FUNGAL P450 SYSTEMS IN BIODEGRADATION OF HIGHER PAHS-R01ES15543-01

描述（由申请人提供）：微生物生物降解是保护人类健康免受高毒性和致癌物质的污染环境的有效途径，如多环芳烃（PAHs）。多环芳烃在这些地点的持久性是由于高毫瓦多环芳烃（4-5环）抵抗天然微生物群的生物降解。这就需要使用适当的生物修复剂和策略来加强这些顽固性多环芳烃的生物降解。我们的重点是腐烂木材的白腐真菌黄孢Phanerochaete chrysosporium。尽管其具有非凡的生物降解潜力，但对这种真菌在环境中耐药多环芳烃生物修复中有效应用所需的酶促过程和条件知之甚少。利用功能基因组学方法，我们已经确定了P. chrysosporium中pah诱导的P450单加氧酶基因的一个子集，与之前表征的过氧化物酶不同，它在更多样化的营养条件下可表达。我们将验证我们的假设，即白腐菌对更高的多环芳烃（4-5环）的有效生物修复活性可以通过双相方法实现，包括优化其有前途的P450单加氧酶（用于初始氧化）的初始表达，然后在木质素降解酶（用于随后的生物转化/矿化到二氧化碳）的表达之前，在营养消耗（如木质纤维素）上。本研究的总体目标是了解P450系统在P450启动的多环芳烃生物降解中的应用，为开发有效的生物修复策略奠定基础。具体目的是：(1)候选P450的功能表达，选择有潜力的pah氧化P450，生成P450真菌生物催化剂；(2)研究开发的P450生物催化剂的催化活性，以及白腐菌对P450氧化产物的生物降解性和毒性降低。(3)优化新鉴定的多环芳烃氧化P450相对于木质素降解过氧化物酶的表达条件；(4)。评价了白腐菌生物修复多环芳烃的最佳条件，开发了P450生物催化剂。这些研究将为开发基于白腐菌对顽固性多环芳烃的有效生物修复技术提供知识基础。公共卫生相关性：微生物生物降解是保护人类健康免受高毒性和致癌性多环芳烃（PAHs）污染环境污染的有效途径。该建议将为多环芳烃的生物修复开发合适的真菌生物催化剂和策略。

DESCRIPTION (provided by applicant): Microbial biodegradation is an effective way to protect human health from polluted environments contaminated with highly toxic and carcinogenic chemicals such as polycyclic aromatic hydrocarbons (PAHs). Persistence of PAHs at these sites is due to the resistance of high m.w. PAHs (4-5 ring) to biodegradation by natural microflora. This necessitates the use of appropriate bioremediation agents and strategies that can enhance biodegradation of these recalcitrant PAHs. Our focus is on the wood-rotting white rot fungus Phanerochaete chrysosporium. Despite its extraordinary biodegradation potential, little is known on the enzymatic processes and conditions required for effective field application of this fungal agent in bioremediation of resistant PAHs in the environment. Using functional genomic approach, we have identified a subset of PAH-inducible P450 monooxygenase genes in P. chrysosporium, which are expressible under more varied nutrient conditions unlike the previously characterized peroxidases. We will test our hypothesis that an effective bioremediation activity of the white rot fungus toward higher PAHs (4-5 ring) can be achieved in a biphasic approach involving optimized initial expression of its promising P450 monooxygenases (for initial oxidation) preceding the expression of ligninolytic enzymes (for subsequent biotransformation/ mineralization to CO2) with nutrient depletion such as on lignocellulose. The overall goal of this research is to gain an understanding of the promising P450 system for use in P450-initiated biodegradation of PAHs in this organism as a basis for developing effective bioremediation strategies. The specific aims are (1) Functional expression of the candidate P450s to select the promising PAH-oxidizing P450(s) and generate P450 fungal biocatalyst(s); (2) Investigate catalytic activity of the developed P450 biocatalyst(s) and further biodegradability and toxicity reduction of the P450-oxidation products of PAHs by white rot fungus. (3) Optimize conditions for enhanced expression of the newly-identified PAH-oxidizing P450 vis-a-vis the ligninolytic peroxidases using varied treatment conditions; (4). Evaluate the optimized conditions and developed P450 biocatalysts for bioremediation of PAHs by the white rot fungus. The studies are expected to provide a knowledgebase for development of effective bioremediation technologies for recalcitrant PAHs based on the use of white rot fungus. PUBLIC HEALTH RELEVANCE: Microbial biodegradation is an effective way to protect human health from polluted environments contaminated with highly toxic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). This proposal will develop appropriate fungal biocatalysts and strategies for PAH bioremediation.