Genomic and mechanistic insights into the biodegradation of organic pollutants Dietmar H Pieper, Vı´tor AP Martins dos Santos and Peter N Golyshin  Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia. Addresses Division of Microbiology, German Research Centre for Biotechnology, Mascheroder Weg 1, Braunschweig, Germany  e-mail: pgo@gbf.de Current Opinion in Biotechnology 2004, 15:215–224 This review comes from a themed issue on Environmental biotechnology Edited by Michael Y Galperin and Alan JM Baker Available online 17th April 2004 0958-1669/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.03.008 Abbreviations 2,4-D 2,4-dichlorophenoxyacetate PAH polycyclic aromatic hydrocarbon PCP pentachlorophenol 2,4,5/6-TCP 2,4,5/6-trichlorophenol Introduction For several decades, significant efforts have been devoted to the study of the biodegradation of organic pollutants. Various aspects need to be studied to obtain a detailed overview of biodegradation processes in the environment and to optimize and predict the performance of degrading microorganisms in situ. Approaches to analyze and assess biodegradation processes have been shifting towards the application of culture-independent methodologies to characterize natural and engineered pollutant-degrading microbial associations. Various new culture-independent tools have become available to analyze microbial com- munity structure and function in natural and engineered environments. In addition, the genomes of several mi- crobes relevant to biodegradation have been published and others are likely be reported soon, providing the opportunity to gain global insights into the evolutionary potential of specific microorganisms and their ability to bioremediate polluted environments. Here, we provide an overview of the most recent advances in the knowledge of the mechanisms of aerobic and anaerobic degradation of organic pollutants, yielded from biochemical and genomic approaches, and give a short outlook on its development from a systems biology perspective. New chloroaromatic metabolic enzymes and pathways Although the metabolism of chlorinated aromatics has been the focus of research for decades, novel metabolic capabilities are still being discovered. The metabolism of 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2- methylphenoxyacetate is initiated by an a-ketogluta- rate-dependent dioxygenase (TfdA), which catalyzes cleavage of the sidechain resulting in the formation of 2,4-dichloromethylphenol and 4-chloro-2-methylphenol, respectively. The tfdA genes have been described from a broad set of 2,4-D-degrading b- and g-Proteobacteria. It has now been shown that tfdA-like tfdAa genes are also present in a-Proteobacteria [1]. As phenoxyacetate rather than 2,4-D seems to be the preferred substrate of TfdAa enzymes, it is assumed that ancestral tfdAa genes served as the origin for the more specific 2,4-D-transforming enzymes. Dichlorprop (2-(2,4-dichlorophenoxy)propio- nate) and mecoprop (2-(4-chloro-2-methylphenoxy)pro- pionate) are chiral molecules that are poor substrates for TfdA. Specific enantioselective a-ketoglutarate-depen- dent dioxygenases that transform (S)- and (R)-mecoprop have now been purified and their genes from Delftia acidovorans MC1 have been sequenced [2]. The respec- tive dioxygenases share only low sequence identity with the previously characterized TfdA genes or proteins. In contrast to 2,4-D, the degradation of 2,4,5-trichlorophe- noxyacetate, mainly elucidated in Burkholderia cepacia AC1100, is initiated by a monooxygense. Evidence has now been reported to show that 2,4-D degradation can also be initiated by a monooxygenase [3  ]. In contrast to 2,4-D, which is transformed to 3,5-dichloro- catechol and further metabolized through the chloroca- techol pathway, 2,4,5-trichlorophenol (2,4,5-TCP) is metabolized through a hydroquinone (quinol) pathway. www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:215–224