Research Project
3296969
The important polyketide family of antibiotics includes such medically important compounds as tetracyclines, anthracyclines, macrolides and others. In their biosynthesis, multifunctional synthases catalyse inerated condensation of thio-esters derived from acetate, propionate or butyrate to yield carbon chains of varying length and carrying different alkyl substitutions. Thelong-term objective of the research to understand how the choice of starter unit and the number and choice of extender units operates in this process of chain assembly to generate part of the wide variation in chemical structure of the polyketides. This knowledge will, in turn, provide a rational basis for the generation of novel antibiotics by site-directed mutagenesis, and in vitro recombination between DNA coding for parts of different polyketide synthases to produce "hybrid" antibiotics. (Novel antibiotics are still very much needed for the treatment of recalcitrant bacterial diseases, as well as those caused by fungi, viruses and cancers.) The specific aims of the research are: to isolate DNA coding for the biosynthesis of polyketides from Streptomyces strains making a selection of chemically distinct polyketides (three isochromanequinones, an anthracycline, two marolides and two polyethers); to locate the approximate limits of the polyketide synthase genes on the cloned DNA by DNA-DNA hybridization to specific restriction fragments of the DNA, by complementation of blocked mutants, and by gene disruption tests; to sequence these segments of the DNA and to recognize the precise limits and structure of the putative synthase genes by computer-aided analysis of the sequences for protein-coding character; and to predict the existence of specific substrate binding sites from the primary structure and postulated secondary structure of the proteins coded by the synthase genes. Over- production of the cloned gene products by cloning on expression vectors will allow the isolation of synthases for biochemical characterization, both to test the conclusions derived from structure comparisons and to predict suitable combinations of gene segments for the construction of recombinants that may produce novel compounds. The research is part of an international collaboration involving specific relationships with three other laboratories and is expected to form part of an even wider development in this timely area of research into the molecular biology of antibiotic biosynthesis.
