, 2008). The lack of austinols can thus be linked directly to an AN8383-encoded function rather than to silencing of another gene caused by chromatin changes provoked by the AN8383 deletion. To confirm the role of AN8383 in austinol production, we constructed selleck compound a new strain that expresses the AN8383 ORF controlled by the inducible alcA promoter from the ectopic locus, IS1 (Hansen et al., 2011). On inductive medium, the subsequent LC-MS analysis showed a large novel peak eluting at ca. 6 min (see Fig. S9). The corresponding compound was

purified and the structure was elucidated by NMR (Fig. S10), identifying 3,5-dimethylorsellinic acid (20), which is in good agreement with the route of synthesis previously proposed for austinol (Fig. 6b; Geris & Simpson, 2009). In a parallel analysis using a strain expressing AN8383 under the control of the constitutive gpdA promoter we obtained the same result (data not shown). Together, the results strongly indicate that AN8383 encodes a PKS producing 3,5-dimethylorsellinic acid and that this compound serves as the first intermediate in the complex biosynthesis of austinol and dehydroaustinol that also involves a yet unidentified prenyl

transferase(s). Based on selleck inhibitor these results, we have named the locus AN8383, ausA. In the present study, we constructed a genome-wide PKS deletion library, which we screened for effects on polyketide production on a variety of media. This analysis has provided Flucloronide novel links between PKS genes and polyketide products demonstrating the strength of this approach. Many PKS genes still remain to be functionally connected to products, as many gene clusters have not yet been activated. As the repertoire of tools and methods to induce gene expression is rapidly increasing, new polyketide compounds will likely soon be uncovered in A. nidulans. To this end, the genome-wide PKS gene deletion

library presented here will undoubtedly serve as a useful resource. This work was supported by the Danish Research Agency for Technology and Production, grant # 09-064967. We thank Lisette Knoth-Nielsen for her dedicated and valuable technical assistance in the laboratory. In addition, we thank Rasmus John Normand Frandsen for suggestions and critical reading of the manuscript. Fig. S1. Eight known metabolites that have been linked to specific PKS genes in Aspergillus nidulans. Fig. S2. A graphical illustration of the procedure used to make the gene targeting fragments for the PKS deletions. Fig. S3. Procedure for diagnostic PCR. Fig. S4. Chromosome map showing the position of the 32 PKS genes. Fig. S5. Verification that the polyketide is absent in selected deletion mutants. Fig. S6. Positive mode extracted ion chromatograms for the mdpGΔ strain cultivated on RTO. Fig. S7. Additional polyketides that were detected in metabolite extracts in this study.