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

描述(申请人提供)：微生物生物降解是保护人类健康免受多环芳烃(PAHs)等剧毒和致癌化学品污染的环境的有效方法。多环芳烃在这些地点的持久性是由于高相对分子质量的抗性所致。多环芳烃(4-5环)可被天然微生物群降解。这就需要使用适当的生物修复试剂和策略，以加强这些顽固的多环芳烃的生物降解。我们的重点是木腐白腐真菌黄孢原毛平革菌。尽管它具有非凡的生物降解潜力，但人们对这种真菌制剂在环境中有效修复抗性多环芳烃所需的酶过程和条件知之甚少。利用功能基因组学方法，我们已经在黄孢霉中鉴定了多环芳烃诱导的P450单加氧酶基因的一个子集，该基因可以在更多样化的营养条件下表达，而不是以前描述的过氧化物酶。我们将检验我们的假设，即白腐菌对较高的PAHs(4-5环)的有效生物修复活性可以通过两相方法实现，包括优化其有望的P450单加氧酶的初始表达(用于初始氧化)，然后表达具有营养消耗的木质素分解酶(用于随后的生物转化/矿化为CO2)，例如在木质纤维素上。这项研究的总体目标是了解P450系统在P450启动的生物降解该生物体中多环芳烃方面的应用前景，作为开发有效的生物修复策略的基础。具体目的是(1)对候选P450进行功能表达，筛选出有潜力的多环芳烃氧化产物P450(S)，生成P450真菌生物催化剂(S)；(2)研究开发的P450生物催化剂(S)的催化活性，并进一步研究白腐菌对多环芳烃P450氧化产物的生物降解性和毒性。(3)利用不同的处理条件优化新发现的多环芳烃氧化P450相对于木质素降解过氧化物酶的表达条件；对白腐菌修复多环芳烃的优化条件和开发的P450生物催化剂进行了评价。这些研究有望为开发基于白腐菌的难降解多环芳烃的有效生物修复技术提供知识基础。与公共健康相关：微生物生物降解是保护人类健康免受高毒性和致癌多环芳烃(PAHs)污染环境的有效方法。该提案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.