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Table of contents Page

Experimental Procedures: Strains and media S3 Gene knock-out in Calcarisporium arbuscula S3 RNA preparation and reverse transcription PCR (RT-PCR) S3 Chemical analysis and characterization of compounds S4 Purification of aurovertin B (1) and D (2) from C. arbuscula wild type S4

Purification of compounds 3-12 from the hdaA mutant S4 Structure characterization of 3-4 S5 Supplementary Tables: Table S1. Summary of polyketide synthase (PKS) S6 Table S2. Summary of non-ribosomal peptide synthase (NRPS) S7 Table S3. Genes involving terpene synthesis S8 Table S4. A domain signatures in contig 2507 NRPS S9 Table S5. A domain signatures in contig 453 NRPS S10 Table S6. Primers for gene knock-out in C. arbuscula S11 Table S7. RT-PCR primers for polyketide synthases (PKS) S12 Table S8. RT-PCR primers for non-ribosomal peptide synthases (NRPS) S13 Table S9. RT-PCR primers of genes for terpene synthesis S14 Table S10. RT-PCR primers for hdaA and house-keeping genes S15 Table S11. NMR data of arbumycin (3) in CDCl3 S16 Table S12. NMR data of arbumelin (4) in CD3OD S17 Table S13. NMR data of arbumelin (4) in DMSO-d6 S18 Table S14. NMR data of arbusculic acid A (11) in DMSO-d6 S19 Table S15. NMR data of arbusculic acid B (12) in CD3OD S20 Supplementary Figures: Figure S1. Gene cluster comparison for the biosynthesis of sterigmatocystin (6) S21 Figure S2. Gene cluster comparison for the biosynthesis of citrinin S22 Figure S3. Deletion of hdaA gene in Calcarisporium arbuscula S23

Figure S4. Phenotype of C. arbuscula wild type and the hdaA mutant S24 Figure S5. RT-PCR analysis of gene expression for PKS, NRPS and terpene synthesis S25 Figure S6. Crystal structure of 3 S26 Figure S7. Deletion of arbA in Calcarisporium arbuscula S27 Figure S8. Gene cluster of contig 453 for the possible biosynthesis of arbumelin (4) S28 Figure S9. Gene cluster of contig 1462 for the biosynthesis of verlamelin (5) S29 Figure S10. Gene cluster of contig 1936 for three diterpene biosynthesis (9, 10, 11) and structure analysis of the terpene cyclase S30 Figure S11. 1H NMR of aurovertin B (1) in CDCl3 (500 MHz) S31 Figure S12. 13C NMR of aurovertin B (1) in CDCl3 (125 MHz) S32 Figure S13. 1H NMR of arbumycin (3) in CDCl3 (500 MHz) S33 Figure S14. 13C NMR of arbumycin (3) in CDCl3 (125 MHz) S34 Figure S15. 1H 1H-COSY spectrum of arbumycin (3) in CDCl3 S35 Figure S16. HSQC of arbumycin (3) in CDCl3 S36 Figure S17. HMBC of arbumycin (3) in CDCl3 S37 Figure S18. NOSEY of arbumycin (3) in CDCl3 S38 Figure S19. 1H NMR of arbumelin (4) in CD3OD (500 MHz) S39 Figure S20. HSQC-TOCSY of arbumelin (4) in CD3OD (500 MHz) S40

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Figure S21. HSQC of arbumelin (4) in CD3OD S41 Figure S22. HMBC of arbumelin (4) in CD3OD S42 Figure S23. 1H NMR of arbumelin (4) in DMSO-d6 (500 MHz) S43 Figure S24. 1H 1H-COSY spectrum of arbumelin (4) in DMSO-d6 S44 Figure S25. HSQC of arbumelin (4) in DMSO-d6 S45 Figure S26. HMBC of arbumelin (4) in DMSO-d6 S46 Figure S27. NOSEY of arbumelin (4) in DMSO-d6 S47 Figure S28. 1H NMR of verlamelin (5) in CD3OD (500MHz) S48 Figure S29. 13C NMR of verlamelin (5) in CD3OD (125 MHz) S49 Figure S30. 1H 1H-COSY spectrum of verlamelin (5) in CD3OD S50 Figure S31. HSQC of verlamelin (5) in CD3OD S51 Figure S32. HMBC of verlamelin (5) in CD3OD S52 Figure S33. 1H NMR of paeciloquinone B (8) in DMSO-d6 (500MHz) S53 Figure S34. 13C NMR of paeciloquinone B (8) in DMSO-d6 (125 MHz) S54 Figure S35. 1H NMR of zythiostromic acid A (9) in CD3OD (500MHz) S55 Figure S36. 13C NMR of zythiostromic acid A (9) in CD3OD (125 MHz) S56 Figure S37. 1H NMR of zythiostromic acid B (10) in CD3OD (500MHz) S57 Figure S38. 13C NMR of zythiostromic acid B (10) in CD3OD (125 MHz) S58 Figure S39. 1H NMR of arbusculic acid A (11) in DMSO-d6 (500 MHz) S59 Figure S40. 13C NMR of arbusculic acid A (11) in DMSO-d6 (125 MHz) S60 Figure S41. 1H 1H-COSY NMR of arbusculic acid A (11) in DMSO-d6 S61 Figure S42. HSQC of arbusculic acid A (11) in DMSO-d6 S62 Figure S43. HMBC of arbusculic acid A (11) in DMSO-d6 S63 Figure S44. NOESY of arbusculic acid A (11) in DMSO-d6 S64 Figure S45. 1H NMR of arbusculic acid B (12) in CD3OD (500MHz) S65 Figure S46. 13C NMR of of arbusculic acid B (12) in CD3OD (125 MHz) S66 Figure S47. 1H 1H-COSY spectrum arbusculic acid B (12) in CD3OD S67 Figure S48. HSQC of arbusculic acid B (12) in CD3OD S68 Figure S49. HMBC of arbusculic acid B (12) in CD3OD S69 Supplementary Schemes: Scheme S1: A proposed pathway for the biosynthesis of arbumelin (4) S70 Scheme S2: Proposed pathways for the biosynthesis of sterigmatocystin (6), paeciloquinone A (7) and B (8) S71 Supplementary References S72

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Experimental procedures: Strains and media

Fungus Calcarisporium arbuscula NRRL 3705 (ATCC 46034) is the wild type strain. The

hdaA and arbA mutants were constructed by replacement of hdaA and arbA by hph (Hygromycin-resistance gene), respectively. Spores of C. arbuscula were collected after 6 days on potato dextrose agar (PDA, BD). PDA was routinely used for production of compounds from C. arbuscula. Other media MEPA (3 % malt extract, 0.3 % soy flour, 2 % agar), Czapek yeast agar (CYA) (3 % sucrose, 0.5 % yeast extract, 0.1 % K2HPO4, 0.01 % (V/V) Czapek concentrate, 2 % agar), YMEG (0.4 % glucose, 0.4 % yeast extract, 1 % malt extract, 0.2 % CaCO3, 2 % agar), YG (0.5 % yeast extract, 2 % glucose, 0.04 % (V/V) trace element) were also tested for compound productivity. Gene knock-out in Calcarisporium arbuscula:

Primers for gene knock-out were listed in Table S6. arbA and hdaA genes were deleted in C. arbuscula according to the hygromycin

split-marker strategy.[1] Hygromycin-resistance gene hph upstream and downstream fragments were amplified from plasmid pAN-7 (Addgene) with primers hph-up F, hph-up R and hph-dn F, hph-dn R, respectively, and digested with NotI/SacII and SacI/NotI to insert into the self-ligated T-vector pTA2 (Toyobo) to create plasmid pXM01 and pXM02, respectively. The primers for upstream and downstream homologous fragments are: arbA-up F, arbA-up R and arbA-dn F, arbA-dn R for arbA gene, hdaA-up F, hdaA-up R and hdaA-dn F, hdaA-dn R for hdaA gene. The amplified upstream and downstream fragments were ligated into pXM01 or pXM02, respectively, after enzyme digestion: KpnI/NotI for arbA-up, BamHI/NotI for hdaA-up, NotI/KpnI for arbA-down and NotI/XbaI for hdaA-down.

