Polyketide Synthase Pathway Discovery from Soil Metagenomic Libraries
Type of Degreethesis
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Polyketides are structurally diverse bacterial secondary metabolites, many of which have antibiotic or anti-cancer activity. Modular polyketide synthase (PKS) enzymatic complexes contain conserved ketoacyl synthase (KS) domains, and most PKS biosynthetic pathways exceed 30 kb in size. A fosmid metagenomic library constructed from soil at the Hancock Agricultural Research Station in Hancock, WI, (18,432 clones, average insert of 42 kb) was spotted onto a nylon membrane. The macroarray was screened using a degenerate DNA probe targeting the KS domain. Thirty-four clones containing KS domains were identified by Southern hybridization; however, only 21 of the 34 PKS-positive clones produced a PCR product. Interestingly, most of the clones (8 out of 13) that were PCR-negative were nontheless KS-positive by Southern blot hybridization. DNA sequences from a KS-containing clone that was consistently PCR-negative have revealed a biosynthetic pathway that is divergent from known pathways, and is hypothesized to have an origin from the newly described bacterial division Acidobacteria that is prevalent within soils yet has few cultured representatives.
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