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1 Supporting Information Engineering of an unnatural natural product by swapping polyketide synthase domains in Aspergillus nidulans Ting Liu, Yi-Ming Chiang, Amber D. Somoza, Berl R. Oakley, and Clay C. C. Wang * Table of Contents Supplemental Methods S2 Supplemental References S5 Table S1. A. nidulans strains used in this study S6 Table S2. Primers used in this study S7 Table S3. 1 H and 13 C NMR data for compound 1 S8 Figure S1. HPLC profiles of extracts of the stca strain S9 Figure S2. Protein sequence homology alignment of PksA and StcA S10 Figure S3. HPLC profiles of extracts of the stca, hybrid (A1-C3) strains S11 Figure S4. UV-Vis and ESIMS spectra of compound 1 S12 Figure S5. HMBC correlations of compound 1 S12 Figure S6. Results of feeding studies in the stca, hybrid B2, stck stcj strain S13 1 H NMR spectrum of compound 1 S14 13 C NMR spectrum of compound 1 S15 S1

2 Supplemental Methods Fungal strains and molecular genetic manipulations A. nidulans strains used in this study are listed in Table S1. All primers used in this study are listed in Table S2. stca was deleted by replacing it with an A. fumigatus pyroa gene cassette (AfpyroA) in TN02A7 1. Deletion of a chromosomal region including two adjacent genes, stcj and stck, was generated by replacing this region with an A. fumigatus ribob gene cassette (AfriboB) in CW1030. Double mutant strains [stca, hybrid (A1-C3)] were generated from CW1001. The AfoE SAT plus a 120bp promoter region immediately upstream of the afoe start codon was replaced with a cassette containing 1) the A. fumigatus pyrg gene (AfpyrG) followed by 2) a 401bp fragment containing the A. nidulans alca promoter [alca(p)] followed by 3) an stca SAT fragment that started from the start codon and ended at the selected swapping sites A/B/C, such that the hybrid coding sequence was placed under the control of the alca promoter. Two ~1kb fragments upstream and downstream of each targeted DNA region were amplified from A. nidulans genomic DNA by PCR and fused together with the replacement cassette by fusion PCR. 2 Protoplast production and transformation were carried out as described. 2 Three to five transformants for each genotype were analyzed by diagnostic PCR with three primer sets. In the case that the external primers in the first round of PCR were used, the difference in the sizes of targeted DNA region before and after replacement allowed the determination of correct gene replacement. In the cases that one of the external primers and the primer located inside the cassette were used, the correct mutant gave the PCR product of the expected size, otherwise no product was present. Synthesis of hexanoyl-snac The synthesis was carried out using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling as described by Boddy et al. 3 To a solution of hexanoic acid in anhydrous dichloromethane was added EDC (2 eq.) at 0 C under nitrogen. After stirring for 20 min, N-acetylcysteamine (1.2 eq.) a few crystals of DMAP was added to the solution, warmed to room temperature and stirred for additional 3 h. The reaction mixture was quenched with water, and then extracted with ethyl acetate. Organic layers were washed with brine, dried over anhydrous Na 2 SO 4, concentrated, and purified by flash chromatography (40% acetone/n-hexanes). Hexanoic acid d-11 (98%, purchased from Cambridge Isotope Laboratories) was used as the precursor of D 11 -hexanoyl-snac. The respective yield of hexanoyl-snac and D 11 -hexanoyl-snac was 98% and 77%. Fermentation and LC-MS analysis 2.5x10 7 spores of each A. nidulans strain were grown at 37 C, shaking at 200 rpm in 25 ml S2

