genetic manipulation of vacuolar protein sorting genes in ?lamentous fungi leads to enhanced production levels of heterologous proteins.

丝状真菌具有较强的蛋白分泌能力及对蛋白的翻译后修饰作用，已作为基因工程宿主菌应用于外源真核蛋白的生产，但其生产外源蛋白的表达水平远低于内源蛋白，无法满足工业发酵规模的需求。而蛋白在丝状真菌分泌途径中的转录后加工（如转运和分泌），正是其瓶颈所在。本项目拟以自主分离筛选的草酸青霉菌株HP7-1为研究材料，根据其转录组测序结果，克隆鉴定其分泌途径中的液泡蛋白分选受体（VPS），并研究这些VPS在细胞中的定位和功能。通过分析比较vps基因缺失突变菌和对照菌的内源蛋白和外源蛋白在细胞中的定位、表达差异，研究VPS对青霉菌株蛋白分泌表达的影响，以明确液泡蛋白分选受体VPS在蛋白分泌过程中的作用。本项目的研究对于弄清调控高尔基体和液泡间蛋白的运输机制具有重要意义，为提高青霉菌株外源蛋白的产量提供理论依据。

丝状真菌具有较强的蛋白分泌能力及对蛋白的翻译后修饰作用，已作为基因工程宿主菌应用于外源真核蛋白的生产，但其生产外源蛋白的表达水平远低于内源蛋白，无法满足工业发酵规模的需求。而蛋白在丝状真菌分泌途径中的转录后加工（如转运和分泌），正是其瓶颈所在。以自主分离筛选的草酸青霉（Penicillium oxalicum）菌株HP7-1为研究材料，研究PoVPS10对青霉菌株蛋白分泌表达的影响。首先，敲除了非同源末端重组途径关键基因PoligD和Poku70提高了草酸青霉基因打靶效率（97%）；其次，在Poku70基因敲除过程中引入了FLP/FRT特定位点重组酶系统，但诱导FLP表达后，抗性筛选标记并未有效删除；克隆鉴定了其分泌途径中的液泡蛋白分选受体Povps10、Povps24和Povps1，其中液泡蛋白分选受体PoVPS10定位于细胞的液泡，PoVPS10与液泡的形成相关，具有协助运输液泡蛋白（如CPYA）至液胞中并分泌至胞外的功能；Povps10基因缺失促进青霉菌株蛋白分泌，影响了木质纤维素水解酶的分泌，∆vps10突变株纤维素酶和淀粉酶的酶活力均有所提高，但木聚糖酶活力下降。本项目的研究对于弄清调控高尔基体和液泡间蛋白的运输机制具有重要意义，为提高青霉菌株外源蛋白（特别是纤维素水解酶）的产量提供理论依据。