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Supporting Information

Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through

Combinatorial Pathway Reassembly in Engineered Yeast

Mancheng Tang1, Hsiao-Ching Lin1, Dehai Li1,4, Yi Zou1, Jian Li3, Wei Xu1, Ralph A. Cacho1, Maureen E. Hillenmeyer3, and Neil K. Garg2, Yi Tang*,1, 2

1 Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA, USA;

2 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA;

3 Stanford Genome Technology Center, Palo Alto, CA, USA;

4 Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of

China, Qingdao, P. R. China

* Corresponding author: E-mail: [email protected].

S1

Table of contents PageExperimental procedures1. Strains and General DNA Manipulation Techniques S42. Phylogenetic analysis methods S43. Construction of the RC01 yeast biosynthetic host S44. Plasmid construction S45. Chemical analysis and compound isolation and characterization S56. Gene annotation and the cDNA sequences of the studied genes S5

Supplementary tablesTable S1. PCR primers for construction of RC01 yeast strain S9Table S2. PCR primers for different IDT genes S9Table S3. Spectral data of compound 1 S11Table S4. Spectral data of paspaline (3) S12Table S5. Spectral data of aflavinine (5) S13Table S6. Spectral data of anominine (6) S14Table S7. Spectral data of compound 8 S15Table S8. Spectral data of compound 10 S16Table S9. Spectral data of emindole SB (11) S17Table S10. Spectral data of compound 12 S18Table S11. Spectral data of compound 13 S19Table S12. Spectral data of compound 14 S20Table S13. Spectral data of compound 15 S21Table S14. Spectral data of compound 16 S22Table S15. Spectral data of compound 17 S23Table S16. Spectral data of compound 19 S24Table S17. Spectral data of compound 20a/b S25

Supplementary figuresFigure S1. HPLC analysis (λ=280 nm) of the extracts of yeast strains expressing different combinations of indole diterpene biosynthetic genes S26Figure S2. The map of the nonclustered indole diterpene cyclase genes and other genes aside S26Figure S3. The UV spectrum (left) and MS data (right) of the characterized indole diterpenes S27Figure S4. 1H NMR of compound 1 in CDCl3 and Figure S5. 13C NMR of compound 1 in CDCl3 S31Figure S6. COSY of compound 1 in CDCl3 and Figure S7. HSQC of compound 1 in CDCl3 S32Figure S8. HMBC of compound 1 in CDCl3 and Figure S9. NOESY of compound 1 in CDCl3 S33Figure S10. 1H NMR of compound 3 in CDCl3 and Figure S11. 13C NMR of compound 3 in CDCl3 S34Figure S12. COSY of compound 3 in CDCl3 and Figure S13. HSQC of compound 3 in CDCl3 S35Figure S14. HMBC of compound 3 in CDCl3 and Figure S15. NOESY of compound 3 in CDCl3 S36Figure S16. 1H NMR of compound 5 in CDCl3 and Figure S17. 13C NMR of compound 5 in CDCl3 S37Figure S18. COSY of compound 5 in CDCl3 and Figure S19. HSQC of compound 5 in CDCl3 S38Figure S20. HMBC of compound 5 in CDCl3 and Figure S21. NOESY of compound 5 in CDCl3 S39Figure S22. 1H NMR of compound 6 in CDCl3 and Figure S23. 13C NMR of compound 6 in CDCl3 S40Figure S24. COSY of compound 6 in CDCl3 and Figure S25. HSQC of compound 6 in CDCl3 S41Figure S26. HMBC of compound 6 in CDCl3 and Figure S27. NOESY of compound 6 in CDCl3 S42

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Figure S28. 1H NMR of compound 8 in CDCl3 and Figure S29. 13C NMR of compound 8 in CDCl3 S43Figure S30. COSY of compound 8 in CDCl3 and Figure S31. HSQC of compound 8 in CDCl3 S44Figure S32. HMBC of compound 8 in CDCl3 and Figure S33. 1H NMR of compound 10 in CDCl3 S45Figure S34. 13C NMR of compound 10 in CDCl3 and Figure S35. COSY of compound 10 in CDCl3 S46Figure S36. HSQC of compound 10 in CDCl3 and Figure S37. HMBC of compound 10 in CDCl3 S47Figure S38. 1H NMR of compound 11 in CDCl3 and Figure S39. 13C NMR of compound 11 in CDCl3 S48Figure S40. COSY of compound 11 in CDCl3 and Figure S41. HSQC of compound 11 in CDCl3 S49Figure S42. HMBC of compound 11 in CDCl3 and Figure S43. NOESY of compound 11 in CDCl3 S50Figure S44. 1H NMR of compound 12 in CDCl3 and Figure S45. 13C NMR of compound 12 in CDCl3 S51Figure S46. COSY of compound 12 in CDCl3 and Figure S47. HSQC of compound 12 in CDCl3 S52Figure S48. HMBC of compound 12 in CDCl3 and Figure S49. NOESY of compound 12 in CDCl3 S53Figure S50. 1H NMR of compound 13 in CDCl3 and Figure S51. 13C NMR of compound 13 in CDCl3 S54Figure S52. COSY of compound 13 in CDCl3 and Figure S53. HSQC of compound 13 in CDCl3 S55Figure S54. HMBC of compound 13 in CDCl3 and Figure S55. NOESY of compound 13 in CDCl3 S56Figure S56. 1H NMR of compound 14 in CDCl3 and Figure S57. 13C NMR of compound 14 in CDCl3 S57Figure S58. COSY of compound 14 in CDCl3 and Figure S59. HSQC of compound 14 in CDCl3 S58Figure S60. HMBC of compound 14 in CDCl3 and Figure S61. NOESY of compound 14 in CDCl3 S59Figure S62. 1H NMR of compound 15 in CDCl3 and Figure S63. 13C NMR of compound 15 in CDCl3 S60Figure S64. COSY of compound 15 in CDCl3 and Figure S65. HSQC of compound 15 in CDCl3 S61Figure S66. HMBC of compound 15 in CDCl3 and Figure S67. NOESY of compound 15 in CDCl3 S62Figure S68. 1H NMR of compound 16 in CDCl3 and Figure S69. 13C NMR of compound 16 in CDCl3 S63Figure S70. COSY of compound 16 in CDCl3 and Figure S71. HSQC of compound 16 in CDCl3 S64Figure S72. HMBC of compound 16 in CDCl3 and Figure S73. NOESY of compound 16 in CDCl3 S65Figure S74. 1H NMR of compound 17 in CDCl3 and Figure S75. 13C NMR of compound 17 in CDCl3 S66Figure S76. COSY of compound 17 in CDCl3 and Figure S77. HSQC of compound 17 in CDCl3 S67Figure S78. HMBC of compound 17 in CDCl3 and Figure S79.

1H NMR of compound 19 in CDCl3 S68Figure S80. 13C NMR of compound 19 in CDCl3 and Figure S81. COSY of compound 19 in CDCl3 S69Figure S82. HSQC of compound 19 in CDCl3 and Figure S83. HMBC of compound 19 in CDCl3 S70Figure S84. 1H NMR of compound 20a/b in CDCl3 S71Figure S85. 13C NMR of compound 20a/b in CDCl3 S71Figure S86. COSY of compound 20a/b in CDCl3 S72Figure S87. HSQC of compound 20a/b in CDCl3 S72Figure S88. HMBC of compound 20a/b in CDCl3 S73Figure S89. NOESY of compound 20a/b in CDCl3 S73Figure S90. Inferred phylogeny of IDTC and homologous proteins from fungi. S74

References S75

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Experimental procedures

1. Strains and General DNA Manipulation Techniques E. coli TOPO10 and E. coli DH10b were used for cloning, following standard recombinant DNA techniques. DNA restriction enzymes were used as recommended by the manufacturer (New England Biolabs, NEB). PCR was performed using Phusion® High-Fidelity DNA Polymerase (NEB). PCR products were confirmed by DNA sequencing. Saccharomyces cerevisiae strain BJ5464-NpgA (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1 pep4::HIS3 prb1 Δ1.6R can1 GAL) and RC01 were used as the yeast biosynthetic hosts.

2. Phylogenetic analysis methods For phylogenetic analysis of IDT cyclases, protein sequences were identified in NCBI protein databases using blastp with AtmB as a query. Sequences were aligned using MUSCLE v3.8.31.1 Bayesian inferred phylogeny was computed using MrBayes v3.2.5,2 with a mixed model of amino acid substitution. The BLOSUM model was identified as the most probable model by MrBayes. The Markov Chain Monte Carlo algorithm was run for one million generations, with default settings (four chains, sampling every 500 generations, burn-in threshold of 25%). The XiaE protein from Streptomyces was identified during BLAST search as a distant bacterial homolog and was therefore selected as an outgroup to root the tree.

