DNA-Based Approaches to the Discovery of New Biologically Active Natural Products
A major focus of the laboratory is discovering new biologically active natural products using DNA-based approaches. Many of the most powerful drugs used today have come from studying the natural products produced by soil bacteria. The continued screening of easily cultured soil microbes for the production of interesting biologically active small molecules has increasingly led to the rediscovery of known molecules. Many lines of evidence now suggest that only a tiny and unrepresentative minority of soil microbes is cultured using conventional approaches. Soil microbes that have not yet been cultured outnumber their cultured counterparts by two to three orders of magnitude, and this uncultured majority no doubt produces secondary metabolites that could serve as molecular probes of biological processes or future therapeutic agents. Uncultivated microorganisms are a very attractive source of potentially new natural products yet they are not amenable to the traditional approaches used to characterize natural products from microbes grown in pure culture. Although there appears to be no easy way to culture this large collection of unstudied microorganisms, it is possible to isolate large fragments of microbial DNA directly from environmental samples (environmental DNA, eDNA). Heterologous expression of eDNA in an easily cultured host could provide access to natural products encoded by this previously inaccessible genetic material.
In the approach our lab uses, the bacteria present in an environmental sample are lysed in situ by heating in the presence of a detergent, and the freed high molecular weight DNA is collected by alcohol precipitation from the centrifuge clarified crude lysate. After gel purification, the high molecular weight eDNA is blunt ended, ligated into a cosmid vector, packaged into lambda phage and transfected into E. coli. Large eDNA cosmid libraries are then either shuttled into other easily cultured bacteria or simply screened in E. coli to identify clones that express interesting natural product dependent phenotypes.
The characterization of antibacterial active clones found using a double antibiotic selection screen that was developed to identify and recover active clones directly from the original library selection plates has led to the characterization of new molecules, new biosynthetic pathways and new microbial biology. These hits form the basis of individual projects within the Clardy lab. Some of these projects are highlighted below.
1. Heterologous expression of environmental DNA in E. coli has led to the discovery of a prominent family of N-acyl amino acids that are synthesized by N-acyl synthases (NAS). The biosynthetic pathway for the production of fatty acid enol esters (fee pathway) contains a NAS as part of a larger biosynthetic gene cluster. This eDNA-based approach linking directly between bacterial metabolites and the biosynthetic machinery offers an opportunity to establish the structural mechanism of an NAS along with interactions between the individual enzymes in the pathway leading up to the NAS. FeeH which putatively catalyzes an unusual transformation of an eneamide to an enol ester is of particular interest and currently under investigation. (The roles of small molecules produced will also be identified.)