Supplementary Materials. Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like

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Supplementary Materials Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis Applied Microbiology and Biotechnology Hsu-Hua Yeh Yi-Ming Chiang Ruth Entwistle Manmeet Ahuja Kuan-Han Lee Kenneth S. Bruno Tung-Kung Wu Berl R. Oakley Clay C. C. Wang Corresponding author: Clay Wang, Pharmaceutical Sciences and Chemistry, Departments of Pharmacology and Pharmaceutical Sciences and Chemistry, University of Southern California. E-mail: clayw@usc.edu. S1

Table S1 Primers used for gene deletion in this study Primer AN3391.4P1 AN3391.4P2 AN3391.4P3 AN3391.4P4 AN3391.4P5 AN3391.4P6 AN3392.4P1 AN3392.4P2 AN3392.4P3 AN3392.4P4 AN3392.4P5 AN3392.4P6 AN3393.4P1 AN3393.4P2 AN3393.4P3 AN3393.4P4 AN3393.4P5 AN3393.4P6 AN3394.4P1 AN3394.4P2 AN3394.4P3 AN3394.4P4 AN3394.4P5 AN3394.4P6 AN3395.4P1 AN3395.4P2 AN3395.4P3 AN3395.4P4 AN3395.4P5 AN3395.4P6 AN3397.4P1 AN3397.4P2 AN3397.4P3 AN3397.4P4 AN3397.4P5 AN3397.4P6 AN3398.4P1 AN3398.4P2 AN3398.4P3 AN3398.4P4 AN3398.4P5 AN3398.4P6 AN3399.4P1 AN3399.4P2 AN3399.4P3 AN3399.4P4 AN3399.4P5 AN3399.4P6 AN3400.4P1 Sequence (5 3 ) AGA CAC GTC CAA AAG GCT GT GGC TTG ACT GGA GCT ATT GA CGA AGA GGG TGA AGA GCA TTG CCG AAC GTG GAG AGA TAA T GCA TCA GTG CCT CCT CTC AGA CAG AAG TGG TGC TGG TAT CTG G AGG GAC TGC CAT TGC GAA G CAA CTA CCA GTA TCC TCA GG GTG GTG CTG GTA TCT GGA TG ACA GGA AAC AGT GTG CAG C CGA AGA GGG TGA AGA GCA TTG ACG TGG GAG ATA CGA CTG C GCA TCA GTG CCT CCT CTC AGA CAG CCA AAT GCT GCT GTC ACA G CGT GTT TTC GTG CCG TAG T GCA CTG GTT GCT GAC AGG A GGC GCT AGA GCA GAT TCA AC CAC CTG CTG GTC CTT TGA TT CGA AGA GGG TGA AGA GCA TTG GAG TAA CTG GGA CTG ACT G GCA TCA GTG CCT CCT CTC AGA CAG GGC ACT GGT TGC TGA CAG G CTG TGA CTG AGG CGA TGA GT GTC CAA ATG CTG CTG TCA C GAA GTC GAT GCA TGT GCC T CAG TTG ATC ACG CAG AGG A CGA AGA GGG TGA AGA GCA TTG CGG GTG TCA AGA TCA ACA G GCA TCA GTG CCT CCT CTC AGA CAG GTG AAG CTG TTT CTT TAA GG GTA TCC CTT CCT GAA TGG G CTC AGC GCG ATT TCA TTG C CGG CAT AGT CAT AGG CCA T GCA CTG AAT TGT GAC GCA G CGA AGA GGG TGA AGA GCA TTG CTC GGA CCT GGG TGT GTC AA GCA TCA GTG CCT CCT CTC AGA CAG TCC ACT CTA CAT TCC TGC C CCT CAA CCA CTG AGC CTA C GCA CGT CAT CCT CTC GAG AA AGC TCT TGC GGA TTT GGA G ACC GCG AAC AAA ATG CTG C CGA AGA GGG TGA AGA GCA TTG AAG ACA GCT TTG TGG TGA AC GCA TCA GTG CCT CCT CTC AGA CAG GTC AAT CAA GAA AAC TGC CTT GTA CGG GGT GGA CCT A GTG GCT ATC TGA ACC ATC G CCT CGA TCA AGC CTG CTG A TAC AGA GCA CTC GAG CTG G CGA AGA GGG TGA AGA GCA TTG GCA AGG GAT AGA CGG GAG A GCA TCA GTG CCT CCT CTC AGA CAG CGA CGC TGA CCA GTA CGA T GAC TCT CGT CAC AGT GCA G GGA TGT GAT TGA TAC CTG GC GCA GTC TGG CCT CTG TTT C ACT TCA CCA GAC GGA ACG G CGA AGA GGG TGA AGA GCA TTG GTT GCA CAG ATT GGA AGA CG GCA TCA GTG CCT CCT CTC AGA CAG TGT CCA AAG TGA CGA CAA G GGA TCT TCA TCT GCT CTT CG CTA CCG CGT ACG CAA TCT C AAT AAC CCC CGA CTT TGA CC S2