Split-marker DNA was introduced into C. arbuscula by protoplast transformation. All the deletion cassettes were amplified with universal primers M13-F and M13-R from the above plasmids. All PCR products were precipitated with ethanol and dissolved in STC buffer (1.2 M sorbitol, 10 mM CaC12, 10 mM Tris-HCI, pH 7.5). C. arbuscula spores were collected on PDA

(potato dextrose agar, Fluka) for 6 days at 25C, and induced to young germ tubes in PDB

(potato dextrose broth, Fluka) at 25C for 7 hours with 180 rpm agitation. Cells were collected, washed twice with osmotic medium (1.2 M MgCl2, 10 mM sodium phosphate, pH 5.8 ) and resuspended in the enzyme cocktail solution (3 mg/ml Lysing Enzymes, 3 mg/ml

Yatalase in osmotic medium) at 30C for 4 hours. After twice wash with STC buffer, 50 l of

protoplasts were gently mixed with 50 l of DNA and incubated for 1 hour on ice. 300 l of PEG 4000 solution (60% PEG 4000, 50 mM CaC12, 50 mM Tris-HCI, pH 7.5) was added for 100

l protoplast mixture, incubated at room temperature for 30 min and plated on the

regeneration selection medium (PDA, 1.2 M sorbitol, 250 g/ml hygromycin B). After incubation at room temperature for about 2 weeks, the transformants were inoculated in PDB medium with stationary incubation for about 1 week.

Mycelia were collected, lyophilized and grounded for cell disruption. Cell lysate was solubilized in LETS buffer (10 mM Tris-HCI, pH 8.0, 20 mM EDTA, 0.5% SDS, 0.1 M LiCl) and extracted twice with phenol/chloroform. Genomic DNA was precipitated with ethanol, and resuspended in TE buffer. The genotypes of all mutants were verified by PCR. RNA preparation and reverse transcription PCR (RT-PCR):

C. arbuscula wild type and the hdaA mutant were grown on solid PDA media (BD) at room temperature for 4 weeks. Mycelia were collected and RNA was prepared by the hot

acid phenol method with minor modifications.[2] Briefly, mycelia were resuspended in 500 l of TES buffer (50 mM Tris-HCl, pH 7.2, 10 mM EDTA, 1% SDS) with 100 mM

-mercaptoethanol and 500 l of acid phenol (phenol/chloroform/isopentanol, pH 6.2,

Ambion). The extraction mixture was incubated at 65C for 1 hour with vortex every 15 min,

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and centrifuged at 15000 rpm for 15 min to save the aqueous phase. The supernatant was extracted twice again with the acid phenol. RNA was precipitated with ethanol and resuspended in RNase-free water. Genomic DNA was further removed by digestion with RNase-free DNase I (Ambion). RNA was purified by acid phenol extraction and ethanol precipitation. RNA integrity was confirmed by electrophoresis on TBE agarose gel, and concentration was determined by Nanodrop (Thermo Scientific).

cDNA was prepared from 500 ng of total RNA by SuperScript® II Reverse Transcriptase (Invitrogen) with random primers as described by the manufacture. PCR was performed with GoTaq® Green Master Mix (Promega) with 2 ng of reverse transcribed RNA. Primers and PCR cycles were listed in Table S7-S10. Chemical analysis and characterization of compounds:

LC-MS was conducted with a Shimadzu 2010 EV liquid chromatography mass spectrometer by using both positive and negative electrospray ionization and a Phenomenex Luna 5 μm, 2.0 mm × 100 mm C18 reverse-phase column. Samples were dissolved in methanol before separated on a linear gradient of 5% to 99% CH3CN (v/v) in H2O at a flow rate of 0.1 mL/min. 1H, 13C and 2D NMR spectra were obtained using CDCl3 or DMSO-d6 as solvent on a Bruker AV500 spectrometer with a 5 mm dual cryoprobe at the UCLA Molecular Instrumentation Center. Purification of aurovertin B (1) and D (2) from C. arbuscula wild type:

C. arbuscula was cultivated in PDB (6 L) at 25 C for 10 d. The culture was filtered through cheesecloth. The mycelia were extracted with acetone for three times. The combined extracts were evaporated to dryness under reduced pressure to afford the residue (15.6 g).The residue was dissolved in H2O (1 L) to form a suspension, followed by extraction with CHCl3 (4 × 500 mL). The combined organics were dried over MgSO4. The crude products (6.0 g) were separated by a RP-18 column (RediSep Rf Gold C18 Column, 20‒40 μ, 86 g,

MeCN-H2O 2:85:5) to afford four fractions (Fr.1−4). Fr.1 was purified by flash

chromatography (MeCN-H2O 1:93:7) to provide aurovertins D (2) (33 mg). Aurovertin B (1) (110 mg) was obtained from Fr. 3 by recrystallization with MeCN-H2O (9:1).

Purification of compounds 3-12 from the hdaA mutant:

C. arbuscula hdaA mutant were grown on 6 liter of solid PDA media (BD) divided into

60 large 15015 mm2 Petri dishes at room temperature for 30 days. The cultures were extracted with 1.0 liter of acetyl acetate and evaporated to dryness (1.5 g). The dried extract was partitioned between hexane and methanol. The MeOH fraction was evaporated to dryness (1.0 g), and purified by ISCO-CombiFlash® Rf 200 (Teledyne Isco, Inc) with a gradient of H2O and CH3CN (linear gradient of 20% to 80% CH3CN over 50 min at 20 mL/min on 100 g reversed phase silica gel (C18) to give 17 fractions (Fr.1~ Fr.17). Fraction Fr.10 was purified by Sephadex LH-20 eluted with MeOH to give compound 8 (4.0 mg). Fraction Fr.11 was purified by Sephadex LH-20 eluted with MeOH to give 14 subfractions (Fr.11-1~ Fr.11-14). Fr.11-3 was further purified by HPLC eluted with 35% CH3CN in H2O at a flow rate of 2.5 mL/min to give compound 9 (4.0 mg, tR= 30.5 min) and 4 (1.0 mg, tR= 30.5 min). Fr.11-6 was further purified by HPLC with a gradient of H2O and CH3CN (linear gradient of 30% to 50% CH3CN over 30 min at 2.5 mL/min) to give compound 12 (1.2 mg, tR= 25.5 min). Fr.11-12 was purified by Sephadex LH-20 eluted with MeOH to give compound 7 (1.1 mg). Fr.14 was further purified by HPLC with a gradient of H2O and CH3CN (linear gradient of 50% to 65% CH3CN over 30 min at 2.5 mL/min) to give 5 (1.5 mg, tR= 14.2 min), 3 (8 mg, tR = 16.2 min), and 10 (1.4 mg, tR= 17.2 min). Pure 3 was crystallized as white needles from MeOH/CHCl3 (3:1) with an initial concentration of 4 mg/ml. Fraction Fr.15 was purified by Sephadex LH-20 eluted with MeOH to give compound 6 (3.0 mg) and 11 (10 mg). 11 was crystallized as white needles from

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CH3CN / H2O (6:4) with an initial concentration of 4 mg/ml.

arbumycin (3), white needles. 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) see Table S11 and attached spectra. ESI-MS m/z 514.2 [M + H]+ and 536.2 [M + Na]+. HRESIMS m/z 514.31077 [M + H]+ (calcd for 514.31284, C25H44N3O8); 512.29621[M - H]- (calcd for 512.29719, C25H42N3O8).

arbumelin (5), white powder; 1H NMR (500 MHz) and 13C NMR (125 MHz) in CD3OD or DMSO-d6 as well as 2D NMR spectra see Table S12-S13 and attached spectra. ESI-MS m/z 910.2 [M + H]+ and 922.2 [M + Na]+. HRESIMS m/z 910.5062 [M + H]+ (calcd for 910.4674, C44H64N9O12); 932.4591[M + H+ (calcd for 932.4494, C44H64N9O12Na).

arbusculic acid A (11) , white needles. 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) see Table S14 and attached spectra. ESI-MS m/z 351.1 [M + H]+ and 349.1 [M - H]-.

arbusculic acid B (12), yellow powder; 1H NMR (500 MHz) and 13C NMR (125 MHz) as well as 2D NMR spectra in CD3OD or DMSO-d6 see Table S15 and attached spectra. ESI-MS m/z 707.2[M + H]+ and 705.2 [M - H]-. HRESIMS m/z 705.2545[M - H]- (calcd for 705.25526, C38H41O13). Structure characterization of 3-4 and absolute configuration of amino acid moieties in arbumelin (4):

