Molecular and biochemical characterization of the synthesis of the polyketides melanin and cercosporin in Cercospora kikuchii

This research has been a molecular genetic and biochemical analysis of the biosynthesis of the polyketides melanin and cercosporin from the plant pathogenic fungus Cercospora kikuchii. Degenerate PCR primers designed to a highly conserved region of polyketide synthase (PKS) genes were used to amplify a product from C. kikuchii cDNA. That 280 bp product was sequenced and identified as an acyl transferase (AT) domain of a polyketide synthase. The product was cloned and used to screen a cosmid library. A single cosmid which hybridized to the product was isolated and a larger fragment of the PKS subcloned. This 1.6 kb fragment contained motifs for the AT domain as well as a ;In other experiments a thin layer chromatographic method was developed for the separation of cercosporin from other organic-extractable compounds, including biosynthetic intermediates. Two compounds were discovered which demonstrate the accumulation patterns expected from cercosporin biosynthetic intermediates. This technique was used to resolve cercosporin in fungal strains which contained a non-functional cercosporin transporter, CFP as well as those containing an antisense-mediated down-regulation of the cfp gene. The effects of the disruption of CFP upon other genes with enhanced transcription when cultures are grown in the light were also assayed. Finally, the cosmid vector pBC4 which contains the bar selectable marker, providing transformed fungi resistance to the herbicide bialaphos, was constructed. This was introduced into C. kikuchii using a protoplast fusion transformation system.