AN3400.4P2 AN3400.4P3 AN3400.4P4 AN3400.4P5 AN3400.4P6 AN3401.4P1 AN3401.4P2 AN3401.4P3 AN3401.4P4 AN3401.4P5 AN3401.4P6 CGA GCA GAT ATG CCT CTT C CGA AGA GGG TGA AGA GCA TTG GAT ATG GCA AGA CTG ACG C GCA TCA GTG CCT CCT CTC AGA CAG CTG GCA CCA AGG CTG CAT A GCT CAA GTC AAG GAG ATG G ACC CAG GAG GCA TCA GTA C ATA TCC CGG CAA ATG TCC G GAA ATT GTC AGC CGC TCG G CGA AGA GGG TGA AGA GCA TTG TGA AAG ATT GAG CGG GGT CG GCA TCA GTG CCT CCT CTC AGA CAG CGT CGT CTT TTC GAG CTA C GGA TGG TGG ACT CGT TCA C ATC GCA AGC ATC TCG GCT C The underlined sequences are tails that anneal to the A. fumigatus pyrg (AfpyrG) fragment during fusion PCR S3

Table S2 Gene deletion strains used in this study Strain Secondary metabolite mutations Genotype CW3191, CW3192 stcjδ, alca(p)-an3396.4, AN3391.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3391.4::AfpyrG CW3126, CW3127, CW3128 stcjδ, alca(p)-an3396.4, AN3392.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3392.4::AfpyrG CW3136, CW3137, CW3138 stcjδ, alca(p)-an3396.4, AN3393.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3393.4::AfpyrG CW3141, CW3142, CW3143 stcjδ, alca(p)-an3396.4, AN3394.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3394.4::AfpyrG CW3146, CW3147, CW3148 stcjδ, alca(p)-an3396.4, AN3395.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3395.4::AfpyrG CW3151, CW3152, CW3153 stcjδ, alca(p)-an3396.4, AN3397.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3397.4::AfpyrG CW3156, CW3157, CW3158 stcjδ, alca(p)-an3396.4, AN3398.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3398.4::AfpyrG CW3161, CW3162, CW3163 stcjδ, alca(p)-an3396.4, AN3399.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3399.4::AfpyrG CW3166, CW3167, CW3168 stcjδ, alca(p)-an3396.4, AN3400.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3400.4::AfpyrG CW3186, CW3188 stcjδ, alca(p)-an3396.4, AN3401.4Δ pyrg89; pyroa4, nkua::argb; ribob2, stcj::afribob; AN3396.4::AfpyroA-alcA(p)-AN3396.4; AN3401.4::AfpyrG S4

S5

Fig. S1 Results of diagnostic PCR for the alca promoter replacement strains and WT (LO2026). PCR fragments were amplified with two primers, P1 from the chromosomal region just outside of 5 flank of the transforming DNA fragment and P6 from just outside of the 3 flank. Strain numbers labeled in red are correct transformations S6

Fig. S2 HRESIMS spectrum of Microperfuranone (1) S7

Fig. S3 1 H NMR spectrum of microperfuranone (1) in CDCl 3 S8

Fig. S4 1 H NMR spectrum of microperfuranone (1) in acetone-d 6 S9

Fig. S5 13 C NMR spectrum of microperfuranone (1) in CDCl 3 S10

Fig. S6 13 C NMR spectrum of microperfuranone (1) in acetone-d 6 S11

Fig. S7 RT-PCR analysis of selected genes. RNA samples were isolated from culture of LO2026 (WT) and mutant (CW3023) strains after 48 hr of cyclopentanone induction. -tubulin was used as a control S12

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