草酸青霉新转录因子POX04513调控固体发酵中纤维素酶基因表达的分子机理研究

微生物纤维素酶产量低是木质纤维素生物炼制产业化的瓶颈之一。草酸青霉菌株HP7-1能分泌完整的纤维素酶系。前期工作鉴定了一个新转录因子POX04513调控草酸青霉固体发酵产纤维素酶的产量，但其分子调控机理未知。本项目拟通过DAP-Seq、比较转录组、突变体构建及酶产量测定、凝胶迁移电泳和定点突变等鉴定POX04513结合的直接靶标基因、下游关键调控基因、关键DNA结合域和结合的DNA序列。构建一系列DNA序列删除/缩短的突变体，鉴定POX04513关键调控结构域/多肽和关键氨基酸。通过串联亲和纯化-质谱(TAP-MS)、Pull down和Co-IP分析等鉴定POX04513相互作用蛋白。通过酵母单杂交、凝胶迁移电泳、突变体构建及酶产量、酶基因转录水平的测定，鉴定调控POX04513基因表达的上游调控蛋白。本项目对解析真菌固体发酵中纤维素酶基因表达调控分子机理具有重要科学意义。

Low yield of microbial cellulases is one of the bottlenecks encountered during the commercialization of lignocellulosic biorefinery. Penicillium oxalicum strain HP7-1 can secret integrative cellulases. Previous works identified a novel transcription factor POX04513 that regulated the production of cellulases in P. oxalicum under solid-state fermentation, however, its regulatory mechanism remains unknown. In this proposal, the key regulatory genes acting downstream of POX04513, the direct targets and DNA-binding domain of POX04513 and POX04513-binding DNA sequence will be identified via DNA affinity purification sequencing (DAP-Seq), sequencing and analyses of transcriptome of the mutant ΔPOX04513 in comparison with that of ΔPoxKu70, mutant construction and measurement of enzymatic production of the resultant mutants, electrophoretic mobility shift assay (EMSA) and site-specific mutagenesis. The key regulatory domain/peptide of POX04513 and amino acids will be further identified via constructing a series of DNA-truncated deletion mutants and measuring their cellulase activities. Moreover, target binding protein(s) of POX04513 and the gene(s) regulating POX04513 expression will be identified via tandem affinity purification-MS and yeast one-hybrid assays, Pull down and Co-IP analyses, EMSA, gene knock-out and measurement of enzymatic production and transcriptional levels of cellulase genes in P. oxalicum. The expected outcome of this project will have great significance in understanding molecular regulatory mechanism of cellulase gene expression in fungi under solid-state fermentation.