Intact Small Molecule Pathways from Large Insert Soil Metagenomic BACs

Soil microorganisms express diverse bioactive natural products, but the majority are inaccessible due uncultivable issues. Novel Type I polyketide synthase (PKS) pathways were discovered by cloning high molecular weight metagenomic DNA from soil into a bacterial artificial chromosome (BAC) vector. Phylogenetic analysis of the metagenomic library, based on 16S rRNA gene sequences, indicates a diverse assemblage of microbial genomes representing nine bacterial phyla. Functional screening for growth inhibition of methicillin-resistant Staphyloccocus aureus (MRSA) yielded 32 anti-MRSA clones, none of which contained recognizable PKS pathways. PCR amplification using degenerate PKS primers from a pool of all 19,200 BACs identified 110 unique KS domain amplicons by sequence analysis, which had a broad range of homology (35-85% similarity) with known KS domains. The PCR amplicons were used as probes to hybridize against a library macroarray, resulting in the discovery of 12 clones, 11 of which contain recognizable but novel PKS pathways. Screening the library manually using single well PCR and gel-based band detection to find additional PKS clones resulted in 500 positive hits, a 2.6% discovery rate. Sequencing and annotation of these BAC clones reveal ~110 kb inserts with numerous novel genes predicted to be involved in various biosynthetic pathways as well as many with little significant GenBank similarity. To better assess the PKS biochemical diversity a new E. coli strain engineered to support secondary metabolite synthesis has been developed. Data showing the diversity of the pathways and preliminary results for bioactivity and structure of metabolites expressed from large-insert clones will be presented.