3 liquid LMM medium 4 supplemented when necessary with uracil (1 g/l) and uridine (10 mm) and/or riboflavin (2.5 mg/l). For alca(p)-inducing conditions, cyclopentanone at a final concentration of 30 mm was added to the medium after 18 h of incubation. For feeding studies, 4 aliquots of inoculated and induced CW1061 were respectively fed with 7 µl DMSO, 7 µl 0.2 mm hexanoyl-snac, 7 µl 0.2 mm D 11 -hexanoyl-snac, 7 µl 0.2 mm 1:1 hexanoyl-snac: D 11 -hexanoyl-snac successively at 20 h, 38 h and 56 h. Culture medium was collected at 66 h by filtration and extracted once with 25 ml of EtOAc. The EtOAc layer was evaporated in vacuo, redissolved in 0.35 ml of 20% DMSO/MeOH, and 10 µl was injected for HPLC-DAD-MS analysis. HPLC-MS analysis was carried out in the negative mode using an Agilent Technologies 1200 series high-resolution mass spectrometer with an RP C 18 column (Alltech Prevail C18 3 µm mm) at a flow rate of 125 µl/min. The solvent gradient for HPLC was 95% MeCN/H 2 O (solvent B) in 5% MeCN/H2O (solvent A), both containing 0.05% formic acid: 0% B from 0 to 5 min, 0 to 100% B from 5 to 35 min, maintained at 100% B from 35 to 40 min, 100 to 0% B from 40 to 45 min, and re-equilibration with 0% B from 45 to 50 min. Conditions for MS included a capillary voltage at 4000 V, a nebulizer pressure at 20 psig, a drying gas flow rate at 10 L/min, and the drying gas temperature at 350 C. Isolation and identification of secondary metabolites For structure elucidation, strain CW1030 was cultivated in 90 aliquots of 30 ml liquid LMM medium at 37 C with shaking at 200 rpm for 66 h and induced with cyclopentanone at 18 h. The culture medium was collected through filtration, extracted twice with an equal volume of EtOAc, and evaporated as described above. The crude extract (449.5 mg) was applied to a Merck Si gel column (230 to 400 mesh; ASTM) and eluted with CHCl 3 /MeOH mixtures of increasing polarity (fraction A, 1:0; fraction B, 49:1; fraction C, 19:1; fraction D, 9:1; and fraction E, 7:3). Fraction C (42.1 mg) was further purified by reverse-phase HPLC with a Phenomenex Luna C 18 column (5-µm particle size; 250 by 21.2 mm) at a flow rate of 10.0 ml/min and measured by a UV detector at 254 nm. The gradient system was MeCN (solvent B) in 5% MeCN/H2O (solvent A), both containing 0.05% trifluoroacetic acid, as follows: equilibration with 20% solvent B from 0 to 5 min, 20 to 80% solvent B from 5 to 30 min, 80 to 100% solvent B from 30 to 33 min, 100% solvent B from 33 to 38 min, 100 to 20% solvent B from 38 to 41 min, and reequilibration with 20% solvent B from 41 to 45 min. Compound 1 (2.2 mg) was eluted at 22.7 min. Compound Identification Optical rotation was measured on a JASCO P-2000 digital polarimeter. Infrared (IR) spectra were recorded on a Nicolet MAGNA-IR 560 spectrometer. 1 H and 13 C nuclear S3

4 magnetic resonance (NMR) spectra were collected on a Varian Mercury Plus 400 spectrometer. Compound 1: yellow solid; [α] 23 D (MeOH, c 0.2); IR (ZnSe) cm , 1653, 1570, 1380, 1340, 1209, 1138, 1074, 977; For UV and ESI-MS data, see Figure S4; For 1 H and 13 C NMR data, see Table S3. S4