3. Construction of the RC01 yeast biosynthetic host We followed previously reported gene integration methods using double crossover homologous recombination for the construction of the S.cerevisiae strain RC01 used in this study.3 S. cerevisiae strain RC01 was constructed by integrating a copy of the Aspergillus terreus cytochrome P450 reductase gene (AtCPR) under the ADH2 promoter into the genome of BJ5464-NpgA. Briefly, the AtCPR gene was amplified from the plasmid pESC-leu2d::AtCPR using the primer pair NdeI-AtCPR and EcoRV-AtCPR-stp. The resulting PCR product was digested with Nde I and EcoR V and ligated into the vector yeplac195-pADH2 to make yeplac195-pADH2-AtCPR. The CPR expression cassette PADH2-atCPR-TADH2 was PCR amplified from the resulting plasmid using primer pair AtCPR-F-ura3 and AtCPR-R-A’-SHR while the selection marker LoxP-TRP1-LoxP was PCR amplified from pXP316 using primer pair pXP316-F-A-SHR and pXP316-R-ura3. Both PCR products were gel purified prior to transformation to S. cerevisiae strain BJ5464-NpgA. The linear DNA fragment was integrated into the ura3-52 locus of BJ5464-NpgA via homologous recombination using the lithium acetate method4

creating RC01-Trp1. TRP1 was removed by expressing CreA recombinase as reported by Fang et al.3. Integration was verified by PCR amplification of the integrated AtCPR expression cassette using primer pairs binding outside the integration site. All the primers are listed in Table S1.

4. Plasmid construction The annotated genes were cloned from the genomic DNA using the primers listed in Table S2. The 2μ-based yeast-E.coli shuttle plasmids with different auxotrophic markers (Ura3, Trp1 and Leu2) were used for construction of the yeast expression plasmids following the standard protocol.5 The gene fragments of atmG, atmC, atmM, atmB, and afB, were cloned from the genomic DNA of Aspergillus flavus NRRL3357. The gene fragments of atS5M, atS5B1, atS5-P450, and atS2B, were cloned from the genomic DNA of Aspergillus tubingensis. To construct the co-expression plasmid of atmG and atmC, the intact atmG and atmC was first cloned into vector pXW06 with TRP1 marker and pXW02 with LEU2 marker yielding plasmids pTMC-1 and pTMC-2, respectively. Then the cassette PADH2-atmC-TADH2 was amplified from pTMC-2 by using the primers AtmC-G-xw06-For/Rev and the PCR product was ligated into pCR Blunt vector, yielding pTMC-3. The plasmid, pTMC-3, was digested with NotI, and the fragment containing the cassette was recovered and inserted into the same site of pTMC-1, yielding the co-expression plasmid pTMC-4 with the TRP1 marker. The same method was also used to construct the co-expression plasmid of atS5B1 and atS5M, and atS2B and atmM yielding the co-expression plasmids pTMC-5 and pTMC-6, respectively.

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5. Chemical analysis and compound isolation and characterization LC-MS was conducted with a Shimadzu LC-MS-2020 liquid chromatography mass spectrometer by using both positive and negative electrospray ionization and a KinetexTM 1.7µm, 100 Å, 100 mm x 2.1 mm, C18 reverse-phase column. Samples were dissolved in methanol before separated on a linear gradient of 15% to 95% CH 3CN (v/v) in H2O (containing 0.1% formic acid) in 10 min followed by 95% CH3CN (0.1% formic acid) for 7 min with a flow rate of 0.3 mL/min. 1H, 13C and 2D NMR spectra were obtained using CDCl3 as solvent on a Bruker AV500 spectrometer with a 5 mm dual cryoprobe at the UCLA Molecular Instrumentation Center. Optical rotations were measured with a Rudolph Autopol III Automatic Polarimeter. High resolution mass spectra were obtained from Thermo Fisher Scientific Exactive Plus with IonSense ID-CUBE DART source at the UCLA Molecular Instrumentation Center. For small scale analysis, the yeast co-expression strain was first inoculated into 2 mL SDCt (uracil, tryptophan, and leucine dropout) medium at 28C, 250 rpm. Then the overnight seed culture was inoculated into 20 mL YPD (2% Glucose) medium and cultured for four more days. The cell pellets were collected from 2 mL culture and extracted with 1 mL acetone and 1 mL ethyl acetate mix. The extracts were evaporated to dryness and re-dissolved in 500 µL methanol for LC-MS analysis. For compound isolation, large-scale fermentation was carried out. The yeast co-expression strain was inoculated into 40 mL SDCt (uracil, tryptophan, and leucine dropout) medium as a seed culture at 28 C, 250 rpm. Two days later, this seed culture was used to inoculate 4 L YPD (1.5% Glucose) medium. Four days later, the culture was spun down. The supernatant was extracted with equal volume of ethyl acetate, and the cell pellets were extracted with 1 L of acetone and 1 L ethyl acetate. The organic phase was combined and evaporated to dryness. The residue was purified by ISCO-CombiFlash®

Rf 200 (Teledyne Isco, Inc) with a gradient of hexane and acetone. After analysis by LC-MS, the fractions contain the target indole diterpene compound were combined and further purified by semi-preparative HPLC using C18 reverse-phase column. The purity of each compound was confirmed by LC-MS, and the structure was solved by NMR.

6. Gene annotation and the cDNA sequences of the studied genes All the studied genes were annotated manually by using blastx and blastp from NCBI website.6 The cDNA sequences of each gene are predicted as follows: atmG: ATGATTTCAGGTGTGCCCGATCGCTGGAAGCTTGTTGCATCCTCGCTCTCTTCAAACCTGGATGCAAGCTATCCCACTCCAAGCTCATTGTCGACCGAGCCCATAGATACTAGAAGTTCATCCCCCCAGGGATCCGCGTCTACGGAAGTTGACAAAGAGAAG (fragment 1)—ATTATTCGGGGGCCTGTGGACTATCTATTGAAATGTCCCGGGAAGGATATCCGTCGCAAGCTCATGCAAGCTTTCAATGAATGGTTAAAGATTCCAGAGGACAGGCTGAACATCATCGCGGAGATTGTTGGTTTACTGCACACAGCGTCTCTCTT (fragment 2)—GATCGATGACATTCAAGATTCGTCTAAACTGCGACGAGGGATACCAGTGGCTCATTCGATATTCGGTGTCGCGCAAACTATTAACTCCGCAAACTACGCCTACTTCGCCGCCCAGGAGAAGCTCAGAGAGCTCAATCGCCCCAAGGCATACGAGATCTTTACTGAGGAGCTACTCCGTTTACATCGGGGACAAGGCATGGATCTATATTGGCGAGACTCTCTGACCTGCCCCACAGAAGAAGAGTACATCGAAATGATCTCGAATAAAACTGGTGGTCTCTTCCGGTTGGCAATCAAGCTAATGCAGTTGGAAAGTGAGGTAACAAG (fragment 3)—CGACTTCCTCGGGCTTGTCGATCTCTTGGGTATCATTTTTCAGATCCGTGACGACTACCAGAATCTCCAAAGTGACCTGTACAGCAAGAATAAAGGGTTTTGTGAAGACCTCACAGAAGGCAAATTTTCCTTCCTGATTATACACAGCATCAACAGTAACCTGGGTAATCAACAACTGCTCAATATCCTCCGACAGAGGAGCGAGGAGGAGTCAGTGAAGCAATACGCTGTGGAATATATTCGGTCAACAGGATCTTTTGCCTATTGCCAGGACAGACTGGCCTCATTGCTGCATGAAGCAAAGATGATGGTCAATGTACTAGAAGAAAATGTTGGGTTTTCTAAAGGTATCTATGATATCTTAGCTTTTTTACTGTGA (fragment 4) atmC: AT

