草酸青霉固态发酵中纤维素酶基因表达调控分子机制研究

Cellulase produced by filamentous fungi can efficiently hydrolyze cellulosic materials, which could help in mitigating serious challenges that mankind faces such as energy crisis, environmental pollution, and food shortage. To date, only limited knowledge has reported on the regulation mechanism of fungal cellulase gene expression, especially under solid-state fermentation. The genome of Penicillium oxalicum HP7-1, a strain that produces high amounts of cellulase under solid-state fermentation, has been sequenced. In addition, a highly efficient gene targeting system that allows rapid genetic manipulation in P. oxalicum HP7-1 has been constructed. In this proposed project, we will deep-sequence and comparatively analyze the transcriptomes of P. oxalicum HP7-1 under different cultivation conditions, and then screen novel candidate regulatory genes involved in the regulation of cellulase gene expression in P. oxalicum HP7-1 under solid-state fermentation. Subsequently, deletion mutants of the candidate genes and complementation of key regulatory gene mutants and their overexpression mutants in P. oxalicum will be established using the constructed gene targeting system, and their cellulase yields will be measured to verify the regulatory roles of the candidate genes. The direct target cellulase genes of the identified regulators and their binding sequences will be determined using electrophoretic mobility shift assay and DNA footprinting. The proposed project will be the first to uncover the regulatory mechanism of cellulase gene expression in P. oxalicum under solid-state fermentation, which will have theoretical and application significances in studying the expression and regulation of cellulase genes in filamentous fungi.

丝状真菌产生的纤维素酶能有效地降解纤维素物质，对于解决能源危机、环境污染和食品短缺等人类面对的严峻挑战具有重要意义。但是，对真菌纤维素酶基因表达调控机制（特别是固态发酵中）研究较少限制了纤维素酶的获得和广泛应用。本实验室获得了固态发酵条件下高产纤维素酶的草酸青霉菌株HP7-1，完成了其基因组测序和遗传操作系统构建。本项目拟以HP7-1为材料，1）通过转录组测定和差异分析，初步确定参与固态发酵中调控纤维素酶基因表达的候选调控基因；2）构建其候选调控基因的缺失突变体和关键调控基因突变体，相应的互补株及高表达菌株，检测纤维素酶活力以验证基因的调控功能；3）通过凝胶阻滞和DNA足迹等实验，确定关键调控因子的直接靶位纤维素酶基因，及所结合的DNA序列。这将首次揭示草酸青霉固态发酵中纤维素酶基因表达的调控机制，对其他丝状真菌固态发酵中纤维素酶基因的调控研究具有理论指导意义和实际应用价值。

丝状真菌产生的纤维素酶能有效地降解纤维素物质，对于解决能源危机、环境污染和食品短缺等人类面临的严峻挑战具有重要意义。但是，对真菌纤维素酶基因表达调理机制（尤其是固体发酵中）认识不足，限制了纤维素酶的获得和广泛应用。.本项目通过测定草酸青霉菌株HP7-1在固态发酵和液体培养条件下的转录组，以及固态发酵高产纤维素酶的突变株EU2101在含有不同碳源的固态发酵条件下的转录组，进行比对分析，筛选得到固体发酵条件下调控纤维素酶基因表达的候选调控基因。在草酸青霉中敲除候选调控基因，获得缺失突变体；测定缺失突变体的纤维素酶产量以明确基因的调控功能。在大肠杆菌中表达关键调控基因，鉴定关键调控蛋白结合的靶基因的启动子区域。.结果表明草酸青霉固体发酵和液体培养条件下，基因表达谱明显不一样，尤其是纤维素酶基因的转录水平。通过不同条件下的转录组对比，筛选得到了80个候选转录调控基因。敲除了40个候选调控基因，发现了21个调控基因，其中9个为新的调控基因，3个是调控固体发酵中纤维素酶基因表达的关键调控基因（POX03016、POX04513和POX08292）。在大肠杆菌中表达了该3个关键调控基因；鉴定了该3个关键调控蛋白POX03016、POX04513和POX08292结合的纤维素酶基因的启动子区域。本项目取得的成果对认识真菌固体发酵中纤维素酶基因的复杂表达机制具有重要的科学意义，为通过理性设计和遗传工程改造真菌以提高固体发酵纤维素酶产量提供理论指导，具有潜在的应用价值。