Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

Bioactivity of fungal endophytes as a function of endophyte taxonomy and the taxonomy and distribution of their host plants.

Fungal endophytes--fungi that grow within plant tissues without causing immediate signs of disease--are abundant and diverse producers of bioactive secondary metabolites. Endophytes associated with leaves of tropical plants are an especially exciting and relatively untapped source of novel compounds. However, one major challenge in drug discovery lies in developing strategies to efficiently recover highly bioactive strains. As part of a 15-year drug discovery project, foliar endophytes were isolated from 3198 plant samples (51 orders, 105 families and at least 232 genera of angiosperms and ferns) collected in nine geographically distinct regions of Panama. Extracts from culture supernatants of >2700 isolates were tested for bioactivity (in vitro percent inhibition of growth, % IG) against a human breast cancer cell line (MCF-7) and the causative agents of malaria, leishmaniasis, and Chagas' disease. Overall, 32.7% of endophyte isolates were highly active in at least one bioassay, including representatives of diverse fungal lineages, host lineages, and collection sites. Up to 17% of isolates tested per assay were highly active. Most bioactive strains were active in only one assay. Fungal lineages differed in the incidence and degree of bioactivity, as did fungi from particular plant taxa, and greater bioactivity was observed in endophytes isolated from plants in cloud forests vs. lowland forests. Our results suggest that using host taxonomy and forest type to tailor plant collections, and selecting endophytes from specific orders or families for cultivation, will markedly increase the efficiency and efficacy of discovering bioactive metabolites for particular pharmaceutical targets.

Identification and characterisation of a Streptomyces sp. isolate exhibiting activity against methicillin-resistant Staphylococcus aureus.

A soil-borne bacterium exhibiting broad antimicrobial activity, including activity against MRSA, was investigated. The bacterium was identified using morphological, biochemical and genetic techniques. The 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces lavendulae and Streptomyces globosus. DNA-DNA hybridisation revealed 80% homology with S. globosus DSM41122. The antibiotic was partially purified from the culture supernatant using a combination of precipitation, charcoal and ion exchange. The antibiotic was found to be water-soluble and highly thermally stable at acidic pH. It inhibited the growth of S. lavendulae but not Streptomyces tenebrarius. Significantly, the partially purified antibiotic displayed activity towards clinical isolates of methicillin-resistant Staphylococcus aureus, representative of the major clonal complexes.