Research ArticleORGANISMAL BIOLOGY

HEx: A heterologous expression platform for the discovery of fungal natural products

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Science Advances  11 Apr 2018:
Vol. 4, no. 4, eaar5459
DOI: 10.1126/sciadv.aar5459
  • Fig. 1 Standard workflow for heterologous expression.

    Aspects in green italics are addressed in this study.

  • Fig. 2 Tools developed for the HEx platform.

    (A) eGFP expression from a series of PADH2-like promoters in cultures grown under both fermentative (YPD) and respiratory (YPE) conditions. All fluorescence intensities are reported as the mean of three biological replicates. Error bars represent 1 SD (n = 3). (B) Four fungal BGCs, two controls and two previously uncharacterized systems, each produce improved titers when heterologously expressed using PADH2-like promoters as compared to strong constitutive promoters. ND, not detected. Error bars represent 1 SD (n = 3). Quantitation for TC1 and TC3 was based on the sum of the integrations of extracted ion counts corresponding to the oxidized sesquiterpenoids outlined in table S4.

  • Fig. 3 Description and characterization of DHY strains.

    (A) Annotated genotype of the DHY yeast background. (B) DHY-derived yeast strain JHY702 shows improved growth, particularly after diauxic shift. Growth curves are representative of six biological replicates. Density plots for fluorescence intensity in multiple backgrounds show significantly improved eGFP expression when driven by both (C) PADH2 and (D) PPCK1. Density plots represent the fluorescence intensity of 104 individual cells.

  • Fig. 4 DNA assembly by yeast homologous recombination.

    (A) DNA assembly from commercially synthesized fragments and genetic parts using yeast homologous recombination. (B) Modified yeast plasmid preparation (exo+) leads to increased number of sequencing reads mapping to plasmid DNA. Dotted line marks the efficiency threshold to allow sequencing of 192 samples on a single MiSeq run. (C) Efficient assembly of up to 14 unique DNA parts can be achieved using the protocol outlined here. Data based on 78 unique assemblies.

  • Fig. 5 PKS BGCs examined for this study.

    All putative gene function abbreviations are listed in table S9. Cladogram was constructed as described in Materials and Methods. All plots are the chromatograms of the specified extracted ion in three biological replicates each of both the strain expressing the BGC and an empty vector control strain. Chromatograms are data collected with electrospray ionization in either positive (ESI+), negative (ESI−), or rapid polarity switching (RPS) mode or with multimode ionization in positive mode (MMI). Expression strains are outlined in table S10, and EICs of novel products are shown in the figs. S7 to S23.

  • Fig. 6 UbiA-type cyclases represent a general class of biosynthetic enzymes.

    Putative enzyme activity abbreviations are listed in table S9. Cladogram generated using UTC cyclase sequence. The cyclases associated with all clusters examined in this study are denoted by orange tips in the cladogram.

