Mechanistic investigations on the pathway of n-alkane oxidation in anaerobic bacteria

烷烃是原油和各种石油产品的主要成分。它们在受污染的含水层或石油储集层等缺氧自然环境中的生物降解在全球碳循环中发挥着重要作用。在过去的15年里，已经分离和鉴定了几种能够利用烷烃作为唯一碳和能源来源的反硝化和硫酸盐还原细菌。基于对代谢产物的鉴定和同位素标记底物的研究，提出了反硝化细菌HxN1中正己烷完全氧化为二氧化碳的途径。在最初的活化反应中，正构烷烃被加到富马酸的双键上，生成(1-甲基戊基)琥珀酸酯(MPS)。MPS通过一系列酶反应进一步转化为支链脂肪酸(4-甲基-辅酶A)，然后通过β氧化降解。该项目的主要目标将是对所提议的降解途径的重要机制方面获得新的见解，并有助于更好地理解正构烷烃与烷基苯氧化所涉及的生化机制可能的根本差异。特别是，关键代谢物的立体化学将通过真实标准的立体选择性合成和分离代谢物的刚性结构指定来建立。厌氧碳氢氧化菌的底物范围将通过与模型基质和原油的共转化实验来表征。

Alkanes are the main constituents of crude oil and various petroleum products. Their biodegradation in anoxic natural environments such as contaminated aquifers or petroleum reservoirs plays an important role in the global carbon cycle. During the last 15 years several denitrifying and sulphate-reducing bacteria capable of utilizing alkanes as sole source of carbon and energy have been isolated and characterized. Based on the identification of metabolites and studies with isotope labeled substrates a pathway for the complete oxidation of n-hexane to carbon dioxide in the denitrifying bacterium strain HxN1 has been proposed. In the initial activation reaction the n-alkane is added to the double bond of fumarate yielding (1-methylpentyl)succinate (MPS). MPS is further transformed via a sequence of enzyme reactions to a branched fatty acid (4-methyloctanoyl-CoA) which then is degraded by β-oxidation. Main goal of this project will be to obtain new insights into important mechanistic aspects of the proposed degradation pathway, and to contribute to a better understanding of possible fundamental differences in the biochemical mechanisms involved in n-alkane versus alkylbenzene oxidation. In particular, the stereochemistry of key metabolites will be established by stereoselective synthesis of authentic standards and rigid structure assignment of isolated metabolites. The substrate range of anaerobic hydrocarbon oxidizing bacteria will be characterized through co-transformation experiments with model substrates and crude oil.