Anaerobic microbial consortia and enzymes for biotransformation of aromatic compounds

这项发现资助计划的重点是阐明广泛使用的工业化学品苯的厌氧微生物转化机制。苯是最简单的未取代芳烃，具有毒性和致癌性，并且是更复杂的多环芳烃（PAHs）的核心结构，也是环境问题。有趣的是，与甲基化苯（甲苯）或羧基化苯（苯甲酸酯）等密切相关的化合物不同，苯在没有氧气的情况下更难生物降解。甲苯和苯甲酸厌氧生物降解的途径和酶的特点。相比之下，厌氧苯氧化的机制仍然是未知的，虽然在过去的几年中取得了重大进展，特别是在维持厌氧转化反应在实验室培养。多年来，我的实验室一直在研究厌氧富集培养物中复杂的细胞相互作用和反应途径，这些厌氧富集培养物在无氧条件下，在厌氧或产甲烷条件下降解苯。我们已经确定了两种不同的生物负责厌氧苯氧化。我们已经从这些生物体中收集了宏基因组序列数据，并列出了可能在苯活化的初始步骤中催化反应的酶的候选基因。转录和蛋白质组学数据进一步缩小了相关酶的选择范围。根据初步数据，这两种生物似乎使用不同的反应机制-其中之一是羧化-尽管尚未得到生物化学证明。这个发现计划的具体目标是生物化学表征参与厌氧苯氧化的酶。这将使用天然聚丙烯酰胺凝胶电泳、厌氧酶测定和候选蛋白的异源表达来实现。这些酶构成了一种新的代谢途径和新的生物化学，用于苯和其他重要化学品的转化或解毒，包括杀虫剂，除草剂，多环芳烃，药物和木质素，这些都是苯的衍生物。

This Discovery Grant proposal is focused elucidating the mechanisms for the anaerobic microbial transformation of the widely used industrial chemical benzene. Benzene is the simplest unsubstituted aromatic hydrocarbon, is toxic and carcinogenic, and is the structure at the heart of more complex polyaromatic hydrocarbons (PAHs) also of environmental concern. Intriguingly, unlike very closely related compounds like methylated benzene (toluene) or carboxylated benzene (benzoate), benzene is remarkably more difficult to biodegrade in the absence of oxygen. The pathways and enzymes for anaerobic toluene and benzoate biodegradation are now well characterized. In contrast, the mechanism for anaerobic benzene oxidation is still unknown, although significant progress has been made in the past few years, particularly in sustaining anaerobic transformation reactions in laboratory cultures. For many years, my laboratory has been studying the complex cellular interactions and reaction pathways in anaerobic enrichment cultures that degraded benzene in the absence of oxygen, under denitrifying or methanogenic conditions. We have identified two distinct organisms responsible for anaerobic benzene oxidation. We have collected metagenomic sequence data from these organisms and have a list of candidate genes for enzymes that may catalyze the reactions in the initial steps of benzene activation. Transcriptional and proteomic data have further narrowed down the options for relevant enzymes. Based on preliminary data, the two organisms appear to use different reaction mechanisms - one of which is a carboxylation - though neither has been biochemically proven yet. The specific objective of this discovery program is to biochemically characterize the enzymes involved in anaerobic benzene oxidation. This will be achieved using native polyacrylamide gel electrophoresis, anaerobic enzyme assays and heterologous expression of candidate proteins. These enzymes constitute a novel metabolic pathway and novel biochemistry for the transformation or detoxification of benzene and other important chemicals, including pesticides, herbicides, polyaromatic hydrocarbons, pharmaceuticals and lignin, that are all derivatives of benzene.