To elucidate the structures new peptides (3-4), a combination of COSY, HSQC, HMBC and HSQC-TOCSY were firstly used to identify amino acids or hydroxyl acids moieties. Sequential assignment was mainly based on the correlations between the proton and HN of amino acid i with the corresponding resonances of amino acids i+1 and i–1, respectively, in the NOESY spectrum, and HMBC correlations from the proton and the NH of amino acid i to the carbonyl of amino acid i and i+1.[3]

0.4 mg of 4 was subjected to acid hydrolysis at 110 °C for 18 h with 6 N HCl (600 L), and

then the hydrolysates were dried under a steam of N2 gas and redissolved in H2O (200 L). To

one portion (50 L) was added 20 L of 1M NaHCO3 and 100 L of 1%

N--(2,4-Dinitro-5-fluorophenyl)-L-alaninamide (FDAA) in acetone, after which the mixtures were heated at 80 °C for 5 min. The reaction mixtures were cooled, neutralized with 1N HCl

(20 L) and diluted with 300 L CH3CN. About 150 L of each solution of FDAA derivatives was analyzed by LC-MS using a C18 reverse-phase column (Phenomenex Luna, 2.0 × 100 mm,

5). Aqueous CH3CN containing 0.1% formic acid was used as mobile phase with linear gradient elution (30-55% for 40 min) at a flow rate of 0.1 ml/min. FDAA-derivatived amino acids were detected by absorption at 340 nm.

The retention times (min) of the FDAA-derivatized standards were 33.9 min for D-Leu (m/z: 384.1 [M+H]+), 29.5 min for L-Leu (m/z: 384.1 [M+H]+), 23.1 min for L-Tyr (m/z: 434.1 [M+H]+), 24.5 min for D-Tyr (m/z: 434.1 [M+H]+), 29.5 min for D-Val (m/z: 370.1 [M+H]+), 25.1 min for L-Val (m/z: 370.1 [M+H]+), 22.0 min for L-Ser (m/z: 358.1 [M+H]+), 22.5 min for D-Ser (m/z: 358.1 [M+H]+), and 16.1 min for Gly (m/z: 328.1[M+H]+).

The retention times of the FDAA-derivatized hydrolysate of 4 were 29.5 min (L-Leu, m/z: 384.1 [M+H]+), 23.1 min(L-Tyr, m/z: 434.1 [M+H]+), 29.5 min (D-Val, m/z: 370.1 [M+H]+), 25.1 min (L-Val m/z: 370.1 [M+H]+), 22.0 min (L-Ser, m/z: 358.1 [M+H]+), 16.1 min (Gly, m/z: 328.1[M+H]+).

Since there is one each of D and L-valine in the peptide, we assigned the position of D-valine in the peptide at position 5 based on the presence of the E domain in module 5. Similarly, since no O-Me-D-Tyr standard was available, we similarly assigned the stereochemistry of this position as D based on the presence of an epimerase domain.

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Table S1. Polyketide synthase (PKS)

No Contig Module organization PKS type Homolog accession number

Species with the homolog

1 5 KS-AT-ACP-TE NR-PKS XP_001225797.1

Chaetomium globosum CBS 148.51

2 157 KS-AT-DH-MT-ER-KR-ACP HR-PKS EJP62832.1 Beauveria bassiana ARSEF 2860

3 517 KS-AT-DH-ER-KR-ACP HR-PKS EGR46571.1 Trichoderma reesei QM6a

4 661 KS-AT-DH-MT-ER-KR-ACP HR-PKS EMR65646.1 Eutypa lata UCREL1

5 692 KS-AT-DH-MT-ER-KR-ACP HR-PKS XP_003235732.1

Trichophyton rubrum CBS 118892

6 840 KS-AT-DH-ER-KR-ACP HR-PKS XP_390724.1 Fusarium graminearum PH-1

7 921 KS-AT-ACP-MT-TR NR-PKS BAD44749.1 Monascus purpureus

8 959 KS-AT-DH-MT-ER-KR-ACP HR-PKS ABQ42548.1 Solorina crocea

9 1048 KS-AT-ACP-MT-TR NR-PKS EFY84249.1 Metarhizium acridum CQMa 102

10 1080 KS-AT-DH-MT-ER-KR-ACP HR-PKS EMR61679.1 Eutypa lata UCREL1

11 1333 KS-AT-ACP-MT-TR NR-PKS EFY89365.1 Metarhizium acridum CQMa 102

12 1452 KS-AT-DH-MT-KR-ACP HR-PKS EFY96172.1 Metarhizium anisopliae ARSEF 23

13 1462 KS-AT-DH-MT-KR-ACP HR-PKS ACM42412.1 Chaetomium chiversii

14 1530 KS-AT-ACP NR-PKS EFY97792.1 Metarhizium anisopliae ARSEF 23

15 1538 KS-AT-DH-ER-KR-ACP-TE HR-PKS EMT73989.1 Fusarium oxysporum f. sp. cubense race 4

16 1693 AT-KS-AT-ACP-ACP-TE NR-PKS ACH72912.1 Aspergillus ochraceoroseus

17 1737 KS-AT-DH-MT-ER-KR-ACP HR-PKS EFY96790.1 Metarhizium anisopliae ARSEF 23

18 1781 KS-AT-DH-MT-ER-KR-ACP HR-PKS EFY94872.1 Metarhizium anisopliae ARSEF 23

19 1936 KS-AT-DH-MT-ER-KR-ACP HR-PKS CCT74300.1 Fusarium fujikuroi IMI 58289

20 1978 KS-AT-DH-MT-ER-KR-ACP HR-PKS AFN68295.1 Alternaria alternata

21 1998 KS-AT-DH-MT-ER-KR-ACP HR-PKS EJP68806.1 Beauveria bassiana ARSEF 2860

22 2016 KS-AT-DH-MT-KR-ACP-P450 HR-PKS EKJ68630.1 Fusarium pseudograminearum CS3096

23 2032 KS-AT-DH-MT-ER-KR-ACP-mevDPdecarb

HR-PKS XP_002485885.1

Talaromyces stipitatus ATCC 10500

24 2051 KS-AT-DH-MT-ER-KR-ACP HR-PKS XP_002486631.1

Talaromyces stipitatus ATCC 10500

25 2134 KS-AT-DH-MT-KR-T-C-A-T-E PKS-NRPS

EME40768.1 Dothistroma septosporum NZE10

26 2142 KS-AT-DH NR-PKS XP_002152245.1

Talaromyces marneffei ATCC 18224

27 2163 KS-AT-DH-MT-ER-KR-ACP HR-PKS KFH43187.1 Acremonium chrysogenum ATCC 11550

28 2174 KS-AT-DH-MT-ER-KR-ACP HR-PKS EHK20022.1 Trichoderma virens Gv29-8

29 2197 KS-AT-ACP NR-PKS ADI24953.1 Penicillium aethiopicum

30 2221 A-T-KS-AT-KR-ACP-TE NRPS-PKS

EMR62731.1 Eutypa lata UCREL1

31 2230 KS-AT-DH-ACP-TE HR-PKS KEY71921.1 Stachybotrys chartarum IBT 7711

32 2290 KS-AT-DH-MT-KR-ACP HR-PKS GAA83278.1 Aspergillus kawachii IFO 4308

33 2305 KS-AT-ACP-TE NR-PKS EHK23001.1 Trichoderma virens Gv29-8

34 2333 KS-AT-DH-ER-KR-ACP HR-PKS EGX94103.1 Cordyceps militaris CM01

35 2361 KS-AT-DH-ER-KR-ACP HR-PKS XP_007786831.1

Endocarpon pusillum Z07020

36 2384 KS-AT-DH-MT-ER-KR-ACP HR-PKS XP_002152245.1

Talaromyces marneffei ATCC 18224

37 2385 KS-AT-DH-MT-KR-C-A-T-TE PKS-NRPS

EMR62487.1 Eutypa lata UCREL1

38 2437 KS-AT-DH-KR-ACP HR-PKS EJP63776.1 Beauveria bassiana ARSEF 2860

39 2521 KS-AT-ACP-MT-TE NR-PKS XP_007596699.1

Colletotrichum fioriniae PJ7

40 2686 KS-AT-DH-MT-KR-ACP HR-PKS EFZ04272.1 Metarhizium anisopliae ARSEF 23

41 2720 KS-AT-DH-ER-KR-ACP-A-T-TR PKS-NRPS

EFY98483.1 Metarhizium anisopliae ARSEF 23

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Table S2. Non-ribosomal peptide synthase (NRPS) (excluding PKS-NRPS hybrids)

