Polyketides are a group of secondary metabolite with a wide diversity of structures and bioactivities. Polyketide synthases (PKS) are classified as type I, II, and III. Type I PKS pathways typically contain a multidomain architecture and require large genetic regions (usually > 40 kb) for a complete biosynthetic pathway. We constructed a large-insert soil metagenomic clone library (~110 kb and 19,200 clones) from an agricultural soil (Cullars Rotation, Auburn, AL) using a broad host range shuttle BAC vector, pSmartBAC-S. A phylogenetic analysis of the metagenomic library, based on 16S rRNA gene sequences, indicates a diverse assemblage of microbial genomes representing nine bacterial phyla. A degenerate primer set targeting a conserved KS domain was used to PCR amplify this domain from a pooled BAC clone library. The KS domain amplicons were cloned and sequenced, resulting in 110 unique KS domain sequences that have a range of only 32.3% - 82.7% amino acid identity with known KS domains in GenBank. These unique KS domains were used to design probes and primers to identity BAC clones that contain a Type I PKS pathway. Next-Generation sequencing revealed complete or nearly complete Type I PKS pathways within the BAC clones, with limited homology to known PKS pathways from Streptomyces and other bacterial taxa. This study demonstrates the potential to obtain complete and novel PKS pathways from a large-insert soil metagenomic library. These pathways may be subsequently expressed in a heterologous host to identify novel polyketide products.