S5

GGGCTTCTTTCATGACTTCCTCTCTCGGCCTACAACCTATGCTATACTGGCCGTGCTGGTTATTCCGGTCACGGCCCTGGCGTGGGACAGGTTGCCGCCTCTGTTGCCTTCTGCAAAGAGGTTATTGGTTGGCAAAAAGAATCCCTCTAAGATAACATCACTAGAATGCCCTTACAGTTACATCCGACAGATATACGGAACGCACCACTGGGCACCATTCGTTGACAAGCTCTCCCCTAGCCTCAAGACAGAGCGGCCGGCAAAGTACCACATGATCCTAGAGATCATGGACGGAATACATCTTTGTCTGATGCTTGTTGATGAT (fragment 1)—ATAAGTGACGGCAGTGACTATCGGAAAGGACGCCCCGCTGCCCATCACATCTATGGTCCCTCGGAGACAGCAAACCGAGCATACTATCGGGTAACCCAGCTACTCAATCGCACGGTGCACGAGTTCCCAGAGCTTGCACCTTGGCTGCTCCAATGTCTCGAAGAGATACTAGAGGGACAAGACCTCTCGCTTCTTTGGCGGCGGGACGGCCTTTCTGCCTTTCCCGTCCAACCAGAGGAGCGAGTAGCGGCATATCGCCAAATGGCATATTTGAAGACAGGTGCCCTCTTCCGCCTAGTGGGCCAGCTTGTACTAGAGAATCAATCTTACGATGATACTTTAAGTACAGTAGC (fragment 2)—ATGGTATTCCCAATTGCAAAATGACTGCAAGAATGTGTACTCCTCTGACTACGCAAAAGCCAAAGGAGCCATTGCCGAAGACCTACGCAATGGCGAGCTTTCTTACCCAATTGTTGTCGCTTTGAATGTCCCAAAGGGACAATATGTGGTGCGAGCATTGGCGTTTCGGTCGCCACATAATATCCGACAAGCTCTGCGAGTCATTCAGAGCGATCAAGTTCGAAATATATGTCTCACAGAAATGAAGAAATCAGCAGTTTCTGTTCAGGACTGGCTCGCACTCTGGGGACGGAATGAAAAGATGGACATGAAGAACGAGAAATGA (fragment 3) atmM: ATGTGTGATAAAGATCGCTTCAAAGTGATTATTGTTGGGGGATCTGTCGCTGGTCTCACCCTGGCGCACTGTCTCCAACGGGCAGGAATAGACCATGTTGTCCTCGAGAAAAATTCAGATCTCTCTCCACAGGTGGGGGCATCAATCGGCATTATCCCCAATGGGGGACGTATTCTAGATCAGTTGGGTCTCTTTGATGCTGTGGAGAAGATGACCTACCCTCTCAGCATGGCTACTATCACATACCCTGACGGATATTCTTTCCGTAACAATTATCCAAAAACTGTCGATGAAAG (fragment 1)—ATTCGGGTATCCCATTGCATTCCTAGATCGACAGAAATTCCTCGAGATTCTGCACACATCGTACCCTGACCCGTCAAATATCCATACAAATTGCCGGGTGACGCATATCCGACGGCATGACAGCCATATGGAAGTTGTTACAAGCTCCGGGCAGGAGTATACTGGCGACCTAGTGGTTGGTGCTGATGGTGTCCATAGCGTTATCCGCTCTGAGATGTGGAAATTGGCGGATGCGCTGGAGCCTGGACGGGTGTCGAAGCGGGAAAAGAGAA (fragment 2)—GCATGAAGGTCGAATACGCCTGTGTTTTCGGTATCTCATCACCCGTTCCAGGCTTAAAGGTCGGAGACCAAGTAAACGCATTTCATGACGGCCTGACTATTATCACCATCCATGGAAAAAATGGACGGGTGTTCTGGTTCGTGATCAAGAAGTTGGACGACATGCACACATACCCGGACACGGTGAGATTTTCTAGCGCTGATGCAGTACGTACTTGTGAGAATATCGCGCACTTCCCACTAGTGAACGGGGCCACTTTCGGCCACGTCTGGGAGAACAGGGAGGTTACGTCTATGACAGCATTAGAGGAGAATATCTTTAACACCTGGTATGCGGACCGCATTGTTTGTATTGGGGACAGTATTCACAAG (fragment 3)—ATGACGCCAAACATCGGACAAGGAGCGAATACTGCCATCGAAGACGCCACAGTCTTAACCAATCTGTTGTATGATAGGCTCTCGAAGAATGGACACAAGAAACTCGCACAGCAAGAGCTGCTGCAGCTTCTTCGGGAATTCCAGTTCCAGCGCTTCCGCCGTGTCAACAAGATCTACCAAGATTCTCGGTTCCTAGTTCGGCTCCACGCACGAGACGGAATCGTTAAATCTCTCCTTGCACGATACATTGTCCCCTATATGACAGAACTCCCGGCAGATCTGGCCTCAAAGTCCATTGCTGATAGTCCCACCATCGGCTTTCTCCCACTCCCTTCGCGCAGTGGACCTGGATGGCTGCAGTGGAGTCGGAAGCAAAGAAGACCTGCTACCCCGTGGATACTGGTACTTCTTGTCATCGTGGTTAGCTTTGGTCTGCATTCACCCGAGCTTGTTATTCCAACGTTCTGGAGTAATTCACTGGTTTCTAAAACGGTTGAGTAA (fragment 4) atmB: ATGGACGGATTTGGCTCATCACAGGCCCCAGCTGCGTATCGTGAAGTGGAATGGATCGCAGATGTTTTCGTCATAGGGATGGGGATCGGTTGGGTTATCAACTATGTCGGCATGGTCTACGGATCGCTCAAGGGCCGTACATATGGAATGGCTATCATGCCACTCTGTTGCAATATTGCATGGGAAATCGTGTATGGCCTTATCTACCCATCCAAGACATTGTATGAGCAAGGGGTCTTTCTAAGCGGTCTTACCATCAACCTGGGCGTCATATACACAGCAATTAAATTTGGGCCCAAGGAATGGACTCATGCGCCTTTGGTGATGCACAATCTGCCCCTGATTTTTATGCTGGGTATACTCGGCTTTCTCACAGGCCACCTTGCCCTAGCCGCGGAGATTGGCCCTGCATTGGCCTATAACTGGGGAGCTGCATTCTGTCAGCTGTT

S6

GCTCAGTGTTGGAGGACTCTGCCAGCTGATCAGTCGCGGAAGCACCCGAGGAGCCTCGTACACGCTCTG (fragment 1)—GCTTTCCCGATTCCTAGGATCTTTCTCCGTGGTGATTTCGGCCTGGTTGCGTTATAAATACTGGCCACAGGCATTTTCGTGGCTGGGAAAACCCCTGATATTGTGGTGCCTTTTTGCCTGGCTCGTTGTCGACGGTTCCTATGGGGTTTGCTTTTATTATGTCAAGCGGTATGAGCGGAGAATTGGGCACGATTCCGATCGGAAGACAGTCTAG (fragment 2) afB: ATGGACAGCTTCGATCTCGCCAACGCACCCCCCGAAATCCGAGCCTACGCCACCCCAATCATCTTGCTGAACCTCTACACCAACGCCAGTTGGCTTTACGTCTACTTCGGCATGGTCTACCGCTCCGTAAAGGACAAGTCCTACGCCATGCCCCTCTACTCCCAATGTCTCAATATCGCCTGGGAGATAACCTACGGTTACATATACGGTGACGATTGGATGCTCTTCGCGACATTCCTCGTTACCTTCCCCACAGACTGCCTCGTCATCTGGGCGGCCATCTACCACGGCGCCAAAGAGTGGGACCGCTCACCCCTGGTGCAGCGCAATCTCCTCTGGTACTATGTGATCGGGACGGGGATCGCTGTTGCCCTGCACATGTGCGCTGCGTCGGAGCTGGGGGTTGAAAAGGCATTCTTTGCCGGTGCGATTGGGTGTCAGGCGGTGTTGAGTGTAGGTTATCTTGGGAATTTGATTCAAATGGGGAGTACAAGGGGGTTTTCAATGCATCTGTG (fragment 1)—GTTCTTCCGCTTCACAGGCTCCCTAACCCTCGTCCCCGAATTCTACCTGCGGGTGAAATACTGGCCCGAGAGGTTCGGCTTCCTGGGCCAGCCCCTCATGCTCTGGTGCTGCGCTGTCTTCCTGGGATTTGATCTGGTCTACGGGATCTTATTCTGGTATATTCGACGACAGGAACGCGAGACGGGGATGCTGCTTGCTGATGGGCGGAAGAGGAAGTGA (fragment 2) atS5M: ATGGCCAATGCCCAGCAACCCCCCTTTCGCGTCCTTATTGTGGGCGGTTCTGTCGCAGGCCTCACCCTTGCGCACTGTCTCGAACGCGCCAATATCGAGTACCTCATACTCGAGAAAGGAGAAGATGTTGCCCCGCAAGTTGGTGCCTCGATAGGTATCATGCCAAATGGCGGACGGATCCTCGAGCAACTGGGCCTATTTGGGGAGATTGAGCGTGTGATCGAGCCGTTGCATCAGGCGAATATCAGCTATCCAGATGGGTTCTGCTTTAGTAACGTCTATCCTAAGGTTCTTGGCGACAG (fragment 1)—GTTCGGATACCCGGTTGCATTCTTGGACCGGCAGAAATTCCTGCAGATTGCGTATGAAGGGCTTAGAAAGAAGCAGAACGTTCTCACTCGAAAAAGGGTAGTCGGCGTGCGACTGACGGAACATGGAACTGCTGTTTCTGTGGCTGATGGAACAGAGTATGAGGCCGATCTCGTGGTTGGTGCTGATGGAGTACATAGTCGGGTGAGAAGTGAAATTTGGAAGATGGCGGAGGAGAATCGGCCTGCATCGGTTTCGACACGTGAAAGAAGAA (fragment 2)—GCATGACTGTTGAATATGTCTGCGTTTTCGGGATCTCATCAGCCATCCCAGGGCTCGAGATAAGCGAACAGATCAACGGGATTTTCGACCATCTATCCATTCTAACAATCCACGGCAGACATGGTCGGGTGTTCTGGTTCGTGATCCAGAAGTTGGATAGGAAGTACGTCTATCCTGATGTCCCGCGATTCTCAGACGAGGATGCTGTACAGCTCTTCGATCGGGTCAAACACGTGCGGTTCTGGAAAAACATCTGCGTGGGGGACTTGTGGAAGAACAGAGAGGTGTCCTCGATGACAGCGCTGGAGGAGGGAGTGTTCGAGACATGGCACCATGATAGGATGGTTTTGATTGGAGATAGCGTTCACAAG (fragment 3)—ATGACGCCCAACTTTGGCCAGGGAGCCAATTCTGCCATCGAGGATGCTGCCGCGCTCTCCTCCCTTCTACATGATCTCGTCAACGCCCGTGGGGTTTGCAAGCCATCAAATGTCCAGATTCAGCATCTCCTCAAGCAGTATCGGGAGACTCGATACACTCGCATGGTAGGCATGTGTCGCACCGCGGCTTCAGTCTCTCGAATTCAGGCCCGAGATGGCATCCTCAACACCGTCTTTGGACGATATTGGGCACCATATGCTGGCAACCTGCCTGCTGACCTGGCATCAAAAGTAATGGCCGATGCAGAGGTTGTTACTTTTCTGCCTTTGCCAGGCCGCTCAGGACCGGGCTGGGAGATGTACAAGCGGAAGGGGAAGAGAGGGCAGGTGCAATGGGTGCTTATAATCTTCACCTTACTTACGATTGGTGGATTGGGCATATGGCTCCAAAGCAATGCGTTGAGTAGATGA (fragment 4) atS5B1: ATGGATGGGTTCGACCATTCTACTGCTCCACCAGAATATAACGAGCTAAAATGGCTCGCCGATATTTTCGTCATCGGAATGGCTGTTGGCTGGGTTGCTCACTATGTGGAGATGATTCACATATCGTTCAAGGACCAAACATACTGCATGA

