Chemical diversity from biodiversity: Natural products from endophytic fungi and uncultured bacteria.

摘要

Natural product extracts have traditionally been a rich source of structurally unique biologically active small molecules. The work reported here has focused on the isolation and characterization of new antimicrobials from endophytic fungi, a relatively unstudied and highly diverse group of plant-associated filamentous fungi, as well as the development of methodologies to access bioactive natural products produced by uncultured soil microbes.;The isolation and structure determination of antimicrobial metabolites from extracts of three endophytic fungi CR377, CR200 and CR115 is described. CR377 yielded a novel pentaketide antifungal agent that is active against strains of Candida albicans. The octaketide-derived cytosporone antibiotics and biochemical induction assay active cytoskyrins were isolated from cultures of CR200. CR115 cultures produce a family of diterpene antibiotics based on a new carbon skeleton given the trivial name, guanacastane.;The isolation and characterization of pantocin B from Escherichia coli cultures transformed with a 20 kb fragment of Erwinia herbicola genomic DNA is described. Pantocin B is a novel peptidic sulfone that inhibits N-acetylornithine transaminase, an enzyme required for arginine biosynthesis. The success of these initial E. herbicola heterologous expression studies suggested that it would be feasible to study the natural products of previously uncultured microorganisms by shotgun cloning eDNA into E. coli.;Nucleic acid based studies of microbial communities indicate that only a small fraction of microorganisms is cultured using standard microbiological methods and the vast majority of microorganisms remain uncultured. To study the natural products produced by uncultured microorganisms, large fragments of DNA extracted directly from environmental samples (eDNA) were introduced into E. coli and these clones were screened for the production of small molecules. A sequential double antibiotic selection scheme was developed to rapidly identify and recover clones with “natural product-like phenotypes” (the production of color or antibiosis) from large eDNA libraries.;Violacein, a dark blue antibiotic, was characterized from a blue eDNA clone and four new families of long chain N-acyl amino acid antibiotics were characterized from seven independent antibacterial hits identified in the antibacterial screen. Heterologous expression directly couples natural products with their biosynthetic genes. Therefore, the characterization of these clones not only led to the identification of new long chain N-acyl amino acid natural products but also to the characterization of the first biosynthesis genes for long chain N-acyl amino acid natural products.