No Contig

Module organization Homolog accession number

Species with the homolog

1 5 ATC-ATEC-ATC-ATC-A(MT)TC-A(MT)TC KFG77713.1 Metarhizium anisopliae ARSEF 23

2 453 ATC-ATC-ATC-ATEC-ATEC-ATC-ATC-ATC-ATC EHK50755.1 Trichoderma atroviride IMI 206040

3 567 ATC-ATC EGR45389.1 Trichoderma reesei QM6a

4 840 ATC-ATEC-ATEC-ATEC-ATC-ATC XP_390720.1 Fusarium graminearum PH-1

5 921 TC-ATC-ATR EHK45804.1 Trichoderma atroviride IMI 206040

6 1462 TC-ATEC-ATEC-ATC-ATC-ATEC-ATC BAO73252.1 Lecanicillium sp. HF627

7 1538 C-ATC-TE EIT81855.1 Aspergillus oryzae 3.042

8 1747 ACT-ATEC-ATC-ATC-ATC EFY94500.1 Metarhizium anisopliae ARSEF 23

9 1811 ATC-ATC-ATC-ATC-ATC EHK21622.1 Trichoderma virens Gv29-8

10 1981 TC-ATEC-ATC XP_007826599.1 Metarhizium anisopliae ARSEF 23

11 2021 C-ATEC-ATC-ATC-ATC-ATC-ATEC EHK21757.1 Trichoderma virens Gv29-8

12 2034 ATC-ATC-TC-ATC-TC-TC EHK26839.1 Trichoderma virens Gv29-8

13 2353 ATC-ATEC-ATC-ATEC-ATC-ATEC EHK20429.1 Trichoderma virens Gv29-8

14 2362 ATEC-ATC-ATR XP_008596990.1 Beauveria bassiana ARSEF 2860

15 2384 TC-ATC-ATC-ATC-ATC-ATEC-ATC-ATC-ATEC-ATC-TE

KFH42120.1 Acremonium chrysogenum ATCC 11550

16 2410 ATC-ATC-ATC-ATC-A(MT)TC-ATEC XP_003044554.1 Nectria haematococca mpVI 77-13-4

17 2474 ATC-TC KEY64460.1 Stachybotrys chartarum IBT 7711

18 2507 ATC-ATC-ATC-ATC-ATC EHK21757.1 Trichoderma virens Gv29-8

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Table S3. Genes involving terpene synthesis

No Contig Putative functions Homolog accession number

Species with the homolog

1 693 Isoprenoid Biosynthesis enzymes, class I CAX94841.1 Trichoderma brevicompactum

2 797 Trans-Isoprenyl Diphosphate Synthase XP_002846999.1 Arthroderma otae CBS 113480

3 1299 Trichodiene synthase (TRI5) AGZ87953.1 Trichoderma brevicompactum

4 1737 ent-copalyl diphosphate synthase KFH47392.1 Acremonium chrysogenum ATCC 11550

5 1789 Trans-Isoprenyl Diphosphate Synthases XP_390273.1 Fusarium graminearum PH-1

6 1930 ent-kaurene synthase XP_002849529.1 Arthroderma otae CBS 113480

7 1936 Copalyl diphosphate synthase KFH47392.1 Acremonium chrysogenum ATCC 11550

8 2327 pentalenene synthase ETS03221.1 Trichoderma reesei RUT C-30

9 2560 Isoprenoid Biosynthesis enzymes, Class 1 EAA31071.2 Neurospora tetrasperma FGSC 2509

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Table S4. A domain signatures in contig 2507 NRPS.

Residues in A domain signature

A1 domain D G L F I G I P I K

A2 domain A G Q F C G T T Y K

A3 domain D V Y T V L A V L K

A4 domain D M H I L M G C F K

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Table S5. A domain signatures in contig 453 NRPS.

Residues in A domain signature Proposed substrate

A1 domain D L Y P I A I P V K L-Leu

A2 domain D A F P A G C P V K L-Tyr

A3 domain D M F Q L I T P V K Gly

A4 domain D M S G I A G P C K D-O-methyl-Tyr

A5 domain D I L Y I G A P A K D-Val

A6 domain D V M L L G V P C K L-Ser

A7 domain D M F Q L I A P C K Gly

A8 domain D M F Q L I S P C K Gly

A9 domain D M F N L G A P F K L-Val

S11

Table S6. Primers for gene knock-out in C. arbuscula. Primer name Primer sequence

hph-up F ACCTGGCGGCCGCTACAACGACCATCAAAGTC

hph-up R ACTGACCGCGTCTGCTGCTCCATACAA

hph-dn F CAGGGAGCTCTCGGAGGGCGAAGAATC

hph-dn R GAATGCGGCCGCAGGATTACCTCTAAACAAGTG

arbA-up F CCTGGTACCGTTGGGATGTG

arbA-up R AAGCGGCCGCTTGGAATTCACA

arbA-dn F AAAGCGGCCGCAACGTCTACGC

arbA-dn R AAGGTACCCTAGATCATCAGCCT

hdaA-up F CGCAAGAAAGTCCAAGCC

hdaA-up R ACCTGGCGGCCGCTCCATCCTAGCGTAGTGAC

hdaA-dn F CCTGGCGGCCGCGGACACAACTGAGGAAGATG

hdaA-dn R ACCTGTCTAGACTGGGGGAATCGTTGGAC

M13-F GTAAAACGACGGCCAGTG

M13-R CCTTTGTCGATACTGGTAC

S12

Table S7. RT-PCR primers for polyketide synthases (PKS) contig sequence cycle number contig sequence cycle number