S7

CCATCGGGGGCCTTTGCATCAATTTTGCCTGGGAAATCATATTCTGCACAATGTATCCTGCCAAAGGGTTTGTCGAGCGGGTTGCCTTTCTCATGGGCATTTCTCTCGATCTTGGGGTTATTTACGCGGGAATCAAGAATGCCCCAAATGAGTGGCACCACACTGCAATGGTGAGGGACCATATGCCCCTCGTCTTCGCAGCGACGACAATTTGTTGTCTGAGCGGTCACATGGCTCTTACTGCCCAGGTTGGTCCCGCACAAGCCTATACGTGGGGGGCAATTGCATGCCAGCTCTTTATCAGCATAGGGAATGTGTTTCAATTGTTGAGTCGGGGAAACACACGAGGGGCGTCATGGACGCTATG (fragment 1)—GACCTCCAGGTTTTTTGGATCAACATCAGCCATTGGCTTTGCTCTTGTTCGATATATTCGCTGGTGGGAGGCTTTTTCTTGGTTGAACTGCCCGCTTGTGTTATGGTCCGTGGTCATGTTCTTTCTGTTTGAAATTCTGTATGGAGCGTTATTCTATTCTGTCAAGCGACAAGAAGAAAGATCCCAGTGTGGAATCAAGCACAAAGAGAGGTAG (fragment 2) atS5-P450: ATGACTCCACCCCTCCTCCCAACAGCCCTCATCCCCGCCATTCTCGGCATCACCGCCCATCTAACCTACTTCATTCACGCCGACCTCGAAAAGCATGTCGTCTTCCTCGCCAACTGCCTCATCTTCTGGCCGTTCCTCGGCTTACTAGCCCTCTTCATCCTCGGATATAAAGAGATATCCCAATGGATCGGACTAGCGATACTAACCTTCCACTCCTCGCTGCTAGCTTCTATAGCGATCTATCGATACTTCTTTCATGCGCTGGGCGGGTTTCCAGGTCCGAAGCTAGCAAGGGTCACAGCGTTGTGGGATTTCAGAAATACAGTGCTGGATGCGAAATGGTACCTTAAAGTGAAGGAGTTGCATGAGGTGTATGGGGATGTTGTTCGGATTA (fragment 1)—AACCTAGGGAGATCTCCATCAACGATCCATCTGCGATAAAAGACATCTACGGCGTGGGTACGACATGCGTCAAGGGACCGTTCTACGATCTACATTATCCGCATCGATCATTGCAGATGGCGAGGGATAAGGCGTTTCATTCGAAGAGGAGGAGGTTATGGGATCGGGGGTTTACGTCGAAGG (fragment 2)—CACTTGCCGGCTACGAACCCTACCTTCTAGAGCACTGCAAAGATATAGTCCAGGTTATTGAATCCCGATCATCGCAAGCACAAGAGACCACAGCGCTAATTGATGGGTTTGCATGGGACTCGATGGGGATATTTGCGTTTGGGAAATCGTTCAATATGCTGCATGGGGAGCCGCCGAAGATGCTGCAGATGATGAGAATGATGGGGCGAGGGGCAAGTGCCTTGTTGAGCTCGATCTGGCTGGTTATATTTATGCGGGGGATGGTGGGTGTGAGGCGGTTTACGGATAGATGGTTGGAGTGGTGTGCGCAGTGTGTGGAGGAGAGGAGTAGG (fragment 3)—GTCGAAACTGATCGTCGGGATCTCTTCAGCTATTTGATCGAGGAGTCAAGCTCCGGGGGAGACCAGTCTATAGGTGGCGTTGATGGGGATCTTGTTCGCGATTCAGAGCTCGCTATTACGGCTGGCAGCGACACGGCCGCCAGTACCCTCAATGCTTTGTTCTATCTCCTGGCACGACATCCGGAGAAGCTGCGCCGGCTGCGGGAGGAGATAGATTCTGCTGTGCCTGCTGGGCAGGAGCTGTGTCATGCGGCATTGGTGAAGAAGCCGTACCTAGAGGGGTGTATCAACGAGAGTCTGCGTCTGTGTCCAGCTGTGTTGAGCGGATTGCAGCGCGAGACAAGACCAGAAGGGCTTCGAACGGCGGGAGTTTATATACCTCCAGGGATGATAGTGTCTGTGCCGACGTATACGATTCAACGAG (fragment 4)—ACTCGCGCAACTTCCCCCGTCCAGACGAGTTCATCCCAGAAAGATGGTCCAGCCAGCCAGAGCTAGTCATCCACAAAGAAGCACTCAACGCATTCTCCAGCGGCACCTACTCCTGCGCAGGAAAGGCATTCGCGATGATGGAGATGCGACTGCTCGTTTCCACGATCGTAAGGCACTTCGACATTAAGTTTCCCCCAGGGGAAGGACAAGAACCGCTAGATAGACTGGAGGGAACAGGTCCATTAGACTGTTTCACAACACATATTCCAGAGTATCATCTACTCTTTACAAAGAGATAG (fragment 4) atS2B: ATGGATGCCTTTGATCTTTCCACTGCTCCCCCGGAATTCGCGTCCTGGGCCACAACACTCTATGCCTGCAATATATACACTAATTTCATATGGCTGTACGTGTATTATGGGATGATCTACCGATCCTACAAGGACAAGTCCTTTGCCATGCCACTCATCTCCCAGTGTCTCAACATAGCGTGGGAAATCGTTTTCGGCTTCTTGTTCTCCCAGGACCACTGGTTTATAACTCTTTCCTTCCAAGCCGCAGTCATATCCAACTGTGGCGTGATCTACGCAGCAATCAAATATGGCGCCCCAGAGTGGAACAGATCACCTATGATACAACGCAACCTGCCCTGGATTTACATAGGTGGAACGCTATTGGCAATTGCAGGCCATCTTGCTCTTGCCACAGAGCTCGGCATGGTGAGGGCTTGCTTTCAAGGCGCAATCGTATGTCAGGCTATCCTAAGTGTGGGATATGTCTGTCAGCTTTTAGTGAGGGGGTCTACGCGTGGTTTCTCTCTTAACTTATG (fragment 1)—GTTCTTCCGATTCACGGGCTCTCTTGTCATGGTTCCGGAATTTTACATTCGTGTCAACTACTGGCCGGACGCTTTTAGCTGGTTGGGTGAGCCTTTCATGCTCTGGTGCTGCTTCATTTATTTGGGCTTTGATCTGGCATACCCGGTGCT

S8

TTTCTGGTATATTCAGAGGCGTGAGAAGGAGGAGGCGTTGGCGAAAAGTATCAAGAGTCTGTAA (fragment 2)

Table S1. PCR primers for construction of RC01 yeast strain

Primer name Primer sequence

Nde I-AtCPR TAAAAACATATGGCTCAACTCGACACTC (the underlined is the Nde I site)

EcoRV-AtCPR-stp TAAAAAGATATCTTAGAGGTCTTCTTCGGAAATCAAC(the underlined is the EcoR V site)

AtCPR-F-ura3 GACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAAAGGCATTATCCGCCAAGTACAA-

GAGCTCGGATCCATTTAGCG

AtCPR-R-A’-SHR GTGCCTATTGATGATCTGGCGGAATGTCTGCCGTGCCATAGCCATGCCTTCACATATAGT-

AGCTCGGTACCCTCGAGG

pXP316-F-A-SHR ACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCGCCAGATCATCAATAGGCAC-

