Microbial technologies for the discovery of novel bioactive metabolites

Abstract

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 50 000 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.

Introduction

The search for new substances of pharmaceutical or agricultural importance can be conducted through screening large collections of diverse chemical entities (generally defined as a library), employing assays designed to detect modulators of pharmacologically or agriculturally relevant targets. Nowadays, screening is mostly conducted in an automated fashion and is referred to as high throughput screening (HTS). HTS is finding increased applications thanks to the availability of large libraries and to the large number of targets that are being identified through genome sequencing. General considerations about HTS can be found in a recent overview (Carrano and Donadio, 1999). A critical role in identifying novel bioactive substances lies in the quality of the library and in the assays employed for screening it. Some considerations about assays and the appropriate libraries to use in the antibacterial field, can be found in another paper in this issue (Donadio et al., 2002). In general terms, the ideal library should consist of as many as possible different chemical entities, without any bias in the type of chemical classes, synthetic routes or biological activities. In addition, it should contain a relevant fraction of unique compounds, unlikely to be found in other libraries. It should also consist of pure substances of known structure and specified concentration. Finally, increased amounts of interesting compounds found through screening should be readily obtained for further tests. Unfortunately, no single library can satisfy all these criteria.

Microbial secondary metabolites represent a large source of compounds endowed with ingenious structures and potent biological activities. Many of the products currently used for human or animal therapy, in animal husbandry and in agriculture are produced by microbial fermentation, or are derived from chemical modification of a microbial product. These products have been obtained after a few decades of intensive screening involving probably millions of microorganisms. These past successes make discovering new bioactive metabolites from microbial sources harder than ever, since thousands of compounds are described in the literature. However, different strains generally produce different compounds. Thus, new bioactive metabolites continue to be identified from microbial sources, thanks to the large variety of existing strains. However, not all microorganisms are equally capable of producing secondary metabolites. In fact, this capability is at the moment restricted to a few groups of bacterial or eukaryotic microbes. In particular, the ability to produce a large number of chemically different secondary metabolites is associated mostly with the filamentous actinomycetes, the myxobacteria, the pseudomonads and the cyanobacteria within the prokaryotic world, and mostly to the filamentous fungi for the eukaryotic microbes (G. Toppo, personal communication).

We believe that novel antibiotics and other bioactive secondary metabolites can still be discovered from microbial sources. In our opinion, the probability of finding novel bioactive compounds depends on a series of critical factors. On the one hand, there is the number of strains screened and their degree of diversity; on the other hand, their uniqueness and their potential to produce secondary metabolites. These last two criteria are extremely important, since intensively screened microbes are less likely to yield novel metabolites than unexploited groups. These critical factors must all be considered when embarking on a screening program for bioactive metabolites from microorganisms.