XENOBIOTIC-OXIDIZING P450 SYSTEM IN A WHITE ROT FUNGUS

产品描述：微生物对污染化学品的解毒是一种有效的方法 减少污染环境对人类健康的危害。鞘丝菌属 一种降解木质素的白色腐真菌--黄孢霉已被用于生物修复 在营养有限（木质素分解）的环境下，基于其表达， 在这些条件下引发的外切过氧化物酶。然而，越来越多的 P450单加氧作用启动外源性催化剂的证据 营养丰富的条件下，当外过氧化物酶被抑制，P450 随后产生的反应产物可与木质素降解产物连接。 因为主要营养素被耗尽。长期目标是 研究是为了更好地了解分解代谢P450系统与 白色腐真菌和森林中外源物质解毒总途径 开发基于酶或基于全细胞的技术， 生物修复具体目标是：（一）完成隔离， 表征的基因的分解代谢P450单加氧酶，（ii）研究其 异生素在不同生理条件下的诱导和调节; (iii)操纵其在异源宿主和天然宿主中的表达， 结合最近表征的P450还原酶基因;（iv）研究其 在体内和体外的催化作用;和（v）评估实际应用 使用优化的P450条件和工程菌株进行生物修复。一 P450基因的部分序列是可用的，并将用于筛选 新构建的基因组和cDNA文库用于基因分离。Xenobiotic 将基于mRNA和酶检查诱导和调节条件 程度.单加氧酶cDNA将与可用的还原酶共表达 cDNA是以酿酒酵母的全细胞和微粒体为基础的 催化测定或在杆状病毒系统中表达，用于酶生产， 体外测定。将根据诱导鉴定酶底物 结果，利用P450无效突变株单倍体和基因扩增株， 为此目的构建的黄孢子菌，和/或使用 表达酶。优化的P450条件和菌株将在 生物修复的台式模型。

DESCRIPTION: Microbial detoxification of pollutant chemical is an effective way to reduce human health hazards from contaminated environments. Phamerochaete chrysosporium, a ligninolytic white rot fungus has been used in bioremediation under nutrient-limited (ligninolytic) environments, based on expression of its exo-peroxidases triggered under these conditions. However, there is increasing evidence that P450 monooxygenation initiates xenobiotic catabolism under nutrient-rich conditions when exo-peroxidases are suppressed, and that P450 reaction products produced subsequently may link up with the ligninolytic pathways as the primary nutrients are depleted. The long-term objective of this research is to better understand the relationship of catabolic P450 systems to the overall pathways of xenobiotic detoxification in white rot fungi and for development of enzyme-based or whole cell-based technologies for bioremediation. The specific aims are to (i) complete the isolation and characterization of the gene for a catabolic P450 monooxygenase, (ii) study its xenobiotic induction and regulation under varied physiological conditions; (iii) manipulate its expression in heterologous hosts and the native host in conjunction with the recently characterized P450 reductase gene; (iv) study its catalytic role in vivo and in vitro; and (v) evaluate the practical application in bioremediation using optimized P450 conditions and engineered strains. A partial sequence of the P450 gene is available and will be used in screening newly constructed genomic and cDNA libraries for gene isolation. Xenobiotic induction and regulatory conditions will be examined based on mRNA and enzyme levels. The monooxygenase cDNA will be coexpressed with the available reductase cDNA is Saccharomyces cerevisiae for whole cell-based and microsome-based catalytic assays or expressed in a baculovirus system for enzyme production for in vitro assays. Enzyme substrate(s) will be identified based on induction results, the use of P450 null mutant haploids and gene amplified strains of P. chrysosporium constructed for this purpose, and/or in vitro assays using expressed enzymes. Optimized P450 conditions and strains will be tested in bench top models of bioremediation.