TGTAAAACGACGGCCAGTGC

pXP316-R-ura3 CTTCTCAAATATGCTTCCCAGCCTGCTTTTCTGTAACGTTCACCCTCTACCTTAGCATCC-

TAGCACGTGATGAATTCGAG

Table S2. PCR primers for different IDT genes

S9

Table S3. Spectral data of

compound 11H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

position δH , mult (J in Hz) δC COSY

S10

Primer name Primer sequenceAtmG-p1-For AACTATCAACTATTAACTATATCGTAATACCATCA TATG ATTTCAGGTGTGCCCGAtmG-p1-Rev TCCACAGGCCCCCGAATAAT CTTCTCTTTGTCAACTTCCGAtmG-p2-For CGGAAGTTGACAAAGAGAAG ATTATTCGGGGGCCTGTGGAtmG-p2-Rev AATCTTGAATGTCATCGATC AAGAGAGACGCTGTGTGCAGAtmG-p3-For CTGCACACAGCGTCTCTCTT GATCGATGACATTCAAGATTCGAtmG-p3-Rev CGACAAGCCCGAGGAAGTCG CTTGTTACCTCACTTTCCAACAtmG-p4-For TTGGAAAGTGAGGTAACAAG CGACTTCCTCGGGCTTGTCAtmG-p4-Rev TGATAATGGAAACTATAAATCGTGAAGGCATGTTT TCACAGTAAAAAAGCTAAGATATCAtmC-p1-For ATCAACTATCAACTATTAACTATATCGTAATACCA TATG GGCTTCTTTCATGACTTCCAtmC-p1-Rev TAGTCACTGCCGTCACTTAT ATCATCAACAAGCATCAGACAAAGAtmC-p2-For GTCTGATGCTTGTTGATGAT ATAAGTGACGGCAGTGACTATCAtmC-p2-Rev TTTGCAATTGGGAATACCAT GCTACTGTACTTAAAGTATCATCGAtmC-p3-For GATACTTTAAGTACAGTAGC ATGGTATTCCCAATTGCAAAATGAtmC-p3-Rev TGATAATGAAAACTATAAATCGTGAAGGCATGTTT TCATTTCTCGTTCTTCATGTCCAtmM-p1-For TGGCTAGCGATTATAAGGATGATGATGATAAGACT ATGTGTGATAAAGATCGCTTCAtmM-p1-Rev ATGCAATGGGATACCCGAAT CTTTCATCGACAGTTTTTGGAtmM-p2-For CCAAAAACTGTCGATGAAAG ATTCGGGTATCCCATTGCAtmM-p2-Rev GGCGTATTCGACCTTCATGC TTCTCTTTTCCCGCTTCGAtmM-p3-For GTCGAAGCGGGAAAAGAGAA GCATGAAGGTCGAATACGAtmM-p3-Rev TGTCCGATGTTTGGCGTCAT CTTGTGAATACTGTCCCCAtmM-p4-For TTGGGGACAGTATTCACAAG ATGACGCCAAACATCGGACAtmM-p4-Rev TGTCATTTAAATTAGTGATGGTGATGGTGATGCAC CTCAACCGTTTTAGAAACCAGAtmB-p1-For ATCAACTATCAACTATTAACTATATCGTAATACCA TATG GACGGATTTGGCTCATCAtmB-p1-Rev ATCCTAGGAATCGGGAAAGC CAGAGCGTGTACGAGGCTCAtmB-p2-For GGAGCCTCGTACACGCTCTG GCTTTCCCGATTCCTAGGATCAtmB-p2-Rev TGATAATGAAAACTATAAATCGTGAAGGCATGTTT CTAGACTGTCTTCCGATCGGAfB-p1-For ATCAACTATCAACTATTAACTATATCGTAATACCA TATG GACAGCTTCGATCTCGCCAfB-p1-Rev AGCCTGTGAAGCGGAAGAAC CACAGATGCATTGAAAACCCCCAfB-p2-For GGGTTTTCAATGCATCTGTG GTTCTTCCGCTTCACAGGCTCAfB-p2-Rev TGATAATGAAAACTATAAATCGTGAAGGCATGTTT TCACTTCCTCTTCCGCCCATCAtmC-G-xw06-For ATAT GCGGCCGC AAAACGTAGGGGC (the underlined is the Not I site)AtmC-G-xw06-Rev ATAT GCGGCCGC CGACGTTGTAAAACGACGGCCAG (the underlined is the Not I site)AtS5B1-p1-For ATCAACTATCAACTATTAACTATATCGTAATACCA TATG GATGGGTTCGACCATTCTACAtS5B1-p1-Rev ATCCAAAAAACCTGGAGGTC CATAGCGTCCATGACGCCCCTCAtS5B1-p2-For GGGGCGTCATGGACGCTATG GACCTCCAGGTTTTTTGGATCAACAtS5B1-p2-Rev TGATAATGAAAACTATAAATCGTGAAGGCATGTTT CTACCTCTCTTTGTGCTTGATTCCAtS5M-p1-For TGGCTAGCGATTATAAGGATGATGATGATAAGACT ATGGCCAATGCCCAGCAACAtS5M-p1-Rev ATGCAACCGGGTATCCGAAC CTGTCGCCAAGAACCTTAGAtS5M-p2-For CCTAAGGTTCTTGGCGACAG GTTCGGATACCCGGTTGCAtS5M-p2-Rev GACATATTCAACAGTCATGC TTCTTCTTTCACGTGTCGAAACAtS5M-p3-For TTCGACACGTGAAAGAAGAA GCATGACTGTTGAATATGTCTGAtS5M-p3-Rev TGGCCAAAGTTGGGCGTCAT CTTGTGAACGCTATCTCCAATCAtS5M-p4-For TTGGAGATAGCGTTCACAAG ATGACGCCCAACTTTGGCCAtS5M-p4-Rev TGTCATTTAAATTAGTGATGGTGATGGTGATGCAC TCTACTCAACGCATTGCTTTGAtS5-p450-p1-For ATCAACTATCAACTATTAACTATATCGTAATACCA TATG ACTCCACCCCTCCTCAtS5-p450-p1-Rev GATGGAGATCTCCCTAGGTT TAATCCGAACAACATCCCCATACACCAtS5-p450-p2-For TGGGGATGTTGTTCGGATTA AACCTAGGGAGATCTCCATCAtS5-p450-p2-Rev GGGTTCGTAGCCGGCAAGTG CCTTCGACGTAAACCCCCGAtS5-p450-p3-For TCGGGGGTTTACGTCGAAGG CACTTGCCGGCTACGAACAtS5-p450-p3-Rev TCCCGACGATCAGTTTCGAC CCTACTCCTCTCCTCCACAtS5-p450-p4-For GTGTGGAGGAGAGGAGTAGG GTCGAAACTGATCGTCGGGAtS5-p450-p4-Rev ACGGGGGAAGTTGCGCGAGT CTCGTTGAATCGTATACGTCAtS5-p450-p5-For GACGTATACGATTCAACGAG ACTCGCGCAACTTCCCCCAtS5-p450-p5-Rev TGATAATGAAAACTATAAATCGTGAAGGCATGTTT CTATCTCTTTGTAAAGAGTAGAtS2B-p1-For ATCAACTATCAACTATTAACTATATCGTAATACCA TATG GATGCCTTTGATCTTTCCAtS2B-p1-Rev AGCCCGTGAATCGGAAGAAC CATAAGTTAAGAGAGAAACCACAtS2B-p2-For GGTTTCTCTCTTAACTTATG GTTCTTCCGATTCACGGGCAtS2B-p2-Rev TGATAATGAAAACTATAAATCGTGAAGGCATGTTT TTACAGACTCTTGATACTTTTCG

1 8.06, brs

2 6.94, s 121.42, CH

3 116.18, C

4 127.61, C

5 7.59, d (7.8) 119.12, CH H6

6 7.10, dd (7.2, 7.8) 119.21, CH H5, H7

7 7.18, dd (7.2, 8.1) 122.00, CH H6, H8

8 7.35, d (8.1) 111.19, CH H7

9 136.64, C

10 3.46, d (7.0) 24.02, CH2 H11

11 5.46, t (7.0) 123.27, CH

12 135.52, C

13 2.06, m 39.71, CH2 H14

14 2.08, m

2.14, m

26.52, CH2

15 5.18, t (6.5) 124.84, CH H14

16 134.28, C

17 2.06, m

2.13, m

36.42, CH2 H18

18 1.59, overlap 27.46, CH2

19 2.70, t (6.2) 63.64, CH H18

20 61.09, C

21 1.39, m

1.63, overlap

38.99, CH2 H22

22 2.06, m 24.07, CH2

23 5.08, t (7.0) 123.84, CH H22

24 132.03, C

25 1.60, s 17.81, CH3

26 1.68, s 25.85, CH3

27 1.75, s 16.21, CH3

28 1.61, s 16.09, CH3

29 1.24, s 16.63, CH3

HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30962.[α]21.8

D +18.67 (c = 0.1, CHCl3)

Table S4. Spectral data of paspaline (3)1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

S11


1 7.76, brs

2 150.99, C

3 118.35, C

4 125.24, C

5 7.42, m 118.52, CH H6

6 7.07, m 119.67, CH

7 7.07, m 120.55, CH

8 7.29, m 111.58, CH H7

9 140.10, C

10 2.33, dd (10.7, 13.2)

2.67, dd (6.3, 13.2)

27.64, CH2

11 2.76, m 48.88, CH H10, H12

12 1.59, m

1.78, m

25.40, CH2 H13

13 1.41, m

1.65, m

22.06, CH2

14 1.47, m 46.53, CH H13

15 36.67, C

16 1.11, m

1.83, m

37.77, CH2

17 1.38, m

1.69, m

22.09, CH2

18 3.21, dd (2.9, 8.6) 84.81, CH H17

19 3.03, dd (3.8, 6.8) 85.83, CH

20 1.68, m 24.75, CH2

21 1.62, m

1.96, m

34.02, CH2

22 40.13, C

23 53.13, C

24 72.09, C

25 1.18, s 23.83, CH3

26 1.19, s 26.23, CH3

27 1.03, s 14.70, CH3

28 1.13, s 20.12, CH3

29 0.88, s 12.79, CH3

HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO2, 422.30536, found 422.30440.[α]21.6

D -42.50 (c = 0.4, CHCl3) ; (reported in literature7 [α]D -23.0 (c = 0.36, CHCl3) )

Table S5. Spectral data of aflavinine (5)1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