  • Table 1 Summary of control and cryptic fungal BGCs examined in this study.
    IDTypeSpecies of originDivisionProductive?
    IDTCtlAspergillus tubingensisAscomycotaY
    DHZCtlHypomyces subiculosusAscomycotaY
    PKS1PKSConiothyrium sporulosumAscomycotaY
    PKS2PKSConiothyrium sporulosumAscomycotaY
    PKS3PKSAcremonium Sp. KY4917AscomycotaN
    PKS4PKSAspergillus nigerAscomycotaY
    PKS5PKSThielavia terrestrisAscomycotaN
    PKS6PKSTrichoderma virensAscomycotaY
    PKS7PKSPseudogymnoascus pannorumAscomycotaN
    PKS8PKSScedosporium apiospermumAscomycotaY
    PKS9PKSMetarhizium anisopliaeAscomycotaN
    PKS10PKSCochliobolus heterostropusAscomycotaY
    PKS11PKSPseudogymnoascus pannorumAscomycotaN
    PKS12PKSPseudogymnoascus pannorumAscomycotaN
    PKS13PKSPseudogymnoascus pannorumAscomycotaY
    PKS14PKSVerruconis gallopavaAscomycotaY
    PKS15PKSMoniliophthora roreriBasidiomycotaY
    PKS16PKSPunctularia strigosozonataBasidiomycotaY
    PKS17PKSHydnomerulius pinastriBasidiomycotaY
    PKS18PKSArthroderma gypseumAscomycotaY
    PKS19PKSSetosphaeria turcicaAscomycotaN
    PKS20PKSPyrenophora teresAscomycotaY
    PKS21PKSCladophialophora yegresiiAscomycotaN
    PKS22PKSTalaromyces cellulolyticusAscomycotaY
    PKS23PKSEndocarpon pusillumAscomycotaY
    PKS24PKSTalaromyces cellulolyticusAscomycotaY
    PKS25PKSMoniliophthora roreriBasidiomycotaN
    PKS26PKSHypholoma sublateritiumBasidiomycotaN
    PKS27PKSCeriporiopsis subvermisporaBasidiomycotaN
    PKS28PKSMoniliophthora roreriBasidiomycotaY
    TC1UTCTrichderma VirensAscomycotaY
    TC2UTCTrichderma VirensAscomycotaN
    TC3UTCBotryotonia cinereaAscomycotaY
    TC4UTCFormitiporia mediterraneaBasidiomycotaY
    TC5UTCHeterobasidion annosumBasidiomycotaY
    TC6UTCGelatoporia subvermisporaBasidiomycotaN
    TC7UTCDichomitus squalensBasidiomycotaN
    TC8UTCPleurotus ostreatusBasidiomycotaN
    TC9UTCSchizophyllum communeBasidiomycotaY
    TC10UTCStereum hirsutumBasidiomycotaN
    TC11UTCStereum hirsutumBasidiomycotaN
    TC12UTCDichomitus squalensBasidiomycotaN
    TC13UTCDacryopinax primogenitusBasidiomycotaN
    Total 43
    Productive 24

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaar5459/DC1

    Supplementary Text

    fig. S1. Characterization of S. cerevisiae PADH2-like promoters.

    fig. S2. Cloning vectors used in this study.

    fig. S3. Improving DNA assembly.

    fig. S4. Schematics of all PKS-containing BGCs examined here.

    fig. S5. UTC-containing BGCs examined here.

    fig. S6. Volcano plot of all spectral features identified in the automated analysis of strains expressing PKS-containing BGCs.

    fig. S8. All features produced by PKS1 in strain 132.

    fig. S9. All features produced by PKS2 in strain 133.

    fig. S10. All features produced by PKS4 in strain 255.

    fig. S11. All features produced by PKS6 in strain 178.

    fig. S12. All features produced by PKS8 in strain 164.

    fig. S13. All features produced by PKS10 in strain 246.

    fig. S14. All features produced by PKS13 in strain 206.

    fig. S15. All features produced by PKS14 in strain 257.

    fig. S16. All features produced by PKS15 in strain 247.

    fig. S17. All features produced by PKS16 in strain 177.

    fig. S18. All features produced by PKS17 in strain 176.

    fig. S19. All features produced by PKS18 in strain 207.

    fig. S20. All features produced by PKS20 in strain 208.

    fig. S21. All features produced by PKS22 in strain 209.

    fig. S22. All features produced by PKS23 in strain 241.

    fig. S23. All features produced by PKS24 in strain 210.

    fig. S24. All features produced by PKS28 in strain 240.

    fig. S25. 1H NMR spectrum of compound 6 in CDCl3.

    fig. S26. 13C NMR spectrum of compound 6 in CDCl3.

    fig. S27. 1H-1H COSY spectrum of compound 6 in CDCl3.

    fig. S28. HSQC spectrum of compound 6 in CDCl3.

    fig. S29. HMBC spectrum of compound 6 in CDCl3.

    fig. S30. 1H NMR spectrum of compound 7 in acetone-d6.

    fig. S31. 13C NMR spectrum of compound 7 in acetone-d6.

    fig. S32. 1H-1H COSY spectrum of compound 7 in acetone-d6.