5 GCGGCAGTCAGGCTTGG 35 2016 ATGCGTATCCCGATGG 35

CTTTGGGCTTGGTCGGAG GCGAGCAGGTATTTCTGG

157 CGCAATTACAGACAAAGAC 35 2032 CACCTCGTCTCAAAATGG 28

GGACGTAGTGAGAGGAC GGGAAGTTGTTCTCGGAC

517 CCGGCGGCAGGAATGGTG 28 2051 GCAAACTTACTCTTGGAC 35

GTCCGGGATACGAACTTTG TGGCATCGGCTTCAATAG

661 CGCGGGATGTGGGTTAC 35 2134 GACTCCACTCAGGCTCTC 35

CGGCGTGTGCGTTTGTG GAAATGACACCTTCGGC

692 GCCGGCATGGGGAAAC 35 2142 GAGATTGCGCAGCCATTG 28

TGGCGCCGGCAGCATAAG GCCCGAGACTGTGACAC

840 TTCCGGCGTCGTCTCC 35 2163 CCGCCCGGCTGGATTG 35

GGCGTGTCACAGGTCC TCCCCTACCTCATCTAAC

921 CGCCTCGGCATACCTGTC 28 2174 TGGTACGAGGGGGTGGG 28

AGTTCGCCCTCAGTCCAG TGAGACTGTGGCGGCAAG

959 GAGCCCGGAGGATGTTAC 28 2197 CGAGAAGAGGTGAAGAC 28

CTGGCCCTTGCGAATGG GCGGAACAATGGAGAGTC

1048 CGACGCCGACGCCTTTG 28 2221 GGTTTAGCGACGGAATC 35

AGAGCCCGTACTGCCGAA CCTGGTGCCAAGTAATG

1080 GCACTGGGCAAATAGAG 35 2230 AGCACAAGCCCCTACCG 28

GAAGCGACCAAGGGGAG AAGGACCAACCACCAAGG

1333 CCTGCCTCTCCCCTTCC 35 2290 TCGCGCTGGTGCCTTGG 35

TCCCCTTCCAGAATGCG TTAGAACCCTGGACAAAAG

1452 CTTCCTTCTCCATCCG 35 2305 CGTGGTTCTATGCTCGC 35

GTCGCGAGTGAACAAG ACGCAAATGGTTCGCAA

1462 CCGTAAGCTCTGTTTCC 28 2333 CGCCGGTCCTCTACAAC 35

CTCCGGCGGCGTAGTTG GTCTTCTTCGGCAAACTC

1530 CTGGTGGAAATACGAC 35 2361 GCAGTGTCCATTCCTTTC 28

AAAACCCGAGCCCTGAG CGTTGTCTGCGAGTAGTC

1538 CCGAAGCCGTCATCTAC 35 2384 GGACCCTCAACAACGAC 28

CTCGTTGAATCTTCTTGGG TACGCCGCCGACGAAG

1693 TTGTCATACACAACCACTG 28 2385 TTGGCAACAGTATCACG 35

CAAGGCTAACTGAACGAC GTCTTTGTGTAACCCTCC

1737 TTGGCGCGGGTACAGG 35 2437 CTCGGGCGCAGTCTTAC 28

CAAATGGTAAGAGCAAAGG CGGATTACCAAGCACGG

1781 GAAGGCGGGCAAGATG 35 2521 TTCCCAAGATTTGACGGC 28

GGACAGAAAACAGAAGC CTGCTGGCGACATTGCG

1936 CTGAAGTTCAGACTATG 35 2686 CACCCTCGTATTCCTGA 35

GCCAGTGATAAGATTCG GACCAATAAGCCAGTAG

1978 GCGGCATACTGTGTTGG 28 2720 CACCGCCAGCCCATTCAC 28

ATTCTTCAGAAACCTGTTG CGAGCGCGACATAGCAGG

1998 CACCCTCTAAATGTCGC 35

CGTCCGAACAGTAATGC

S13

Table S8. RT-PCR primers for non-ribosomal peptide synthases (NRPS) contig sequence cycle number

5 CAGGCGCGTATGTAATG 35

CGGGCTTGTTCAGTATC

453 GGCTCAGCTCCCTATTC 28

GTCCCCTAGACCCAGAG

567 CTGGCTGCTGCTGCGAG 28

GAGCCGTAAGTCATTGTTG

840 GAACTCCAATGCTCTGAC 35

ACCTCGGCCAAACCCTG

921 CGAGTCATCCATTTGCTC 28

AGGGCTGTGAGATTTTGATA

1462 GAAGTTCTAAAGCAGGTC 28

CATCACAAGGCTCTCTAC

1538 CCATATCTAACCTACCAG 35

ACACTGCTGTTTCCGATG

1747 TCCAACCGCATTAGCCC 28

ATCCTCGAGCCCGTCAA

1811 TTAGGCGGAGAAGAAGTC 28

CGTCGGCCGGATTGTATT

1981 GCCCAAGACAGCCCAAG 35

CAAGCTGCGTGCCCACA

2021 GGTCCCGGCCTTTTCAC 35

TGTAGGTAGTCGTCAAG

2034 CAGCAATATCGGCAGGC 28

CGTGATACAATAGTAGCAA

2353 CTCCTGGGACGGCGAAC 35

CGTGAGCAGCGATACTC

2362 CCCTCCCTCTTGCGAC 35

CGAGTTATCCTGACCATC

2384 TTCGGGCATGGTCCTAC 28

GCGCCGCAGAGAAACAC

2410 CTGCACCGCCCTGGAGA 28

GAGCGCAGATATGAGAAC

2474 TTGTTCGCCCAGCCCTC 35

GAAGTTGCTCTGTCTAAATAC

2507 TGCCGGCTGTATTCAAGA 28

CCAGTAGAAGATTTATCG

S14

Table S9. RT-PCR primers of genes for terpene synthesis contig sequence cycle number in RT-PCR

693 CCACTGCGTCGGGTCTAC 28

CGCAATCTGGAATAACTC

797 CAGCGAAGCCAACGATG 28

CCAGAGCAAGGAAGTAGAC

1299 GGTGGCGTCTGGTAAAC 28

ATCGCGCTCGGCATTAAAC

1737 CGGCTCTTTTACATCAACC 35

GAAACCGACACCACGAC

1789 GACGGGCGATAAGGAACG 28

GCCAGAAGAGGTCCATCC

1930 GCCCGAGAAACTTGAGCA 28

CGAGCGGTACAGAGCAG

1936 GGAGAAGCGTAGCAAGC 28

AGCGGACCCAGGCGTATG

2327 GCTCTGTGACTGGGGCAAC 35

CGGCTCCGATAGAAACAC

2560 AGACGCGGCCCCTCAAC 28

CGAAGATGTACCCCACC

S15

Table S10. RT-PCR primers for hdaA and house-keeping genes contig sequence cycle number in RT-PCR

hdaA ACGCAGAATACAACTCGC 28

ATCCTGCCGAAATAATGAC

actA AAGGCCGGTTTCGCTGG 28

GGACGGCCTGGATGGAG

tubC ACAACTGGGCCAAGGGTC 28

CGGGGAAACGCAGGCAGG

gpdA CATCGTCTTCCGCAATG 28

CTGCCGTCGTAGGTCTTC

S16

Table S11. NMR data of arbumycin (3) in CDCl3

amino acids

No. H, mult, (J in Hz) C COSY HMBC

Ala C=O 1

172.76 C

4.21, t (7.6) 51.58 CH H13, NH C1, C3, C13

1.56, d (7.2) 17.33 CH3 H2 C1

7.52 d (8.3)

H2 C2, C3

HMVA-1 C=O 3

171.39 C

4 4.67, d (7.3) 80.66 CH H4 C3, C14, C15

14 1.92, m 36.32 CH H4, H15, H16

15 1.00, d (7.0) 14.68 CH3 H14

16 1.62, m 24.89 CH2 H14, H17

1.10, m

17 0.91, d (7.0) 11.33 CH3 H16

Ile C 5

172.70 C

6 4.62, dd (6.3, 10.0) 57.52 CH H18, NH C5, C18, C19

18 2.04, m 35.01 CH H6, H19, H20

19 0.98, d (7.0) 16.18 CH3 H18

20 1.45, m 24.92 CH2

1.21, m

21 0.91, d (7.0) 11.47 CH3 H20

7.77, d (10.0)

H6 C7 HMVA-2 C 7

170.56 C

8 H15', d (4.2) 79.73 CH H22 C9, C22, C23

22 1.97, m 36.69 CH H8, H23, H24

23 1.0, d (7.0) 14.52 CH3 H22

24 1.46, m 25.91 CH2

1.26, m

25 0.91, d (7.0) 11.64 CH3 H24

Thr-9 C=O 9

170.29 C

10 4.43, (7.1) 59.33 CH H11, NH C12

11 4.47, m 68.18 CH

C9

12 1.27, d (6.5) 20.8 CH3

7.44, brs

S17

Table S12. NMR data of arbumelin (4) in CD3OD

no amino acids

δH, mult. (J in Hz) δc HMBC

1 Leu C=O

174.3, C

2

4.60, dd (4.1,12.2) 49.6, CH C1, C3 3

1.64, ddd (4.1, 12.2, 14.4) 38.0, CH2

1.07, m

4

1.38, m 24.3, CH

5

0.82, d (6.6) 19.4, CH3

6

0.79, d (6.6) 22.6, CH3

7 Tyr1 C=O

173.2, C

8

4.23, dd (5.5, 6.3) 58.8, CH C7, C9, C10 9

3.03, d (4.0) 35.5, CH2 C7

10

Ph-1

128.0, C

11

Ph-2, 6 7.26,d (8.6) 129.8, CH 2 C12, C13, C11

12

Ph-3, 5 6.72, d (8.6) 115.0, CH 2 C10, C12, C13

13

Ph-4

156.0, C

14 Gly1 C=O

172.4, C

15

4.44 d (17.2) 41.7, CH2 C14

3.56, d (17.2)

C14

16 Tyr2 C=O

172.4, C

17

4.38, dd (12.1, 4.0) 56.8, CH C16, C18, C20 18

3.22, dd (4.3, 14.4) 35.0, CH2 C16

2.67, dd (12.3, 14.6)

C16

19

Ph-1

129.4, C

20

Ph-2, 6 7.18, d (8.8) 128.7, CH2 C19,C20, C22

21

Ph-3, 5 6.87, d (8.8) 113.9, CH2 C10, C21, C22 22

Ph-4

158.9, C

23

Ph-4-OCH3 3.76, s 54.3, CH3 C22

24 Val2 C=O

173.7, C

25

3.74, d (6.1) 63.2, CH C24 26

1.89, m 28.9, CH C24, C25, C28

27

0.85, d (6.7) 18.0, CH3 C25, C26 28

0.76, d (6.7) 17.6, CH3 C25, C26

29 Ser C=O

174.7, C

30

4.74, dd (10.5, 5.9) 53.0, CH C29, C31, C32 31

3.99, t (10.0) 61.7, CH2 C29

3.82, dd (10.0, 6.0)