5 Supplemental References (1) Nayak, T.; Szewczyk, E.; Oakley, C. E.; Osmani, A.; Ukil, L.; Murray, S. L.; Hynes, M. J.; Osmani, S. A.; Oakley, B. R. Genetics 2006, 172, (2) Szewczyk, E.; Nayak, T.; Oakley, C. E.; Edgerton, H.; Xiong, Y.; Taheri-Talesh, N.; Osmani, S. A.; Oakley, B. R. Nat. Protoc. 2006, 1, (3) Sharma, K. K.; Boddy, C. N. Bioorg. Med. Chem. Lett. 2007, 17, (4) Bok, J. W.; Chiang, Y.-M.; Szewczyk, E.; Reyes-Domingez, Y.; Davidson, A. D.; Sanchez, J. F.; Lo, H.-C.; Watanabe, K.; Strauss, J.; Oakley, B. R.; Wang, C. C. C.; Keller, N. P. Nat. Chem. Biol. 2009, 5, S5

6 Table S1. A. nidulans strains used in this study strain related mutation(s) genotype TN02A7 None pyrg89; pyroa4; nkua::argb; rbiob2 CW1001 to stca pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa CW1005 CW1006 to CW1010 stca ; hybrid A1 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)1::AfpyrG-alcA(p)-SAT(stcA)A CW1011 to CW1015 stca ; hybrid A2 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)2::AfpyrG-alcA(p)-SAT(stcA)A CW1016 to CW1020 stca ; hybrid A3 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)3::AfpyrG-alcA(p)-SAT(stcA)A CW1021 to CW1025 stca ; hybrid B1 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)1::AfpyrG-alcA(p)-SAT(stcA)B CW1026 to CW1030 stca ; hybrid B2 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)2::AfpyrG-alcA(p)-SAT(stcA)B CW1031 to CW1035 stca ; hybrid B3 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)3::AfpyrG-alcA(p)-SAT(stcA)B CW1036 to CW1040 stca ; hybrid C1 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)1::AfpyrG-alcA(p)-SAT(stcA)C CW1041 to CW1045 stca ; hybrid C2 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)2::AfpyrG-alcA(p)-SAT(stcA)C CW1046 to CW1050 stca ; hybrid C3 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)3::AfpyrG-alcA(p)-SAT(stcA)C stca ; hybrid B2; stcj stck CW1057 to CW1061 pyrg89; pyroa4; nkua::argb;rbiob2; stca::afpyroa; SAT(afoE)2::AfpyrG-alcA(p)-SAT(stcA)B; stcjstck::afribob S6

