XENOBIOTIC-OXIDIZING P450 SYSTEM IN A WHITE ROT FUNGUS

描述：微生物脱毒是污染化学物质的有效途径 以减少受污染环境对人类健康的危害。裸毛目动物 黄孢霉是一种能分解木质素的白腐菌，已被用于生物修复 在营养受限(木质素分解)环境下，基于其表达 在这些条件下触发的外源过氧化物酶。然而，有越来越多的人 P450单氧代谢启动异种生物分解代谢的证据 当外源过氧化物酶被抑制时，营养丰富的条件，以及P450 随后产生的反应产物可能与木质素分解物相连接。 作为主要营养物质的通路被耗尽。这样做的长期目标是 研究是为了更好地了解分解代谢P450系统与 白腐菌和白腐菌异物解毒的总体途径 基于酶或全细胞技术的研究进展 生物修复。具体目标是：(一)完成隔离和 分解代谢P450单加氧酶基因的鉴定(II)研究 异源生物在不同生理条件下的诱导与调控； (Iii)操纵其在异源寄主和本地寄主中的表达 与最近鉴定的P450还原酶基因结合；(Iv)研究其 在体内和体外的催化作用；以及(V)评价实际应用 利用优化的P450条件和工程菌株进行生物修复。一个 获得了P450基因的部分序列，并将用于筛查 新构建的用于基因分离的基因组和cDNA文库。异种生物 将基于mRNA和酶来检查诱导和调节条件 级别。单加氧酶cdna将与现有的还原酶共表达。 酿酒酵母基因是以全细胞和微体为基础的 催化分析或在杆状病毒系统中表达以产生酶 体外试验。酶底物(S)将根据诱导进行鉴定 结果利用P450缺失突变单倍体和基因扩增菌株。 为此目的构建的黄孢子菌，和/或使用 表达的酶。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.