C29

32 Gly2 C=O

170.0, C

33

4.13, d (17.2) 42.3, CH2 C32

3.46, d (17.2)

C32

34 Gly3 C=O

171.1, C

35

3.91, d (17.2) 43.6, CH2 C34,C36

3.69, d (17.2)

C36, C34

36 Val-1 C=O

175.8, C

37

3.66, m 62.5, CH C36 38

2.17, m 28.2, CH C37, C39

39

1.07, d (6.3) 19.4, CH3 C37, C38 40

0.94, d (6.3) 18.2, CH3 C37, C38

S18

Table S13. NMR data of arbumelin (4) in DMSO-d6

no amino acids

δH, mult. (J in Hz) δc COSY HMBC NOESY

1 Leu C=O

174.3, C

2

4.37, ddd (13.0, 9.1, 4.2)

49.8, CH 2-NH,

C1 2-NH,37-NH

3

1.53, ddd (3.6, 11.1, 15.0)

38.2, CH2

1.08, d (15.0)

4

1.33, m 24.6, CH

5

0.73, d (6.4) 20.9, CH3

6

0.71, d (6.8) 23.5, CH3

2-NH

7.80, d (8.8)

C7 H4, H3, H6

7 Tyr1 C=O

171.7, C

8

4.05, dd (6.6, 6.6) 58.5, CH 8-NH, H9

C7 2-NH, 8-NH, H11

9

2.86, d (6.9) 36.1, CH2 H8 C7 2-NH, 8-NH, H11 8-NH

7.45, d (6.6)

C14 H9, H15

10

Ph-1

128.2, C

11

Ph-2, 6 7.13,d (8.5) 130.3, CH 2 H12 C12

12

Ph-3, 5 6.63, d (8.5) 115.6, CH 2 H11 C10, C12, C13

13

Ph-4

156.5, C

14 Gly1 C=O

171.5, C

15

4.15 dd (13.4, 9.2) 42.4, CH2 15-NH,

C14 15-NH, 8-NH

3.38, overlap (m)

15-NH C16

15-NH

7.41, m

H15, H17

16 Tyr2 C=O

171.2, C

17

4.17, m 57.2, CH 17-NH C16 17-NH, H20, 15-NH

18

3.03, dd (13.7, 3.7) 35.3, CH2 H17

17-NH

2.67, dd (13.7, 13.7)

H17 C16 17-NH

17-NH

7.846, d (6.3)

19

Ph-1

129.9, C

20

Ph-2, 6 7.17, d (8.5) 130.4, CH 2 H21 C20, C22

21

Ph-3, 5 6.81, d (8.5) 114.2, CH 2 H20 C19, C21, C22

22

Ph-4

158.5, C

23

Ph-4-OCH3 3.69, s 55.5, CH3

C22

24 Val2 C=O

173.9, C

25

3.64, dd (11.5, 5.9) 63.1, CH 25-NH C24

26

2.00, m 28.4, CH

27

0.93, d (6.4) 20.4, CH3

28

0.84, d (6.9) 19.5, CH3

25-NH

9.87, brs

H25

H30, H25,

29 Ser C=O

174.6, C

30

4.72, dd (15.5, 9.2) 53.1, CH 30-NH C29 25-NH, 30-NH 31

3.68, m, overlap 62.4, CH2

C29

3.60, dd (11.2, 6.5)

30-NH

7.06, d (7.4)

H30, 30-NH

32 Gly2 C=O

168.9, C

33

3.85, dd (17.1, 5.5) 43.0, CH2 33-NH C32 33-NH, 30-NH

3.30, m, overlap

C32, C34 33-NH

33-NH

7.87, t (6.0)

H33 C34

34 Gly3 C=O

169.6, C

35

3.73, dd (17.1, 5.5) 43.6, CH2 35-NH C34 35-NH, 33-NH

3.57, dd (17.1, 5.5)

C36 35-NH

35-NH

9.22, brs

H35 C36

S19

36 Val-1 C=O

174.8, C

37

3.66, m 61.7, CH 37-NH

38

1.87, m 28.9, CH

39

0.74 d (6.4) 19.0, CH3

40

0.70, d (6.9) 19.1, CH3

37-NH

7.31, d (5.9)

H37 C1 H2, H37

Table S14. NMR data of arbusculic acid A (11) in DMSO-d6

no δH, mult, (J in Hz) δc COSY HMBC NOSEY

1.65, d (13.0) 26.0, CH2 H2, C2 1.40, m, overlap

2 2.18, dt (13.3, 3.5) 26.5, CH2 , ,

H3

1.57, d (14.0)

,

3 3.95, t (7.5) 72.1, CH C5, C4, C1 4

49.8, C

5

78.4, C

2.12, ddd (13.3, 4.0, 4.0)

28.9, CH2 C7

1.61, m

1.72, ddd (13.3, 13.3,4.2)

24.4, CH2

7 1.08, d (12.8)

, C8, C6 8 1.45, t (11.0) 36.6, CH H9

H20

9 2.41, d (10.9) 41.2, CH H8 C8, C11, C10, C20 10

41.9, C

11 5.61, d (10.0) 130.0, CH H12 C8, C10, C13 12 5.91,dd (10.0, 2.2) 130.4, CH H1' C9, C13, C14 H15 13

141.7, C

14 2.45, m 33.9, CH H17 C8,C12, C13, C15, C17

H8, H16

15 5.19, t (6.8) 126.5, CH C12, C13, C14, C16 H16 4.04, m 57.2, CH2 H15, 16OH C13, C15 H14 3.96, m

H1

17 0.78, d (7.0) 13.9, CH3 H14 C8, C13, C14 H9 18

177.5, C

19 1.25, s 20.4, CH3

C3, C4, C5,C18 H3, H20, 5-OH 20 0.72, s 16.1, CH3

C5, C1, C9 H2, H8, H19

3-OH 5.76, d () / H3 C2, C3, C4 H3 5-OH 5.40, s /

C4, C5, C6 H9, 19-OH

16-OH 4.51, t (5.3) / H16 C15, C16

S20

Table S15. NMR data of arbusculic acid B (12) in CD3OD no mult, (J in Hz) C COSY

162.2, C

119.7, C

163.1, C

7.28, s 108.9, CH

C2, C3, C10, C9a

133.7, C

7.08, d (2.5) 107.98, CH H7 C6, C7, C10

165.5, C

6.49, d (2.5) 107.7, CH H5 C5, C6, C8

165.1, C

108.9, C

189.4, C

109.1, C

181.7, C

135.2, C

4.44,dd (5.4, 9.1) 36.5, CH C1, C2, C12, C13, C14

2.52, m 31.4, CH2 C2, C11, C13, C14 2.16,m

, H13

3.58, m 59.7, CH2 , C11, C12

172.0, C

1.30, m 26.0, CH2 H2', C5' 1.01, m

H2'

2' 2.27, m 22.8, CH2

, ,

1.81, d (14.0)

,

3' 5.30, t (2.5) 77.4, CH C14, C5', C4', C1' 4'

50.4, C

5'

77.6, C

2.15, m 28.3, CH2 ,

1.59 ,m

C5'

1.53, m 23.9, CH2 C5', C10' 0.98, m

8' 1.42, m 36.3, CH H9', H14'

9' 1.94, 40.7, CH H12', H11', H8'

10'

41.0, C

11' 5.34, d (10.1) 128.9, CH H12', H9' C8', C9', C13' 12' 5.73,dd (10.1, 2.7) 129.6, CH H11', H9' C9', C14' 13'

143.5, C

14' 2.32, m 33.8, CH H17' C8' 15' 5.13, dd (6.8, 6.8) 123.2, CH , C10', C12', C14'

4.11, dd (7.4, 12.8) 57.2, CH2 H15' C13', C15' 4.01,dd (6.8, 12.8)

H15'

17' 0.38, d (7.2) 12.2, CH3 H14' C8', C13' C14' 18'

177.4, C

19' 1.17, s 18.5, CH3

C3', C4', C18' 20' 0.73, s 15.5, CH3

C5', C1', C10'

S21

Figure S1. Gene cluster comparison of sterigmatocystin (6) from Calcarisporium arbuscula and Aspergillus ochraceoroseus. Protein identities are shown on each homologous enzymes.

