GENE AND FUNCTIONAL DIVERSITY OF CYTOCHROME P450 MOLECULAR SPECIES IN THE LIGNIN-DEGRADING BASIDIOMYCETE

白腐菌是唯一已知的能够降解木质素的生物。木质素是最丰富的、可再生的芳香族聚合物，被认为是地球上最具生命力的生物材料之一。最初，真菌利用非特异性氧化酶如木质素和锰过氧化物酶在细胞外攻击聚合木质素，导致形成各种芳香片段。这些芳香底物的共同特征之一是高电离电位;因此细胞外氧化酶不能启动某些底物的代谢反应。细胞色素P450（s）参与了白腐真菌对柠檬酸酯类芳香化合物的降解。最近，美国DOE/JGI完成了白腐菌属黄孢囊革菌的基因组注释工作，并将其数据公开（http：//www.jgi.doe. gov/programs/whiterot.htm）。利用他们的注释数据和我们的cDNA克隆数据，获得了154个细胞色素P450基因组序列。本研究采用功能基因组学、蛋白质组学、同源模建以及常规生化方法对真菌P450基因多样性及其功能调控进行了研究。从基因组研究中，发现122个革兰氏菌P450基因具有微外显子（仅编码3-10个氨基酸）。同源性建模表明，这些微外显子在多样化的底物识别位点的结构中发挥作用。成功地进行了代谢和表达研究，以确定2个基因的功能。还发现P450表达、NADPH产生和血红素生物合成之间的独特途径串扰。

White-rot basidiomycetes are only known organism to be capable of degrading lignin. Lignin is the most abundant, renewable aromatic polymer and is known as one of the most recalcitrant biomaterials on the earth. Initially, fungi extracellularly attack polymeric lignin utilizing nonspecific oxidative enzymes, such as lignin and manganese peroxidases, resulting in the formation of a variety of aromatic fragments. One of the common features for these aromatic substrates is a high ionization potential ; thus extracellular oxidative enzymes could not initiate the metabolic reactions for certain substrates. The involvement of cytochrome P450(s) has been indicated for the degradation of recalcitrant aromatic compounds by white-rot fungi. Hydroxylation of aromatic moieties dramatically reduces their ionization potentials, forming more susceptible structures for further oxidative metabolism.Recently, genome annotations of the white-rot basidiomycete, Phanerochaete chrysosporium, were almost completed by US DOE/JGI and the data were made available to the public (http://www.jgi.doe. gov/programs/whiterot.htm). Utilizing their annotation data and our cDNA cloning data, 154 cytochrome P450 genomic sequences were obtained. The gene diversity and the functional regulation of fungal P450s were studied via functional genomics, proteomics, homology modeling as well as conventional biochemical methods. From genomic studies, 122 Phanerochaete P450 genes were found to possess microexons (coding only 3-10 amino acids). Homology modeling indicated that these microexons play a role in diversifying the structure of substrate recognition sites. Metabolic and expression studies were successfully achieved to identify the function of 2 genes. A unique pathway crosstalk between P450 expressions, NADPH production, and heme biosynthesis were also found.