Natural products have proven to be a productive source of lead structures for the development of new antimicrobial agents. Culture independent analyses of environmental samples suggest that traditional approaches used to identify microbial metabolites from laboratory grown microorganisms have likely missed the vast majority of bacterial natural products that exist in nature.[2,3] In most environments, microbes that have not yet been cultured are thought to outnumber their cultured counterparts by at least two to three orders of magnitude.[2–5] If the diversity of molecules discovered from cultured bacteria is any indication, as yet uncultured bacteria are likely to be a very rewarding source of previously unknown biologically active small molecules that could serve as novel anti-infective agents. New strategies using both culture-dependent and culture-independent methods are now being developed to access this untapped reservoir of chemical diversity. This review focuses primarily on recent culture-independent, or metagenomic, efforts to identify bioactive natural products and the biosynthetic gene clusters from which they are derived.
The foundation of all metagenomic approaches is the isolation and subsequent examination of DNA extracted directly from naturally occurring microbial populations (environmental DNA, eDNA), which avoids the difficulties associated with culturing environmental microbes (Figure 1). Metagenomics is particularly appealing to natural product researchers because the genetic information needed to encode for the production of bacterial secondary metabolites is typically clustered on bacterial chromosomes. It is therefore possible to envision capturing complete small molecule biosynthetic gene clusters on individual or, at most, a small number of overlapping eDNA clones. Both expression-dependent (functional) and expression-independent (homology) screening strategies have been used to identify eDNA clones that produce bioactive small molecules. In functional metagenomic studies, eDNA libraries are examined in simple high throughput assays designed to identify clones that have phenotypes traditionally associated with the production of small molecules, while in homology-based studies, libraries are probed to identify clones that contain conserved sequences traditionally associated with secondary metabolite biosynthesis. Hits identified in these initial high throughput assays are subsequently examined for the ability to confer the production of small molecules to model cultured heterologous hosts.