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A metagenomic approach was taken to retrieve catabolic operons for aromatic compounds from activated sludge used to treat coke plant wastewater. Metagenomic DNA extracted from the sludge was cloned into fosmids and the resulting Escherichia coli library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, yielding 91 EDO-positive clones. Based on their substrate specificity for various catecholic compounds, 38 clones were subjected to sequence analysis. Each insert contained at least one EDO gene, and a total of 43 EDO genes were identified. More than half of these belonged to new EDO subfamilies: I.1.C (2 clones), I.2.G (20 clones), I.3.M (2 clones) and I.3.N (1 clone). The fact that novel I.2.G family genes were over-represented in these clones suggested that these genes play a specific role in environmental aromatic degradation. The I.2.G clones were further classified into six groups based on single-nucleotide polymorphisms (SNPs). Based on the combination of the SNPs, the evolutionary lineage of the genes was reconstructed; further, taking the activities of the clones into account, potential adaptive mutations were identified. The metagenomic approach was thus used to retrieve novel EDO genes as well as to gain insights into the gene evolution of EDOs.

Biphenyl dioxygenases (BP Dox) from different organisms, which are involved in the initial oxygenation and subsequent degradation of polychlorinated biphenyls (PCB), are similar in structure but have different functions. The large subunit of BP Dox, encoded by the bphA1 gene, is crucial for substrate selectivity. Using the process of DNA shuffling, we randomly recombined the bphA1 genes of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 and selected for genes that expressed proteins with altered function. Upon expression in Escherichia coli, some of these evolved genes exhibited enhanced degradation capacity, not only for PCB and related biphenyl compounds, but for single aromatic hydrocarbons such as benzene and toluene, which are poor substrates for the original BP Dox.

A major research goal in microbial ecology is to understand the relationship between gene organization and function involved in environmental processes of potential interest. Given that more than an estimated 99% of microorganisms in most environments are not amenable to culturing, methods for culture-independent studies of genes of interest have been developed. The wealth of metagenomic approaches allows environmental microbiologists to directly explore the enormous genetic diversity of microbial communities. However, it is extremely difficult to obtain the appropriate sequencing depth of any particular gene that can entirely represent the complexity of microbial metagenomes and be able to draw meaningful conclusions about these communities. This review presents a summary of the metagenomic approaches that have been useful for collecting more information about specific genes. Specific subsets of metagenomes that focus on sequence analysis were selected in each metagenomic studies. This 'targeted metagenomics' approach will provide extensive insight into the functional, ecological and evolutionary patterns of important genes found in microorganisms from various ecosystems.

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