Microbial technologies for the discovery of novel bioactive metabolites.

Soil microbes represent an important source of biologically active compounds. These molecules present original and unexpected structure and are selective inhibitors of their molecular targets. At Biosearch Italia, discovery of new bioactive molecules is mostly carried out through the exploitation of a proprietary strain collection of over 50000 strains, mostly unusual genera of actinomycetes and uncommon filamentous fungi. A critical element in a drug discovery based on microbial extracts is the isolation of unexploited groups of microorganisms that are at the same time good producers of secondary metabolites. Molecular genetics can assist in these efforts. We will review the development and application of molecular methods for the detection of uncommon genera of actinomycetes in soil DNA and for the rapid dereplication of actinomycete isolates. The results indicate a substantial presence in many soils of the uncommon genera and a large diversity of isolated actinomycetes. However, while uncommon actinomycete strains may provide an increased chance of yielding novel structures, their genetics and physiology are poorly understood. To speed up their manipulation, we have developed vectors capable of stably maintaining large segments of actinomycete DNA in Escherichia coli and of integrating site specifically in the Streptomyces genome. These vectors are suitable for the reconstruction of gene clusters from smaller segment of cloned DNA, the preparation of large-insert libraries from unusual actinomycete strains and the construction of environmental libraries.