S22

Figure S2. Gene cluster comparison of citrinin from Calcarisporium arbuscula (contig 921) and Monascus purpureus. Protein identities are shown on each homologous enzymes.

S23

Figure S3. Deletion of hdaA gene in C. arbuscula.

S24

Figure S4. Phenotype of C. arbuscula wild type and the hdaA mutant grown on PDA for 7 days.

S25

Figure S5. RT-PCR analysis of gene expression for biosynthetic core genes in PKS (a), NRPS (b), terpene synthesis (c) and internal controls (d) from Calcarisporium arbuscula wild type and the

hdaA mutant. The house-keeping genes actA, tubC and gpdA were the internal controls. rRNA were shown for loading control and RNA integrity. Up-regulated genes were shown in red, down-regulated genes in green and non-regulated genes in black.

S26

Figure S6. ORTEP representation of the asymmetric unit of the crystal of 3. Ellipsoids displayed at 50% probability.

S27

Figure S7. Deletion of arbA in C. arbuscula.

S28

Figure S8. Gene cluster of contig 453 for the possible biosynthesis of arbumelin (4).

S29

Figure S9. Gene cluster of contig 1462 for the biosynthesis of verlamelin (5) in C. arbuscula. The protein sequence identity to Lecanicillium sp. HF627 homologs responsible for verlamelin biosynthesis are also shown.

S30

Figure S10. a) Gene cluster of contig 1936 for the possible biosynthesis of three diterpenes (9, 10, 11). b) Structural modeling of taxadiene synthase (Taxus brevifolia) and the predicted terpene cylcase (C. arbuscula) on SWISS-MODEL server. The structure of terpene cylcase (C. arbuscula) is modeled based on taxadiene synthase (Taxus brevifolia). c) Domain organization of the predicted

terpene cylcase (C. arbuscula) based on the structural modeling. Three putative , , domains are shown, and the conserved motifs from class I and class II terpene cyclases are labeled together with the amino acid positions. The highly conserved residues are bold.

S31

Figure S11: 1H NMR of aurovertin B (1) in CDCl3 (500 MHz)

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

1.0694

1.0845

1.0994

1.1819

1.2604

1.6276

1.6360

1.6408

1.6509

1.6559

1.6649

1.6711

1.6799

1.6914

1.7065

1.9457

1.9571

2.1553

2.1629

2.2426

2.2568

2.2727

3.8302

3.9079

3.9167

3.9258

3.9346

4.1184

4.1326

4.1476

4.7974

5.4867

5.5037

5.8952

5.9078

5.9238

5.9362

6.3322

6.3617

6.3843

6.4064

6.4276

6.4348

6.4564

6.4784

6.4847

6.5062

7.1490

7.1710

7.1788

7.2012

7.2555

S32

Figure S12: 13C NMR of aurovertin B (1) in CDCl3 (125 MHz)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

8.90

11.76

15.05

16.45

20.16

20.80

56.17

76.39

77.95

80.54

82.73

83.40

85.53

88.84

108.07

119.57

131.71

132.13

134.13

135.67

137.04

154.31

163.68

169.91

170.60

S33

Figure S13: 1H NMR of arbumycin (3) in CDCl3 (500 MHz)

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.8246

0.8383

0.8888

0.9028

0.9132

0.9277

0.9406

0.9547

0.9849

0.9984

1.0098

1.0234

1.1833

1.1961

1.2272

1.2406

1.2559

1.2821

1.2949

1.3147

1.4206

1.4347

1.4521

1.4662

1.4788

1.4924

1.5069

1.5719

1.5864

1.9017

1.9298

1.9397

1.9575

1.9761

1.9891

2.0450

2.0575

3.7537

4.2277

4.2423

4.4413

4.4557

4.6320

4.6393

4.6509

4.6758

4.6893

5.1408

5.1490

7.2552

7.5241

7.5382

7.7851

7.8041

S34

Figure S14: 13C NMR of arbumycin (3) in CDCl3 (125 MHz)

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

172.70

11.25

11.33

11.47

11.64

14.52

14.68

16.18

17.33

20.80

24.89

24.92

25.91

35.01

36.32

36.69

51.58

57.52

57.74

59.23

68.19

79.73

80.66

170.29

170.56

171.39

172.76

S35

Figure S15: 1H 1H-COSY spectrum of arbumycin (3) in CDCl3

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

9

8

7

6

5

4

3

2

1

0

S36

Figure S16: HSQC of arbumycin (3) in CDCl3

ppm

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0 ppm

10

20

30

40

50

60

70

80

90

100

S37

Figure S17: HMBC of arbumycin (3) in CDCl3

S38

Figure S18: NOSEY of arbumycin (3) in CDCl3

ppm

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5 ppm

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

S39

Figure S19: 1H NMR of arbumelin (4) in CD3OD (500 MHz)

8 7 6 5 4 3 2 1 0 ppm

0.7780

0.7851

0.7985

0.8221

0.8350

0.8473

0.8609

0.9442

0.9577

1.0705

1.0833

1.2298

1.9263

1.9939

2.0029

2.6483

3.0342

3.0444

3.0522

3.2175

3.2380

3.2460

3.3056

3.3381

3.4492

3.4840

3.5453

3.5800

3.6530

3.6732

3.7074

3.7387

3.7508

3.7631

3.8221

3.9116

3.9461

3.9903

4.1213

4.1561

4.4278

4.4626

4.5925

4.6090

4.7316

4.7407

4.7523

4.8050

4.9231

6.7130

6.7299

6.8685

6.8856

7.1774

7.1945

7.2503

7.2672

S40

Figure S20: HSQC-TOCSY of arbumelin (4) in CD3OD (500 MHz)

ppm

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

S41

Figure S21: HSQC of arbumelin (4) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

S42

Figure S22: HMBC of arbumelin (4) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

190

180

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

0

S43

Figure S23: 1H NMR of arbumelin (4) in DMSO-d6 (500 MHz)

10 9 8 7 6 5 4 3 2 1 ppm

0.6881

0.7020

0.7156

0.7267

0.7394

0.8322

0.8456

0.9227

0.9353

1.2107

1.7306

1.9658

2.4964

2.8623

2.8759

3.1440

3.2419

3.2532

3.2634

3.2748

3.3044

3.3137

3.3203

3.3311

3.3365

3.3468

3.3578

3.3928

3.4089

3.4228

3.4366

3.5846

3.5955

3.6065

3.6144

3.6262

3.6374

3.6489

3.6772

3.7333

4.0455

4.1616

4.1760

6.6200

6.6368

6.8015

6.8187

7.1195

7.1363

7.1659

7.1831

7.4422

7.4531

7.5860

7.8164

7.8578

S44

Figure S24: 1H 1H-COSY spectrum of arbumelin (4) in DMSO-d6

ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

11

10

9

8

7

6

5

4

3

2

1

S45

Figure S25: HSQC of arbumelin (4) in DMSO-d6

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

S46

Figure S26: HMBC of arbumelin (4) in DMSO-d6

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

190

180

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

0

S47

Figure S27: NOSEY of arbumelin (4) in DMSO-d6

ppm

11 10 9 8 7 6 5 4 3 2 1 ppm

11

10

9

8

7

6

5

4

3

2

1

S48

Figure S28: 1H NMR of verlamelin (5) in CD3OD (500MHz)

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.8939

0.8983

1.2502

1.2628

1.2872

1.2902

1.3010

1.3154

1.3381

1.3518

1.5534

1.5597

1.5702

1.9648

1.9783

1.9911

1.9991

2.0053

2.2233

2.2500

3.2928

3.2961

3.2993

3.3026

3.3059

3.3382

3.6174

3.6341

3.8858

3.8995

4.2035

4.3763

4.3909

4.5689

4.6205

4.6342

6.6744

6.6783

6.6877

6.6915

7.0636

7.0807

S49

Figure S29: 13C NMR of verlamelin (5) in CD3OD (125 MHz)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

18.31

19.28

20.53

22.32

24.30

25.22

26.69

29.02

29.20

29.34

29.66

31.64

31.77

32.82

34.08

35.02

37.59

48.46

53.55

55.19

57.99

59.74

61.06

67.01

70.04

73.75

114.91

127.58

130.02

155.97

171.31

171.56

172.20

172.36

172.74

172.89

174.61

176.11

S50

Figure S30: 1H 1H-COSY spectrum of verlamelin (5) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