    fig. S33. HSQC spectrum of compound 7 in acetone-d6.

    fig. S34. HMBC spectrum of compound 7 in acetone-d6.

    fig. S35. 1H NMR spectrum of compound 8 in acetone-d6.

    fig. S36. 13C NMR spectrum of compound 8 in acetone-d6.

    fig. S37. 1H-1H COSY spectrum of compound 8 in acetone-d6.

    fig. S38. HSQC spectrum of compound 8 in acetone-d6.

    fig. S39. HMBC spectrum of compound 8 in acetone-d6.

    fig. S40. 1H NMR spectrum of compound 9 in CDCl3.

    fig. S41. 13C NMR spectrum of compound 9 in CDCl3.

    fig. S42. 1H-1H COSY spectrum of compound 9 in CDCl3.

    fig. S43. HSQC spectrum of compound 9 in CDCl3.

    fig. S44. HMBC spectrum of compound 9 in CDCl3.

    fig. S45. 1H NMR spectrum of compound 10 in CDCl3.

    fig. S46. 13C NMR spectrum of compound 10 in CDCl3.

    fig. S47. 1H-1H COSY spectrum of compound 10 in CDCl3.

    fig. S48. HSQC spectrum of compound 10 in CDCl3.

    fig. S49. HMBC spectrum of compound 10 in CDCl3

    fig. S50. 1H NMR spectrum of compound 11 in CDCl3.

    fig. S51. 13C NMR spectrum of compound 11 in CDCl3.

    fig. S52. 1H NMR spectrum of compound 12 in CDCl3.

    fig. S53. 13C NMR spectrum of compound 12 in CDCl3.

    fig. S54. 1H-1H COSY spectrum of compound 12 in CDCl3.

    fig. S55. HSQC spectrum of compound 12 in CDCl3.

    fig. S56. HMBC spectrum of compound 12 in CDCl3.

    fig. S57. NOESY spectrum of compound 12 in CDCl3

    fig. S58. 1H NMR spectrum of compound 13 in CDCl3.

    fig. S59. 13C NMR spectrum of compound 13 in CDCl3.

    fig. S60. 1H-1H COSY spectrum of compound 13 in CDCl3.

    fig. S61. HSQC spectrum of compound 13 in CDCl3.

    fig. S62. HMBC spectrum of compound 13 in CDCl3.

    fig. S63. NOESY spectrum of compound 13 in CDCl3.

    fig. S64. 1H NMR spectrum of compound 14 in CDCl3.

    fig. S65. 13C NMR spectrum of compound 14 in CDCl3.

    fig. S66. 1H-1H COSY spectrum of compound 14 in CDCl3.

    fig. S67. HSQC spectrum of compound 14 in CDCl3.

    fig. S68. HMBC spectrum of compound 14 in CDCl3.

    fig. S69. 1H NMR spectrum of compound 15 in CDCl3.

    fig. S70. 13C NMR spectrum of compound 15 in CDCl3.

    fig. S71. 1H-1H COSY spectrum of compound 15 in CDCl3.

    fig. S72. HSQC spectrum of compound 15 in CDCl3.

    fig. S73. HMBC spectrum of compound 15 in CDCl3.

    fig. S74. HMBC spectrum of compound 15 in CDCl3.

    fig. S75. 1H NMR spectrum of compound 16 in CDCl3.

    fig. S76. 13C NMR spectrum of compound 16 in CDCl3.

    fig. S77. 1H-1H COSY spectrum of compound 16 in CDCl3.

    fig. S78. HSQC spectrum of compound 16 in CDCl3.

    fig. S79. HMBC spectrum of compound 16 in CDCl3.

    table S1. Ion source parameters used in this study.

    table S2. Expression data for selected promoters drawn from genome-wide expression studies.

    table S3. Sequences of HEx promoters

    table S4. Background strains used throughout this study.

    table S5. Features integrated for the determination of sesquiterpenoid titer in Fig. 2B.

    table S6. Order of promoters and terminators used in the expression of all cryptic fungal BGCs examined in this study.

