(A) Organization of the asperfuranone synthase gene cluster in A. nidulans. Each arrow indicates the direction of transcription and relative sizes of the ORFs deduced from analysis of the nucleotide sequences. Black ORF’s, AN1029.3 (transcriptional activator), AN1031.3 (efflux pump), AN1032.3 (oxydoreductase), AN1033.3 (salicylate hydroxylase), AN1034.3 (NR-PKS), AN1035.3 (oxygenase/oxidase), AN1036.3 (HR-PKS) are required for asperfuranone biosynthesis. Grey ORF’s are genes not involved in asperthecin biosynthesis according to our deletion analysis. (B) Analysis of the effects on asperfuranone production of deletions of genes surrounding the asperfuranone PKSs. Positive mode extracted ion chromatograms (EIC) at m/z 333 of AN1027.3Δ (a), AN1028.3Δ (b), AN1030.3Δ (c), AN1031.3Δ (d), AN1032.3Δ (e), AN1033.3Δ (f), AN1034.3Δ (g), AN1035.3Δ (h), AN11287.3Δ (i), AN1036.3Δ (j), AN1037.3Δ (k), AN11288.3Δ (l). The Y-axis of each profile was at the same order of magnitude. (C) Detailed analysis of the effects on asperfuranone production of deletions of genes surrounding the asperfuranone PKSs. UV at 254 nm of AN1031.3Δ (a), AN1032.3Δ (b), AN1033.3Δ (c), AN1034.3Δ (d), AN1035.3Δ (e), AN11287.3Δ (f), AN1036.3Δ (g). The Y-axis of each profile was at the same order of magnitude. (D) Hemiacetal formation of 3 and 4 from compound 2.