S12


1 8.03, brs

2 6.89, d (2.2) 121.40, CH

3 118.72, C

4 127.69, C

5 7.43, d (7.9) 119.74, CH H6

6 7.09, ddd (7.1, 7.9, 1.0) 119.52, CH H5, H7

7 7.19, ddd (7.1, 8.1, 1.0) 121.92, CH

8 7.37, d (8.1) 111.15, CH H7

9 135.98, C

10 125.57, C

11 141.22, C

12 2.22, m 20.55, CH2 H13

13 1.82, m

2.10, m

21.88, CH2

14 42.51, C

15 4.48, t (3.0) 71.25, CH H16

16 1.75, m

2.03, m

30.24, CH2

17 1.22, m

1.70, m

25.50, CH2

18 2.03, m 31.39, CH H17

19 38.56, C

20 1.18, m

1.54, m

27.70, CH2

21 1.15, m

1.63, m

25.75, CH2

22 1.97, m 29.23, CH

23 2.43, d (5.5) 43.70, CH H22

24 2.58, m 31.09, CH

25 0.96, d (6.9) 22.03, CH3 H24

26 0.83, d (6.95) 20.92, CH3 H24

27 0.76, d (7.3) 15.87, CH3 H16

28 0.99, s 18.17, CH3

29 1.08, d (7.5) 18.20, CH3 H22


D +38.67 (c = 0.3, CHCl3); (reported in literature8 [α]D +24.9 (c = 0.98, CHCl3) )

Table S6. Spectral data of anominine (6) 1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

S13

position

δH , mult (J in Hz) δC COSY

1 7.90, brs2 6.95, brs 121.40, CH3 117.20, C4 127.84, C5 7.61, d (7.8) 118.70, CH H66 7.12, dd (7.2, 7.8) 119.28, CH H5, H77 7.18, dd (7.2, 8.1) 121.99, CH8 7.34, d (8.1) 111.20, CH H79 136.01, C10 3.02-3.14, m 21.49, CH2 H1111 3.24, d (9.9) 45.97, CH12 149.08, C13 2.10, m

2.21, m33.62, CH2 H14

14 1.52, m 34.52, CH2

15 40.97, C16 2.45, m 31.24, CH H17, H2817 1.33, m

1.70, m25.51, CH2

18 1.64, m, overlap1.93, m

28.86, CH2 H17

19 4.54, brs 70.11, CH H1820 47.99, C21 1.68, m, overlap 29.28, CH2

22 2.21, m 23.95, CH2

23 5.18, t (6.6) 125.96, CH H2224 131.43, C25 1.72, s 25.91, CH3

26 1.69, s 17.97, CH3

27 4.82, brs4.93, brs

107.89, CH2

28 0.83, d (6.6) 16.78, CH3 H1629 1.02, s 18.84, CH3


D +46.00 (c = 0.1, MeOH); (reported in literature9 [α]D +23.6 (c = 0.1, MeOH) )

Table S7. Spectral data of compound 81H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

S14


1 7.92/7.99, brs

2 6.95, brs 121.31, CH

3 116.30/116.28, C

4 127.64, C

5 7.59, d (7.9) 119.19/119.18, CH H6

6 7.10, dd (7.1, 7.9) 119.26/119.24, CH H5, H7

7 7.18, dd (7.1, 8.1) 122.06/122.04, CH

8 7.35, d (8.1) 111.16, CH H7

9 136.64, C

10 3.46, d (7.1) 24.12/24.11, CH2

11 5.45, t (7.1) 123.07/123.11, CH H10

12 135.75/135.69, C

13 1.98, m, overlap

2.06, m, overlap

39.87/39.83, CH2

14 2.07, m

2.12, m

26.73/26.65, CH2

15 5.12-5.20, m, overlap 124.51/124.56, CH H14

16 135.03/135.02, C

17 1.98, m, overlap

2.06, m, overlap

39.79/39.81, CH2

18 1.99, m

2.07, m

26.75/26.69, CH2

19 5.12-5.20, m, overlap 125.15/125.35, CH

20 134.16, C

21 1.99, m

2.20, m

36.46/36.98, CH2 H22

22 1.48, m

1.58, m, overlap

30.12/29.72, CH2

23 3.91, dd (3.2, 10.1)/3.34,

dd (1.8, 10.4)

84.92/78.49, CH H22

24 81.46/73.14, C

25 1.32/1.19, s 28.36/26.54, CH3

26 1.20/1.15, s 23.31/23.44, CH3

27 1.76, s 16.25/16.22, CH3

28 1.60, s, overlap 16.17/16.13, CH3

29 1.60, s, overlap 16.16/16.03, CH3

HRMS-ESI (m/z) [M+H]+ calcd for C28H42NO2, 424.32101, found 424.32040.

Table S8. Spectral data of compound 10 1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

S15


1 8.32, brs

2 6.95, brs 121.71, CH

3 116.00, C

4 127.62, C

5 7.58, d (7.9) 119.06, CH H6

6 7.09, dd (7.1, 7.9) 119.12, CH H5, H7

7 7.17, dd (7.1, 8.1) 121.90, CH

8 7.34, d (8.1) 111.19, CH H7

9 136.67, C

10 3.45, d (7.2) 23.87, CH2

11 5.47, t (7.2) 123.30, CH H10

12 135.54, C

13 2.10, m 39.69, CH2 H14

14 2.16, m 26.30, CH2

15 5.21, t (6.5) 124.86, CH H14

16 135.06, C

17 2.04, m

2.26, m

36.83, CH2

18 1.35, m

1.60, m

30.41, CH2

19 3.57, br d (10.4) 77.16, CH H18

20 86.55, C

21 1.48, m

2.10, m

31.50, CH2 H22

22 1.90, m 27.11, CH2

23 3.83, t (7.4) 84.54, CH H22

24 72.29, C

25 1.15, s, overlap 25.62, CH3

26 1.27, s 27.77, CH3

27 1.15, s, overlap 24.00, CH3

28 1.62, s 15.91, CH3

29 1.73, s 16.22, CH3

HRMS-ESI (m/z) [M+H]+ calcd for C28H42NO3, 440.31592, found 440.31526.

Table S9. Spectral data of emindole SB (11)1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3

S16


1 7.70, brs

2 151.05, C

3 118.46, C

4 125.25, C

5 7.42, m 118.56, CH H6

6 7.07, m 119.72, CH

7 7.07, m 120.60, CH

8 7.29, m 111.56, CH H7

9 140.08, C

10 2.34, dd (10.6, 13.2)

2.68, dd (6.4, 13.2)

27.60, CH2 H11

11 2.73, m 48.93, CH

12 1.61, m

1.77, m

25.27, CH2 H11, H13

13 1.42, m

1.64, m

22.87, CH2

14 1.74, m 39.99, CH H13

15 41.37, C

16 3.58, dd (7.2, 9.0) 73.46, CH H17

17 1.81, m 27.60, CH2

18 1.57, m

1.92, m

33.63, CH2

19 39.41, C

20 53.25, C

21 1.31, m

1.52, m

37.64, CH2 H22

22 1.88, m

1.96, m

21.50, CH2

23 5.13, m 124.75, CH H22

24 131.54, C

25 1.70, s 25.90, CH3

26 1.64, s 17.82, CH3

27 1.02, s 14.79, CH3

28 1.11, s 19.33, CH3

29 0.82, s 16.58, CH3


D -22.50 (c = 0.4, CHCl3)


S17


1 7.91, brs

2 6.94, d (1.8) 122.01, CH

3 116.20, C

4 127.93, C

5 7.58, d (7.9) 119.28, CH H6

6 7.09, ddd (7.1, 7.9, 1.0) 119.17, CH H5, H7

7 7.17, ddd (7.1, 7.6, 1.0) 121.92, CH

8 7.35, d (7.6) 111.17, CH H7

9 136.51, C

10 2.29, m

3.09,

27.04, CH2 H11

11 1.48, m 47.80, CH H10, H20

12 40.08, C

13 148.35, C

14 5.45, t (3.2) 112.51, CH H15

15 2.08, m

2.28, m

31.45, CH2

16 3.56, dd (5.1, 7.1) 71.99, CH H15

17 39.07, C

18 2.15, m 40.44, CH H19

19 1.03, m overlap

1.67, m, overlap

28.49, CH2 H18, H20

20 1.20, m

1.67, m, overlap

28.49, CH2

21 1.25-1.33, m 37.20, CH2 H22

22 1.95, m 22.20, CH2

23 5.10, m 125.16, CH H22

24 131.52, C

25 1.68, s 25.87, CH3

26 1.61, s 17.78, CH3

27 1.36, s 26.17, CH3

28 1.04, s 22.28, CH3

29 0.78, s 17.31, CH3


D -40.33 (c = 0.4, CHCl3)ECD (CH2Cl2) λmax (mdeg) 228 (-3.31), 284 (+1.80) nm.


S18


1 7.95, brs

2 6.95, d (1.8) 122.09, CH

3 116.25, C

4 127.93, C

5 7.58, d (7.8) 119.28, CH H6

6 7.10, dd (7.2, 7.8) 119.17, CH H5, H7

7 7.18, dd (7.2, 8.1) 121.91, CH

8 7.35, d (8.1) 111.20, CH H7

9 136.53, C

10 2.29, dd (14.0, 11.0)

3.09, br d (14.0)

27.11, CH2 H11

11 1.45, m 46.76, CH H10, H23

12 39.69, C

13 146.95, C

14 5.54, br d (6.0) 114.49, CH H15

15 1.97, m

2.07, m

28.97, CH2

16 3.22, dd (5.5, 10.4) 79.95, CH H15

17 3.14, dd (2.6, 11.4) 83.97, CH H18

18 1.42, m

1.48, m

22.24, CH2

19 1.19, m

1.88, m

36.42, CH2

20 34.58, C

21 1.91, m 45.64, CH H22

22 0.92, m

1.62, m

25.60, CH2

23 1.23, m

1.68, m

27.30, CH2

24 72.02, C

25 1.17, s 26.13, CH3

26 1.15, s 23.85, CH3

27 1.36, s 25.78, CH3

28 1.01, s 23.41, CH3

29 0.70, s 13.34, CH3


D -33.93 (c = 1.0, CHCl3)ECD (CH2Cl2) λmax (mdeg) 228 (-1.23), 284 (+1.52) nm.


