草酸青霉纤维素酶基因表达调控机制的研究

Many microorganisms produce cellulases. The cellulases in practical uses at present are mainly produced by fungi. The low yield of fungal cellulases is one of the technological bottlenecks in the limited commercialization of biorefinery by using lignocellulose as feedstock. The cellulase yield of fungi can be elevated through genetic manipulation based on the indepth study on the mechanismes of the regulation of expression of fungal cellulase genes.In this proposed project, the transcriptomes of a high cellulase producing Penicillium oxalicum strain HP7-1 and its derived mutant EU2106 producing 3.3-fold filter paper activity as the wild-type HP7-1 will be sequenced and comparatively analyzed.Novel candidate genes involved in regulation of expression of cellulase genes in P. oxalicum will be predicted through comparative analyses of the transcriptomes of the wild-type and the mutant.The promoter region of the known major cellulase genes and regulatory genes in the wild-type and mutant will be cloned and sequenced to know whether alterations occurred in the promoter of tested genes in the mutant. The deletion mutants for the candidate regulatory genes in P. oxalicum will be generated by deleting the genes in the wild-type fungal strain. Cellulase activity of the deletion mutants will be measured to verify the regulatory role of the candidate genes.The identified regulatory genes will be over-expressed and the translated products will be purified. The binding region and binding sequence in the promoter of target genes by the regulatory proteins will be identified. This proposed project is of great theoretical significance in understanding the mechanism of regulation of expression of fungal cellulase genes. Meanwhile，it will provide solid basis for directed genetic manipulation of fungal strains to increase the yield of cellulases.

许多微生物产生纤维素酶，目前生产上使用的纤维素酶主要来自于真菌。真菌纤维素酶产量低是以木质纤维素为原料进行生物炼制不能商业化的技术瓶颈之一。在深入研究真菌纤维素酶基因表达调控机制的基础上，可通过遗传操作的办法提高真菌的纤维素酶产量。本项目拟通过对自主分离的高产纤维素酶活力的草酸青霉野生型菌株HP7-1和其滤纸酶活力提高了3.3倍的突变体EU2106的转录组比较分析，发现新的调控纤维素酶基因表达的候选基因。克隆野生型和突变体中已知的主要纤维素酶基因及其调控基因的启动子区域、测序分析，明确启动子序列是否发生突变。缺失草酸青霉中的候选调控基因得到突变体，检测突变体的纤维素酶活力以验证基因的调控功能。过量表达鉴定的调控基因并纯化其翻译产物，鉴定调控蛋白结合的靶基因启动子区域及其结合序列。本项目对弄清真菌纤维素酶基因表达调控机制具有重要的理论意义，将为定向改造真菌以提高纤维素酶产量提供理论依据。

许多微生物产生纤维素酶，生产上使用的纤维素酶主要来自丝状真菌，如木霉和青霉。但是，真菌纤维素酶的产量远远不能满足木质纤维素工业规模生物炼制的要求，导致生物炼制的成本较高。深入研究真菌纤维素酶基因表达调控机制，可为理性设计、遗传改造真菌以提高纤维素酶产量提供理论指导。. 本项目通过测定、比较分析草酸青霉野生型菌株HP7-1及其高产纤维素酶突变体EU2106的基因组序列、转录组和分泌组，以及菌株HP7-1在纤维素酶产生被阻遏和被诱导条件下的转录组序列，筛选得到调控纤维素酶基因表达的候选调控基因。敲除草酸青霉中的候选调控基因得到突变体，检测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。. 结果表明菌株HP7-1 的基因组序列大小为30.62 Mb，注释了9834个编码蛋白基因。和野生型菌株HP7-1的基因组序列相比，EU2106基因组中有274个单核苷酸突变和12个插入或缺失突变。筛选得到42个候选调控转录因子基因。敲除了35个候选调控基因，发现14个基因突变体的纤维素酶产量发生显著改变，说明该14个基因为纤维素酶基因的调控基因，其中12个为新的调控基因，证实5个新基因（POX01167、POX02484、POX04420、POX08415和POX08522）在草酸青霉生长过程中能调控纤维素酶基因和木聚糖酶基因的表达，发现其中的3个基因（POX01167、POX04420和POX08415）是关键调控基因。在大肠杆菌中表达了该3个关键调控基因；鉴定了1个关键调控蛋白POX01167结合的靶基因即纤维素酶基因的启动子区域。对认识真菌纤维素酶基因的表达调控机制具有重要的科学意义，为理性设计定向改造真菌以提高纤维素酶产量提供理论依据。