    table S7. Standard part plasmids and expression vectors used for the assembly of cryptic BGCS in this study.

    table S8. Coordinates of native loci from which all clusters examined here were derived along with the IDs of plasmids expressing the engineered cluster versions.

    table S9. Abbreviations for the functional gene annotations used in Figs. 2, 5, and 6.

    table S10. Strains expressing cryptic fungal BGCs analyzed here.

    table S11. NMR data of compound 6.

    table S12. NMR data of compound 7.

    table S13. NMR data of compound 8.

    table S14. NMR data of compound 9.

    table S15. NMR data of compound 10.

    table S16. NMR data of compound 11.

    table S17. NMR data of compound 12.

    table S18. NMR data of compound 13.

    table S19. NMR data of compound 14.

    table S20. NMR data of compound 15.

    table S21. NMR data of compound 16.

    References (7073)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Text
    • fig. S1. Characterization of S. cerevisiae PADH2-like promoters.
    • fig. S2. Cloning vectors used in this study.
    • fig. S3. Improving DNA assembly.
    • fig. S4. Schematics of all PKS-containing BGCs examined here.
    • fig. S5. UTC-containing BGCs examined here.
    • fig. S6. Volcano plot of all spectral features identified in the automated analysis
      of strains expressing PKS-containing BGCs.
    • fig. S8. All features produced by PKS1 in strain 132.
    • fig. S9. All features produced by PKS2 in strain 133.
    • fig. S10. All features produced by PKS4 in strain 255.
    • fig. S11. All features produced by PKS6 in strain 178.
    • fig. S12. All features produced by PKS8 in strain 164.
    • fig. S13. All features produced by PKS10 in strain 246.
    • fig. S14. All features produced by PKS13 in strain 206.
    • fig. S15. All features produced by PKS14 in strain 257.
    • fig. S16. All features produced by PKS15 in strain 247.
    • fig. S17. All features produced by PKS16 in strain 177.
    • fig. S18. All features produced by PKS17 in strain 176.
    • fig. S19. All features produced by PKS18 in strain 207.
    • fig. S20. All features produced by PKS20 in strain 208.
    • fig. S21. All features produced by PKS22 in strain 209.
    • fig. S22. All features produced by PKS23 in strain 241.
    • fig. S23. All features produced by PKS24 in strain 210.
    • fig. S24. All features produced by PKS28 in strain 240.
    • fig. S25. 1H NMR spectrum of compound 6 in CDCl3.
    • fig. S26. 13C NMR spectrum of compound 6 in CDCl3.
    • fig. S27. 1H-1H COSY spectrum of compound 6 in CDCl3.
    • fig. S28. HSQC spectrum of compound 6 in CDCl3.
    • fig. S29. HMBC spectrum of compound 6 in CDCl3.
    • fig. S30. 1H NMR spectrum of compound 7 in acetone-d6.
    • fig. S31. 13C NMR spectrum of compound 7 in acetone-d6.
    • fig. S32. 1H-1H COSY spectrum of compound 7 in acetone-d6.
    • fig. S33. HSQC spectrum of compound 7 in acetone-d6.
    • fig. S34. HMBC spectrum of compound 7 in acetone-d6.
    • fig. S35. 1H NMR spectrum of compound 8 in acetone-d6.
    • fig. S36. 13C NMR spectrum of compound 8 in acetone-d6.
    • fig. S37. 1H-1H COSY spectrum of compound 8 in acetone-d6.
    • fig. S38. HSQC spectrum of compound 8 in acetone-d6.
    • fig. S39. HMBC spectrum of compound 8 in acetone-d6.
    • fig. S40. 1H NMR spectrum of compound 9 in CDCl3.
    • fig. S41. 13C NMR spectrum of compound 9 in CDCl3.
    • fig. S42. 1H-1H COSY spectrum of compound 9 in CDCl3.
    • fig. S43. HSQC spectrum of compound 9 in CDCl3.