S19


1 7.91, brs

2 7.05, d (1.8) 122.96, CH

3 116.87, C

4 127.59, C

5 7.5, d (7.4) 118.53, CH H6

6 7.17, ddd (7.1, 7.4, 1.1) 119.19, CH H5, H7

7 7.11, ddd (7.1, 8.0, 1.1) 121.82, CH

8 7.35, d (8.0) 111.29, CH H7

9 136.12, C

10 3.69, dd (5.4, 13.0) 34.70, CH H11, H23

11 2.61, m 38.74, CH H10, H12

12 1.45, m 29.91, CH

13 0.86, m

1.64, m

29.03, CH2 H12, H14

14 1.17, m

1.51, m

28.25, CH2

15 39.37, C

16 2.17, m 31.51, CH H28, H17

17 1.37, m

1.75, m

25.39, CH2 H18

18 1.89, m

2.19, m

30.21, CH2

19 4.85, t (2.5) 69.19, CH H18

20 44.16, C

21 1.84, m

2.04, m

24.72, CH2 H22

22 1.74, m

1.82, m

28.01, CH2

23 3.19, m 43.72, CH H10, H22

24 150.51, C

25 4.66, m

4.79, d (2.2)

111.20, CH2

26 1.52, s 18.49, CH3

27 1.25, d (7.3) 22.02, CH3 H12

28 0.78, d (6.8) 15.98, CH3 H16

29 0.98, s 18.58, CH3


D -0.73 (c = 1.0, CHCl3) ; (reported in literature8 [α]D -1.2 (c = 0.5, CHCl3) )


S20


1 8.02, brs

2 6.97, d (2.0) 123.12

3 114.98

4 128.53

5 7.55, d (7.8) 118.19 H6

6 7.14, dd (7.2, 7.8) 119.41 H5, H7

7 7.21, dd (7.2, 8.1) 121.93

8 7.39, d (8.1) 111.25 H7

9 135.79

10 4.21, brs 36.13 H23

11 144.07

12 5.67, m 120.26 H13

13 2.48, m, overlap

2.34, m

27.66

14 43.45

15 4.31, brs 70.67

16 1.83, m

2.20, m, overlap

29.23

17 overlap with H2O signal

1.38, m

25.66

18 2.21, m, overlap 30.56

19 38.99


1.20, m

28.66


0.88, m

27.75

22 1.42, m 29.50

23 2.48, m, overlap 39.16 H10, H22

24 2.61, m 29.92 H25, H26

25 1.08, d (6.8), overlap 20.92

26 0.74, d (6.8) 23.46

27 0.77, d (6.8) 16.09 H18

28 0.94, s 18.49

29 1.08, d (6.8), overlap 16.27 H22


D +14.00 (c = 0.1, CHCl3)


S21


1 7.71, brs

2 150.95, C

3 118.47, C

4 125.23, C

5 7.42, m 118.56, CH H6

6 7.07, m 119.73, CH

7 7.07, m 120.63, CH

8 7.29, m 111.57, CH H7

9 140.09, C

10 2.33, dd (10.6,

13.2)

2.67, dd (6.4, 13.2)

27.60, CH2 H11

11 2.75, m 48.90, CH H10, H12

12 1.61, m

1.77, m

25.27, CH2

13 1.44, m

1.63, m

22.87, CH2 H12, H14

14 1.73, m 40.07, CH H13

15 41.37, C

16 3.58, dd (7.2, 9.0) 73.46, CH H17

17 1.82, m 27.72, CH2 H16, H18

18 1.58, m

1.91, m

33.52, CH2

19 39.41, C

20 53.26, C

21 1.34, m

1.57, m

37.28, CH2 H22

22 1.93, m

2.03, m

21.13, CH2

23 5.43, t (6.9) 126.52, CH H22

24 134.81, C

25 4.01, s 69.12, CH2

26 1.70, s 13.83, CH3

27 1.03, s 14.82, CH3

28 1.11, s 19.33, CH3

29 0.83, s 16.55, CH3


D -4.0 (c = 0.2, CHCl3)

Table S15. Spectral data of compound 17S22

1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3


1 7.93, brs

2 6.95, brs 122.10, CH

3 116.24, C

4 127.91, C

5 7.58, d (7.9) 119.28, CH H6

6 7.09, dd (7.2, 7.9) 119.20, CH H5, H7

7 7.17, dd (7.2, 8.1) 121.94, CH

8 7.35, d (8.1) 111.21, CH H7

9 136.54, C

10 2.29, dd (14.0, 11.0)

3.09, brd (14.0)

27.11, CH2 H11

11 1.44, m 46.87, CH H10, H23

12 39.73, C

13 147.60, C

14 5.54, m 114.50, CH H15

15 1.95, m

2.02, m

29.26, CH2

16 3.59, dd (5.6, 10.1) 73.56, CH H15

17 3.70, overlap 77.21, CH H18

18 1.71, m

1.78, m

19.69, CH2

19 1.45, m

1.64, m

33.55, CH2

20 34.86, C

21 1.95, m 46.10, CH H22

22 0.93, m

1.60, m

25.59, CH2 H21, H23

23 1.20, m, overlap

1.67, m

27.34, CH2

24 75.41, C

25 3.42, d (10.8)

3.70, overlap

68.11, CH2

26 1.20, s 21.11, CH3

27 1.35, s 25.80, CH3

28 1.01, s 23.43, CH3

29 0.74, s 15.81, CH3


D -28.83 (c = 0.4, CHCl3)

Table S16. Spectral data of compound 19S23



1 7.92, brs

2 6.94, brs 122.06, CH

3 116.15, C

4 127.91, C

5 7.57, d (7.8) 119.27, CH H6

6 7.09, dd (7.0, 7.8) 119.19, CH H5, H7

7 7.17, dd (7.0, 8.1) 121.94, CH

8 7.35, d (8.1) 111.20, CH H7

9 136.52, C

10 2.27, m

3.09, br d (13.9)

27.05, CH2 H11

11 1.45, m 47.60, CH

12 40.06, C

13 148.19, C

14 5.47, brs 112.86, CH H15

15 2.07, m

2.24, m

31.67, CH2

16 3.56, dd (5.2, 7.2) 72.50, CH H15

17 39.02, C

18 2.07, m 40.70, CH H19

19 1.01, m, overlap

1.66, m

27.97, CH2

20 1.19, m

1.66, m

28.28, CH2 H19, H11

21 1.40, m

1.59, m

34.05, CH2

22 1.27, m 22.84, CH2

23 3.48, overlap 79.04, CH

24 74.07, C

25 3.48, overlap

3.82, d (11.1)

67.79, CH2

26 1.12, s 21.09, CH3

27 1.36, s 26.12, CH3

28 1.02. s 22.48, CH3

29 0.77, s 16.76, CH3


D -23.33 (c = 0.2, CHCl3)

Table S17. Spectral data of compound 20a/bS24



1 7.90, brs

2 6.95, brs 121.42, CH

3 117.11, C

4 127.81, C

5 7.61, d (7.3) 118.67, CH H6

6 7.12, dd (6.8, 7.3) 119.33, CH H5, H7

7 7.18, dd (6.8, 8.0) 122.03, CH

8 7.34, d (8.0) 111.22, CH H7

9 136.02, C

10 3.04-3.13, m 21.52, CH2 H11

11 3.24, d (9.8) 45.97, CH

12 149.05, C

13 2.12, m

2.21, m

33.61, CH2

14 1.52, m 34.54, CH2 H13

15 41.00, C

16 2.47, m 31.23, CH H17, H28

17 1.36, m

1.70, m

25.46, CH2

18 1.65, m

1.92, m

29.03, CH2

19 4.54, brs 70.11, CH H18

20 47.96, C

21 1.73, m, overlap 29.03, CH2

22 2.30, m 23.58, CH2 H22, H21

23 5.48, t (6.6) 127.62, CH H22

24 134.55, C

25 4.04, s 69.18, CH2

26 1.74, s 13.95, CH3

27 4.82, brs

4.93, brs

107.95, CH2

28 0.83, d (6.6) 16.76, CH3

29 1.02, s 18.86, CH3


D +3.50 (c = 0.4, CHCl3)

Figure S1. HPLC analysis (λ=280 nm) of the extracts of yeast strains expressing different combinations of indole diterpene S25

biosynthetic genes

Figure S2. The map of the nonclustered indole diterpene cyclase genes and other genes around

Note: HP means hypothetical protein

Figure S3. The UV spectrum (left) and MS data (right) of the characterized indole diterpenes

S26

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00Inten. (x1,000,000)

406

454

438388 476464421 492447410370

500305 317 352339327

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU

10.762/ 1.00

224

196

281

482

206

254

486

361

Figure S3. The UV spectrum (left) and MS data (right) of the characterized indole diterpenes (continued)

S27

200 300 400 500 nm

-200

-100

0

100

200

300mAU

10.763/ 1.00

226

275

482

540

267

317

195

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00Inten. (x1,000,000)