S51

Figure S31: HSQC of verlamelin (5) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

20

30

40

50

60

70

80

90

100

110

120

130

140

S52

Figure S32: HMBC of verlamelin (5) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

180

160

140

120

100

80

60

40

20

S53

Figure S33: 1H NMR of paeciloquinone B (8) in DMSO-d6 (500MHz)

13 12 11 10 9 8 7 6 5 4 3 2 1 ppm

1.1491

1.1636

1.2008

1.8460

1.8641

2.2333

2.2468

2.2575

2.2652

2.3621

2.3815

2.4716

2.4858

3.1441

3.3223

3.9028

3.9125

3.9220

3.9318

6.5535

6.5582

7.0783

7.0829

7.1501

12.1550

12.7140

S54

Figure S34: 13C NMR of paeciloquinone B (8) in DMSO-d6 (125 MHz)

200 180 160 140 120 100 80 60 40 20 ppm

23.43

29.46

30.19

31.16

40.87

49.06

108.52

109.05

109.24

120.27

133.17

135.42

162.45

164.61

165.41

174.45

181.82

189.09

206.99

208.35

S55

Figure S35: 1H NMR of zythiostromic acid A (9) in CD3OD (500MHz)

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.7663

0.7916

0.8168

1.0795

1.0982

1.1052

1.2014

1.2975

1.3678

1.3883

1.4189

1.6161

1.6216

1.6490

1.7193

1.7480

2.0785

2.0832

2.1050

2.1096

2.2020

2.2111

2.3196

2.3388

2.3621

2.3680

2.3777

2.3844

2.4037

2.5387

2.5700

3.2937

3.2969

3.3001

3.3033

3.3065

3.3389

3.8055

4.1814

4.1867

4.5257

4.6252

4.6283

4.8748

4.9397

4.9438

4.9741

4.9782

5.0984

5.1027

5.1188

5.1232

5.6404

5.6747

S56

Figure S36: 13C NMR of zythiostromic acid A (9) in CD3OD (125 MHz)

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

14.92

18.94

26.37

26.93

29.00

33.43

35.63

40.38

40.61

44.39

47.10

48.13

50.96

54.87

72.16

72.23

79.81

105.34

115.48

139.89

151.36

178.07

S57

Figure S37: 1H NMR of zythiostromic acid B (10) in CD3OD (500MHz)

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

0.8600

0.9271

1.2568

1.2797

1.2876

1.3070

1.3130

1.3391

1.3693

1.6007

1.6280

1.6359

1.6734

1.6779

1.6837

1.6941

1.7107

1.7183

1.7372

1.7445

1.8233

2.0254

2.1466

2.2552

2.2641

2.2834

2.2923

2.3828

2.3890

2.4087

3.2145

3.2936

3.3068

3.3390

3.6297

4.0866

4.0918

4.5079

4.5104

4.6098

4.6135

4.6173

4.9218

4.9259

4.9562

4.9603

5.0834

5.0880

5.1040

5.1085

5.6175

5.6329

5.6376

5.6517

S58

Figure S38: 13C NMR of zythiostromic acid B (10) in CD3OD (125 MHz)

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

181.45

14.62

19.14

26.23

26.53

26.72

26.88

28.22

35.88

40.85

41.68

45.78

54.78

70.14

72.59

79.30

105.23

115.35

139.97

151.41

S59

Figure S39: 1H NMR of arbusculic acid A (11) in DMSO-d6 (500 MHz)

13 12 11 10 9 8 7 6 5 4 3 2 1 ppm

1.6

121

1.6

388

1.6

522

1.6

598

1.6

840

1.7

108

1.7

185

1.7

369

1.7

443

2.1

089

2.1

284

2.1

360

2.1

559

2.1

860

2.4

027

2.4

245

3.1

366

3.1

380

3.1

471

3.1

485

3.9

572

3.9

673

3.9

804

3.9

924

4.0

163

4.0

292

4.0

413

4.0

536

4.0

642

4.4

982

4.5

085

4.5

191

5.1

757

5.1

889

5.2

020

5.4

064

5.5

947

5.6

147

5.7

507

5.7

605

5.9

051

5.9

094

5.9

253

5.9

294

12.3

487

6.3

6

1.0

73

.00

2.0

34

.14

1.9

6

3.4

3

1.0

8

1.0

41

.05

1.0

11

.09

1.0

6

1.0

0

S60

Figure S40: 13C NMR of arbusculic acid A (11) in DMSO-d6 (125 MHz)

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

13

.91

16

.17

20

.34

24

.38

26

.02

26

.57

28

.94

33

.92

36

.64

40

.07

40

.24

40

.41

41

.22

41

.90

49

.79

57

.21

72

.11

78

.39

12

6.5

11

30

.00

13

0.4

7

14

1.7

3

17

7.5

0

S61

Figure S41: 1H 1H-COSY NMR of arbusculic acid A (11) in DMSO-d6

ppm

0.51.01.52.02.53.03.54.04.55.05.56.06.5 ppm

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

S62

Figure S42: HSQC of arbusculic acid A (11) in DMSO-d6

ppm

1.01.52.02.53.03.54.04.55.05.56.0 ppm

20

30

40

50

60

70

80

90

100

110

120

130

140

S63

Figure S43: HMBC of arbusculic acid A (11) in DMSO-d6

ppm

1.01.52.02.53.03.54.04.55.05.56.0 ppm

180

160

140

120

100

80

60

40

20

S64

Figure S44: NOESY of arbusculic acid A (11) in DMSO-d6

ppm

0.51.01.52.02.53.03.54.04.55.05.56.06.5 ppm

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

S65

Figure S45: 1H NMR of arbusculic acid B (12) in CD3OD (500MHz)

9 8 7 6 5 4 3 2 1 ppm

0.3738

0.3880

0.4032

0.4173

0.7218

0.7349

0.7665

0.8115

0.8221

0.8273

1.0131

1.1726

1.2307

1.2450

1.2665

1.2798

1.2992

1.9264

1.9436

1.9477

2.0034

2.1584

3.1545

3.1578

3.1610

3.3191

3.3225

3.4372

3.5612

3.5759

3.5891

3.6028

3.6283

3.6505

4.0133

4.0840

4.0981

4.1127

4.4388

4.4496

4.4567

4.4674

4.5820

4.7965

4.8151

4.8975

5.1354

5.2984

5.3529

6.4963

6.4982

6.5011

7.0823

7.0871

7.2856

8.4974

S66

Figure S46: 13C NMR of of arbusculic acid B (12) in CD3OD (125 MHz)

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

18.52

22.86

23.89

25.95

26.06

28.29

31.36

33.78

36.35

36.54

36.84

40.70

40.94

41.03

41.32

48.21

48.44

50.43

56.92

57.21

59.69

77.39

77.62

107.79

107.98

108.90

109.07

119.68

121.72

123.16

128.96

129.65

130.47

133.76

135.26

143.56

162.05

163.05

165.05

165.52

168.42

171.80

177.35

181.71

189.37

S67

Figure S47: 1H 1H-COSY spectrum arbusculic acid B (12) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

9

8

7

6

5

4

3

2

1

0

S68

Figure S48: HSQC of arbusculic acid B (12) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

170

160

150

140

130

120

110

100

90

80

70

60

50

40

30

20

10

0

S69

Figure S49: HMBC of arbusculic acid B (12) in CD3OD

ppm

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

200

180

160

140

120

100

80

60

40

20

0

S70

Scheme S1. A proposed pathway for the biosynthesis of arbumelin (4).

S71

Scheme S2. Biosynthesis of sterigmatocystin (6), paeciloquinone A (7) and B (8).

S72

Supplementary References: [1] F. N. Gravelat, D. S. Askew, D. C. Sheppard, Methods Mol. Biol. 2012, 845, 119-130. [2] M. A. Collart, S. Oliviero, Curr. Protoc. Mol. Biol. 2001, Chapter 13, Unit13 12. [3] T. Prasch, T. Naumann, R. L. Markert, M. Sattler, W. Schubert, S. Schaal, M. Bauch, H. Kogler, C.

Griesinger, Eur. J. Biochem. 1997, 244, 501-512.