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化学品（如多环芳烃（PAH））污染环境的有效方法。多环芳烃在这些地点的持久性是由于高分子量的阻力。多环芳烃（4-5环），以生物降解的天然微生物。这就需要使用适当的生物修复剂和策略，可以提高这些难降解的多环芳烃的生物降解。我们的重点是木材腐烂的白色腐烂真菌黄孢原毛革菌。尽管其非凡的生物降解潜力，很少有人知道的酶的过程和条件，需要有效的现场应用这种真菌剂在环境中的耐多环芳烃的生物修复。使用功能基因组学方法，我们已经确定了一个子集的PAH诱导P450单加氧酶基因在P. chrysosporium，这是在更多样化的营养条件下表达不同于以前的特点过氧化物酶。我们将测试我们的假设，一个有效的生物修复活性的白色腐真菌对更高的多环芳烃（4-5环），可以实现在一个双相的方法，包括优化的初始表达的有前途的P450单加氧酶（初始氧化）之前的木质素分解酶的表达（随后的生物转化/矿化为CO2）与营养耗尽，如木质纤维素。本研究的总体目标是获得一个有前途的P450系统用于P450启动的生物降解多环芳烃在这种生物体的基础上，制定有效的生物修复策略的理解。具体目的是：（1）对候选P450进行功能性表达，筛选出有前景的多环芳烃氧化P450，并制备P450真菌生物催化剂;（2）考察所开发的P450生物催化剂的催化活性，以及白色真菌对P450氧化多环芳烃产物的降解和毒性降低。(3)使用不同的处理条件优化新鉴定的PAH-氧化P450维斯木质素分解过氧化物酶的增强表达的条件;（4）.评价了利用白色真菌对多环芳烃进行生物修复的优化条件和开发的P450生物催化剂。研究结果为开发利用白色真菌对柠檬酸类多环芳烃的有效生物修复技术奠定了基础。公共卫生相关性：微生物降解是保护人类健康免受高毒性和致癌性多环芳烃污染的有效途径。该建议将开发合适的真菌生物催化剂和多环芳烃生物修复策略。

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.