406

454

438388 476464421 492447410370

500305 317 352339327

350.0 375.0 400.0 425.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

Inten. (x100,000)

406

388444428 436410

422 448392361 402380352 368

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU

9.089/ 1.00

213

281

577

551

502

196

257

543

533

513

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0Inten. (x100,000)

406388

438

420410

454479

496366 386 474323 354334300

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x1,000,000)

446424

496406482441 497

462 472335 345 388361308 409377319

200 300 400 500 nm

0

100

200

300

400

500

600

700

800mAU

9.938/ 1.00

225

194

282

483

207

255

530

356

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

0.5

1.0

1.5

2.0

Inten. (x100,000)

406388

438

444

422410 454 479

464304495337 379354323

200 300 400 500 nm

0

500

1000

1500

2000

2500mAU

10.183/ 1.00

226

201

282

208

258

411

571

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

1.25Inten. (x100,000)

422

404

454499

438

485389 410312 337 488376 450302

472355324 366

200 300 400 500 nm

0

250

500

750

mAU 9.917/ 1.00

224

197

281

483

545

206

253

496

571

350

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU

10.762/ 1.00

224

196

281

482

206

254

486

361


S28

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

Inten. (x100,000)

406

438

388422

410 447499

465 483 496305 334 348 360 380

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

Inten. (x1,000,000)

440

462

422

442 472 494478305 404361323 419393336 380351 370

200 300 400 500 nm

0

250

500

750

1000

1250

1500

1750

mAU 10.246/ 1.00

226

196

281

490

468

208

252

481

567

361

200 300 400 500 nm

0

250

500

750

mAU 8.800/ 1.00

223

197

281

483

207

252

356

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x1,000,000)

422

444

470

439 476404 492460305 328 343 377362 396 419

300.0 325.0 350.0 375.0 400.0 425.0 450.0 m/z0.00

0.25

0.50

0.75

1.00Inten. (x1,000,000)

444

404

420

454437

472463

418312479

365300

386330 377340

200 300 400 500 nm-150

-100

-50

0

50

100

150

200

250

mAU 9.496/ 1.00

225

281

542

263

485

323

195

200 300 400 500 nm

0

250

500

750

1000

1250

mAU 9.813/ 1.00

218

197

282

544

454

207

250

489

571

355

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU

9.089/ 1.00

213

281

577

551

502

196

257

543

533

513

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

1.25

1.50

Inten. (x1,000,000)

446424

496406482441 497

462 472335 345 388361308 409377319

200 300 400 500 nm

0

100

200

300

400

500

600

700

mAU 10.055/ 1.00

224

196

282

483

544

206

253

347

355

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

Inten. (x100,000)

406388

438 450420

410 476 500460

465 494386338322 365349308


S29

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU

7.637/ 1.00

224

195

282

482

565

207

251

356

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

Inten. (x1,000,000)

438

460

420

470442300

362 486497409 476321 392344 381

200 300 400 500 nm

0

100

200

300

400

500

600

700mAU

7.987/ 1.00

229

281

482

417

565

205

254

526

558

366

350.0 375.0 400.0 425.0 m/z0.0

2.5

5.0

7.5

Inten. (x100,000)

404422

438386

439397 419 427

449357 409362 372 391382

200 300 400 500 nm

-100

-50

0

50

100

150

200

250

300

mAU 9.980/ 1.00

225

282

483

56526

2

356

195

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

1.0

2.0

3.0

4.0

5.0Inten. (x10,000)

388

406

444

420 492438 471454369346334499

384313

300.0 325.0 350.0 375.0 400.0 425.0 450.0 m/z0.00

0.25

0.50

0.75

1.00Inten. (x1,000,000)

444

404

420

454437

472463

418312479

365300

386330 377340

200 300 400 500 nm

0

250

500

750

1000

1250

mAU 9.813/ 1.00

218

197

282

544

454

207

250

489

571

355

200 300 400 500 nm

0

250

500

750

1000

mAU 9.723/ 1.00

226

194

283

577

502

207

253

533

526

513

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

2.5

5.0

7.5

Inten. (x100,000)

406

388

420 438

447474

499

456 476 492418386362343321 374330307

S30

200 300 400 500 nm

0

100

200

300

400

500mAU

7.705/ 1.00

226

283

565

207

253

554

355

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0Inten. (x100,000)

422404

444

386

476460387

474 492441 499

312 418372357336

350.0 375.0 400.0 425.0 450.0 475.0 500.0 525.0 m/z0.0

1.0

2.0

3.0

4.0

5.0

Inten. (x1,000,000)

478

438

420456 510 519488

526436 460 538402549351

362 391377

200 300 400 500 nm

0

100

200

300

400

500

600

mAU 6.068/ 1.00

225

195

282

483

545

206

251

487

354

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU 7.065/ 1.00

224

195

282

482

565

207

252

562

355

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.0

1.0

2.0

3.0

4.0

Inten. (x1,000,000)

460

438

494302

420470

330 479416362343 452497

332 402311 392

200 300 400 500 nm

0

100

200

300

400

500

600

700

800

900mAU

7.637/ 1.00

224

195

282

482

565

207

251

356

300.0 325.0 350.0 375.0 400.0 425.0 450.0 475.0 m/z0.00

0.25

0.50

0.75

1.00

Inten. (x1,000,000)

438

460

420

470442300

362 486497409 476321 392344 381

Figure S4. 1H NMR of compound 1 in CDCl3

Figure S5. 13C NMR of compound 1 in CDCl3

S31

Figure S6. COSY of compound 1 in CDCl3

Figure S7. HSQC of compound 1 in CDCl3.S32

Figure S8. HMBC of compound 1 in CDCl3

Figure S9. NOESY of compound 1 in CDCl3

S33

Figure S10. 1H NMR of compound 3 (paspaline) in CDCl3

Figure S11. 13C NMR of compound 3 (paspaline) in CDCl3

S34

Figure S12. COSY of compound 3 (paspaline) in CDCl3

Figure S13. HSQC of compound 3 (paspaline) in CDCl3.S35

Figure S14. HMBC of compound 3 (paspaline) in CDCl3

Figure S15. NOESY of compound 3 (paspaline) in CDCl3

S36

Figure S16. 1H NMR of compound 5 (aflavinine) in CDCl3

Figure S17. 13C NMR of compound 5 (aflavinine) in CDCl3

S37

Figure S18. COSY of compound 5 (aflavinine) in CDCl3

Figure S19. HSQC of compound 5 (aflavinine) in CDCl3

S38

Figure S20. HMBC of compound 5 (aflavinine) in CDCl3

Figure S21. NOESY of compound 5 (aflavinine) in CDCl3

S39

Figure S22. 1H NMR of compound 6 (anominine) in CDCl3

Figure S23. 13C NMR of compound 6 (anominine) in CDCl3

S40

Figure S24. COSY of compound 6 (anominine) in CDCl3

Figure S25. HSQC of compound 6 (anominine) in CDCl3

S41

Figure S26. HMBC of compound 6 (anominine) in CDCl3

Figure S27. NOESY of compound 6 (anominine) in CDCl3

S42



S43


Figure S31. HSQC of compound 8 in CDCl3

S44



S45



S46



S47

Figure S38. 1H NMR of compound 11 (emindole SB) in CDCl3

Figure S39. 13C NMR of compound 11 (emindole SB) in CDCl3

S48

Figure S40. COSY of compound 11 (emindole SB) in CDCl3

Figure S41. HSQC of compound 11 (emindole SB) in CDCl3

S49

Figure S42. HMBC of compound 11 (emindole SB) in CDCl3

Figure S43. NOESY of compound 11 (emindole SB) in CDCl3

S50



S51



S52



S53



S54



S55



S56



S57



S58



S59



S60



S61



S62



S63



S64



S65



S66



S67



S68



S69



S70

Figure S84. 1H NMR of compound 20a/b in CDCl3

Figure S85. 13C NMR of compound 20a/b in CDCl3

S71

Figure S86. COSY of compound 20a/b in CDCl3

Figure S87. HSQC of compound 20a/b in CDCl3

S72

Figure S88. HMBC of compound 20a/b in CDCl3

Figure S89. NOESY of compound 20a/b in CDCl3

S73

Figure S90. Inferred phylogeny of IDTC and homologous proteins from fungi.

S74

References:1. Edgar, R. C. Nucleic Acids Res. 2004, 32, 1792.2. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D. L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M. A.;

Huelsenbeck, J. P. Syst. Biol. 2012, 61, 539.3. Fang, F.; Salmon, K.; Shen, M. W. Y.; Aeling, K. A.; Ito, E.; Irwin, B.; Tran, U. P. C.; Hatfield, G. W.; Da Silva, N. A.;

Sandmeyer, S. Yeast 2011, 28, 123. 4. Gietz, R. D.; Schiestl, R. H.; Willems, A. R.; Woods, R. A. Yeast 1995, 11, 355. 5. Mutka, S. C.; Bondi, S. M.; Carney, J. R.; Da Silva, N. A.; Kealey, J. T. FEMS Yeast Res 2006, 6, 40. 6. Altschul, S. F.; Madden, T. L.; Schäffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J. Nucleic Acids Res. 1997, 25, 3389. 7. Fehr, T.; Acklin, W. Helv. Chem. Acta 1966, 49, 1907. 8. TePaske, M. R.; Gloer, J. B. Tetrahedron 1989, 45, 4961. 9. Gloer, J. B.; Rinderknecht, B. L. J. Org. Chem. 1989, 54, 2530.

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