    • fig. S44. HMBC spectrum of compound 9 in CDCl3.
    • fig. S45. 1H NMR spectrum of compound 10 in CDCl3.
    • fig. S46. 13C NMR spectrum of compound 10 in CDCl3.
    • fig. S47. 1H-1H COSY spectrum of compound 10 in CDCl3.
    • fig. S48. HSQC spectrum of compound 10 in CDCl3.
    • fig. S49. HMBC spectrum of compound 10 in CDCl3.
    • fig. S50. 1H NMR spectrum of compound 11 in CDCl3.
    • fig. S51. 13C NMR spectrum of compound 11 in CDCl3.
    • fig. S52. 1H NMR spectrum of compound 12 in CDCl3.
    • fig. S53. 13C NMR spectrum of compound 12 in CDCl3.
    • fig. S54. 1H-1H COSY spectrum of compound 12 in CDCl3.
    • fig. S55. HSQC spectrum of compound 12 in CDCl3.
    • fig. S56. HMBC spectrum of compound 12 in CDCl3.
    • fig. S57. NOESY spectrum of compound 12 in CDCl3.
    • fig. S58. 1H NMR spectrum of compound 13 in CDCl3.
    • fig. S59. 13C NMR spectrum of compound 13 in CDCl3.
    • fig. S60. 1H-1H COSY spectrum of compound 13 in CDCl3.
    • fig. S61. HSQC spectrum of compound 13 in CDCl3.
    • fig. S62. HMBC spectrum of compound 13 in CDCl3.
    • fig. S63. NOESY spectrum of compound 13 in CDCl3.
    • fig. S64. 1H NMR spectrum of compound 14 in CDCl3.
    • fig. S65. 13C NMR spectrum of compound 14 in CDCl3.
    • fig. S66. 1H-1H COSY spectrum of compound 14 in CDCl3.
    • fig. S67. HSQC spectrum of compound 14 in CDCl3.
    • fig. S68. HMBC spectrum of compound 14 in CDCl3.
    • fig. S69. 1H NMR spectrum of compound 15 in CDCl3.
    • fig. S70. 13C NMR spectrum of compound 15 in CDCl3.
    • fig. S71. 1H-1H COSY spectrum of compound 15 in CDCl3.
    • fig. S72. HSQC spectrum of compound 15 in CDCl3.
    • fig. S73. HMBC spectrum of compound 15 in CDCl3.
    • fig. S74. HMBC spectrum of compound 15 in CDCl3.
    • fig. S75. 1H NMR spectrum of compound 16 in CDCl3.
    • fig. S76. 13C NMR spectrum of compound 16 in CDCl3.
    • fig. S77. 1H-1H COSY spectrum of compound 16 in CDCl3.
    • fig. S78. HSQC spectrum of compound 16 in CDCl3.
    • fig. S79. HMBC spectrum of compound 16 in CDCl3.
    • table S1. Ion source parameters used in this study.
    • table S2. Expression data for selected promoters drawn from genome-wide expression studies.
    • table S3. Sequences of HEx promoters.
    • table S4. Background strains used throughout this study.
    • table S5. Features integrated for the determination of sesquiterpenoid titer in Fig. 2B.
    • table S6. Order of promoters and terminators used in the expression of all cryptic fungal BGCs examined in this study.
    • table S7. Standard part plasmids and expression vectors used for the assembly of cryptic BGCS in this study.
    • table S8. Coordinates of native loci from which all clusters examined here were derived along with the IDs of plasmids expressing the engineered cluster versions.
    • table S9. Abbreviations for the functional gene annotations used in Figs. 2, 5, and 6.
    • table S10. Strains expressing cryptic fungal BGCs analyzed here.
    • table S11. NMR data of compound 6.
    • table S12. NMR data of compound 7.
    • table S13. NMR data of compound 8.
    • table S14. NMR data of compound 9.
    • table S15. NMR data of compound 10.
    • table S16. NMR data of compound 11.
    • table S17. NMR data of compound 12.
    • table S18. NMR data of compound 13.
    • table S19. NMR data of compound 14.
    • table S20. NMR data of compound 15.
    • table S21. NMR data of compound 16.
    • References (70–73)

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