7 Table S2. Primers used in this study. primer sequence (5 3 ) AN7825.1P1 TCCGCCGTTCTAGCATAAG AN7825.1P2 CGTAAGCTGGCAGACTATTTC AN7825.1P3 CGAAGAGGGTGAAGAGCATTG TGCGAAAATAGTATGGTCTG AN7825.1P4 GCATCAGTGCCTCCTCTCAGACAG TGGCAACGACTGAATATGAC AN7825.1P5 GGTAAACGATTGGCCTATTC AN7825.1P6 GGCAGAAAGGATGGTATTGA alca-an7825.1f1 ATCCTATCACCTCGCCTCAAA ATGGCCAGTCACGCTGAGC alca-an7825.1r1 CCGCCCGTGATAAAGTAGGTG pyrgf3 GAGTTATTCTGTGTCTGACG AN7825SAT.PA CTTTATTCCTTCGGTAACCAG AN7825SAT.PB GGTCGGAGGTTCTGCTGG AN7825SAT.PC GTCAGACCCTGGACGGTGG SATSWAP.P1 GCAAGTACGCCATCTTCGAT SATSWAP.P2 GCTTGCCCTGAAGACTTACA SATSWAP.P3 CGAAGAGGGTGAAGAGCATTG AGCGAGCGGGTCAAGAG SATSWAP.P4A1 CTGGTTACCGAAGGAATAAAG GAAGACCTTCCCAAAGACC SATSWAP.P4A2 CTGGTTACCGAAGGAATAAAG CAGCTTCAACCGGTCCTGC SATSWAP.P4A3 CTGGTTACCGAAGGAATAAAG CGCGTCGAGCCAGTCATG SATSWAP.P4B1 CCAGCAGAACCTCCGACC GAAGACCTTCCCAAAGACC SATSWAP.P4B2 CCAGCAGAACCTCCGACC CAGCTTCAACCGGTCCTGC SATSWAP.P4B3 CCAGCAGAACCTCCGACC CGCGTCGAGCCAGTCATG SATSWAP.P4C1 CCACCGTCCAGGGTCTGAC GAAGACCTTCCCAAAGACC SATSWAP.P4C2 CCACCGTCCAGGGTCTGAC CAGCTTCAACCGGTCCTGC SATSWAP.P4C3 CCACCGTCCAGGGTCTGAC CGCGTCGAGCCAGTCATG SATSWAP.P5 CGTCGTTAGCAGTGACCTTG SATSWAP.P6.1 CCCTCTGTATGCCCAATATG SATSWAP.P6.2 CGCCCTCTGTATGCCCAATA SATSWAP.P6.3 CCCCTCGCGCATCATCAT AN7814.1AN7815.1P1 GCCATCTCACACGTTCTC AN7814.1AN7815.1P2 AAGCGACAAGAGAACTACAC AN7814.1AN7815.1P3 CGAAGAGGGTGAAGAGCATTG TGAAATGGAAGGTACACGTA AN7814.1AN7815.1P4 GCATCAGTGCCTCCTCTCAGACAG CCGCCAAAATGATTACCTAA AN7814.1AN7815.1P5 TCATACCAGGAGCTGATACTA AN7814.1AN7815.1P6 AGGCACTCTTCTTCGGAAAT Blue and red sequences are tails that anneal to the A. fumigatus pyroa (AfpyroA) fragment during fusion PCR. Green sequence is a tail that anneals to the alca promoter fragment during fusion PCR. S7

8 17 O OH 16 HO O 7 1 OH Table S3. NMR data for compound 1 (400 and 100 MHz) in CD 3 OD. position δ C δ H (d) 8.06 (1H, s) (s) (s) (s) (s) (s) (s) (d) 7.36 (1H, s) (s) (s) (d) 5.02 (1H, dd, 4.4, 3.2) (t) 1.66, 1.74 (each 1H, m) (t) (t) (t) (q) 0.89 (3H, t, 6.4) (q) 1.99 (3H, s) S8

9 Figure S1. HPLC profiles of extracts of A. nidulans wild-type strain (a) and stca mutant (b) under noninducing (A) and inducing (B) conditions as detected by UV absorption at 254 nm. Wild type produces terrequinone (TQ) as well as sterigmatocystin (ST). The y axis of each profile was at the same order of magnitude. *: metabolites that are non-specific to this study. S9

10 Figure S2. Protein sequence homology alignment of PksA and StcA (only the first 400 amino acids are shown). *: PksA SAT domain cloning sites, which correspond to K357, T363 and D379 on StcA. S10

11 Figure S3. HPLC profiles of extracts of the stca, hybrid (A1-C3) strains. S11

12 Figure S4. UV-Vis and HRESIMS spectra (negative mode) of compound 1. Figure S5. Key HMBC correlations of compound 1. S12

13 Figure S6. High-resolution MS spectra (negative mode) of hybrid AfoE-derived metabolites (a-i) detected by respectively feeding an equal volume of (A) DMSO, (B) 0.2 mm hexanoyl-snac, (C) 0.2 mm D 11 -hexanoyl-snac, (D) 0.2mM 1:1 hexanoyl-snac: D 11 -hexanoyl-snac to the stca, hybrid B2, stck stcj strain. Deuterium-incorporated forms are highlighted in blue and non-incorporated forms are highlighted in pink. The y axis (relative abundance) of (a), which is compound 1, is normalized to 1 based on abundance of highlighted peak in B(a). The y axis of (b)- (e) and (f)- (i) is respectively at 10 and 20 magnitude of that of (a). S13

14 S14

15 S15