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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].
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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
position δH , mult (J in Hz) δC COSY
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
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30919.[α]21.8
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30953.[α]21.4
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
position δH , mult (J in Hz) δC COSY
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
position δH , mult (J in Hz) δC COSY
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
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30909.[α]21.8
D -22.50 (c = 0.4, CHCl3)
Table S10. Spectral data of compound 121H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
S17
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30978.[α]21.8
D -40.33 (c = 0.4, CHCl3)ECD (CH2Cl2) λmax (mdeg) 228 (-3.31), 284 (+1.80) nm.
Table S11. Spectral data of compound 131H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
S18
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO2, 422.30536, found 422.30649.[α]21.6
D -33.93 (c = 1.0, CHCl3)ECD (CH2Cl2) λmax (mdeg) 228 (-1.23), 284 (+1.52) nm.
Table S12. Spectral data of compound 141H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
S19
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30961.[α]22.2
D -0.73 (c = 1.0, CHCl3) ; (reported in literature8 [α]D -1.2 (c = 0.5, CHCl3) )
Table S13. Spectral data of compound 151H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
S20
position δH , mult (J in Hz) δC COSY
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
20 overlap with H2O signal
1.20, m
28.66
21 overlap with H2O signal
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO, 406.31044, found 406.30952.[α]21.8
D +14.00 (c = 0.1, CHCl3)
Table S14. Spectral data of compound 161H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
S21
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO2, 422.30536, found 422.30433.[α]21.6
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
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO3, 438.30027, found 438.29945.[α]21.6
D -28.83 (c = 0.4, CHCl3)
Table S16. Spectral data of compound 19S23
1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H42NO4, 456.31084, found 456.30866.[α]21.8
D -23.33 (c = 0.2, CHCl3)
Table S17. Spectral data of compound 20a/bS24
1H NMR spectrum (500 MHz), 13C NMR spectrum (125 MHz), CDCl3
position δH , mult (J in Hz) δC COSY
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
HRMS-ESI (m/z) [M+H]+ calcd for C28H40NO2, 422.30536, found 422.30442.[α]21.8
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
Figure S3. The UV spectrum (left) and MS data (right) of the characterized indole diterpenes (continued)
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
Figure S3. The UV spectrum (left) and MS data (right) of the characterized indole diterpenes (continued)
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
Figure S28. 1H NMR of compound 8 in CDCl3
Figure S29. 13C NMR of compound 8 in CDCl3
S43
Figure S30. COSY of compound 8 in CDCl3
Figure S31. HSQC of compound 8 in CDCl3
S44
Figure S32. HMBC of compound 8 in CDCl3
Figure S33. 1H NMR of compound 10 in CDCl3
S45
Figure S34. 13C NMR of compound 10 in CDCl3
Figure S35. COSY of compound 10 in CDCl3
S46
Figure S36. HSQC of compound 10 in CDCl3
Figure S37. HMBC of compound 10 in CDCl3
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
Figure S44. 1H NMR of compound 12 in CDCl3
Figure S45. 13C NMR of compound 12 in CDCl3
S51
Figure S46. COSY of compound 12 in CDCl3
Figure S47. HSQC of compound 12 in CDCl3
S52
Figure S48. HMBC of compound 12 in CDCl3
Figure S49. NOESY of compound 12 in CDCl3
S53
Figure S50. 1H NMR of compound 13 in CDCl3
Figure S51. 13C NMR of compound 13 in CDCl3
S54
Figure S52. COSY of compound 13 in CDCl3
Figure S53. HSQC of compound 13 in CDCl3
S55
Figure S54. HMBC of compound 13 in CDCl3
Figure S55. NOESY of compound 13 in CDCl3
S56
Figure S56. 1H NMR of compound 14 in CDCl3
Figure S57. 13C NMR of compound 14 in CDCl3
S57
Figure S58. COSY of compound 14 in CDCl3
Figure S59. HSQC of compound 14 in CDCl3
S58
Figure S60. HMBC of compound 14 in CDCl3
Figure S61. NOESY of compound 14 in CDCl3
S59
Figure S62. 1H NMR of compound 15 in CDCl3
Figure S63. 13C NMR of compound 15 in CDCl3
S60
Figure S64. COSY of compound 15 in CDCl3
Figure S65. HSQC of compound 15 in CDCl3
S61
Figure S66. HMBC of compound 15 in CDCl3
Figure S67. NOESY of compound 15 in CDCl3
S62
Figure S68. 1H NMR of compound 16 in CDCl3
Figure S69. 13C NMR of compound 16 in CDCl3
S63
Figure S70. COSY of compound 16 in CDCl3
Figure S71. HSQC of compound 16 in CDCl3
S64
Figure S72. HMBC of compound 16 in CDCl3
Figure S73. NOESY of compound 16 in CDCl3
S65
Figure S74. 1H NMR of compound 17 in CDCl3
Figure S75. 13C NMR of compound 17 in CDCl3
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Figure S76. COSY of compound 17 in CDCl3
Figure S77. HSQC of compound 17 in CDCl3
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Figure S78. HMBC of compound 17 in CDCl3
Figure S79. 1H NMR of compound 19 in CDCl3
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Figure S80. 13C NMR of compound 19 in CDCl3
Figure S81. COSY of compound 19 in CDCl3
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Figure S82. HSQC of compound 19 in CDCl3
Figure S83. HMBC of compound 19 in CDCl3
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Figure S84. 1H NMR of compound 20a/b in CDCl3
Figure S85. 13C NMR of compound 20a/b in CDCl3
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Figure S86. COSY of compound 20a/b in CDCl3
Figure S87. HSQC of compound 20a/b in CDCl3
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Figure S88. HMBC of compound 20a/b in CDCl3
Figure S89. NOESY of compound 20a/b in CDCl3
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Figure S90. Inferred phylogeny of IDTC and homologous proteins from fungi.
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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.;
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