1. Biochemistry and Chemical Biology
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A terpene synthase-cytochrome P450 cluster in Dictyostelium discoideum produces a novel trisnorsesquiterpene

  1. Xinlu Chen
  2. Katrin Luck
  3. Patrick Rabe
  4. Christopher QD Dinh
  5. Gad Shaulsky
  6. David R Nelson
  7. Jonathan Gershenzon
  8. Jeroen S Dickschat
  9. Tobias G Köllner
  10. Feng Chen  Is a corresponding author
  1. University of Tennessee, United States
  2. Max Planck Institute for Chemical Ecology, Germany
  3. University of Bonn, Germany
  4. Baylor College of Medicine, United States
  5. University of Tennessee Health Science Center, United States
Research Article
Cite this article as: eLife 2019;8:e44352 doi: 10.7554/eLife.44352
7 figures and 6 additional files

Figures

Figure 1 with 4 supplements
Structure elucidation of DdTPS8 product and biosynthetic mechanism.

(A) Structure of discoidol. (B) Contiguous spin systems indicated by bold lines observed in discoidol by 1H,1H-COSY NMR, single headed arrows indicate diagnostic HMBC correlations. (C) important NOESY correlations that are indicated by double headed arrows observed in discoidol. (D) biosynthetic mechanism from farnesyl diphosphate (FDP) to discoidol catalyzed by DdTPS8. See also Figure 1—figure supplements 14.

https://doi.org/10.7554/eLife.44352.002
Figure 1—figure supplement 1
Determination of the absolute configuration of discoidol.
https://doi.org/10.7554/eLife.44352.003
Figure 1—figure supplement 2
Investigation of the reprotonation step in the cyclization mechanism of discoidol.
https://doi.org/10.7554/eLife.44352.004
Figure 1—figure supplement 3
Investigation of the 1,2-hydride migration by incubation of (3–13C,2–2H)FDP with DdTPS8.
https://doi.org/10.7554/eLife.44352.005
Figure 1—figure supplement 4
Incubation experiments with (12–13C)FDP and (13–13C)FDP and DdTPS8.
https://doi.org/10.7554/eLife.44352.006
Figure 2 with 1 supplement
DdTPS8 insertional mutant and its volatile profile.

(A) Schematic presentation of DdTPS8 gene with an insert of 1.6 kb. (B) Volatiles were collected from the headspace of the cultures and analyzed using GC-MS. Total ion chromatograms are shown. 1, unknown compound; 2, unidentified compound; 3, unidentified sesquiterpene hydrocarbon; 4, unidentified compound; 5, β-maaliene; 6, aristolene; 7, calarene; 8–10, unidentified sesquiterpene hydrocarbons; 11, nerolidol. See also Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.44352.007
Figure 2—figure supplement 1
Verification of DdTPS8 insertion mutant.
https://doi.org/10.7554/eLife.44352.008
Cytochrome P450 (CYP) genes associated with DdTPS8.

(A) Expression pattern of three CYP genes that showed highest level of coexpression coefficient with DdTPS8. The cartoons show the six stages of multicellular development of D. discoideum: individual cells (0 hr), streaming (8 hr), loose aggregate (10 hr), slug (16 hr), Mexican hat (20 hr) and fruiting bodies (24 hr). (B) DdTPS8 and CYP521A1 are neighbor genes. The number above the black line indicates the length of the intergenic region in base pairs.

https://doi.org/10.7554/eLife.44352.009
Figure 4 with 1 supplement
Volatile profiles of E. coli Bl21-DE3-Star expressing different combinations of CYP521A1, CYP508C1, the P450 reductase gene RedB, and the terpene synthase gene DdTPS8.

Volatiles were collected from the headspace of the induced bacterial cultures using PDMS tubes and analyzed using GC-TDU-MS. The extracted ion chromatograms for m/z 105 (A) and the mass spectra of the DdTPS8 product (B) and CYP521A1 product (C) are shown. See also Figure 4—figure supplement 1.

https://doi.org/10.7554/eLife.44352.010
Figure 4—figure supplement 1
Activity of DdTPS8 and CYP521A1 in cell-free enzyme assays in the presence of (E,E)-FDP.
https://doi.org/10.7554/eLife.44352.011
Figure 5 with 1 supplement
Mass spectra (A–C) and structures (D) of DdTPS8 products and CYP521A1 products derived from unlabeled farnesyl diphosphate (FDP) (A), 13C15-FDP (B), and 1,1–2 H2,11–13C-FDP (C + D).

The genes were coexpressed in E. coli Bl21-DE3-Star together with the P450 reductase gene RedB. Crude protein extracts were incubated with unlabeled or labeled (E,E)-FDP and volatile enzyme products were collected from the headspace of the assays using PDMS tubes. Product analysis was performed with GC-TDU-MS. See also Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.44352.012
Figure 5—figure supplement 1
Formation of discodiene by CYP521A1.
https://doi.org/10.7554/eLife.44352.013
Developmental phenotype of the DdTPS8 knockout mutant.

Wild type AX4 cells (WT) and mutant DdTPS8 cells (mutant) were grown separately in HL5 to the log phase, washed in buffer, and plated clonally on dark nitrocellulose filters. The filters were cut in half and placed next to each other in one dish. The cells were incubated in the dark at 22°C and photographed from above with a dissecting microscope at the indicated times. This experiment was independently performed three times with same results and the data shown represent one of the replicates.

https://doi.org/10.7554/eLife.44352.014
TPS-CYP gene clusters in D. discoideum (A) and D. purpureum (B).

Green blocks depict TPS genes, red blocks indicate CYP genes. The blue block indicates a non-TPS/CYP gene. The numbers above the black lines indicate length in base pairs.

https://doi.org/10.7554/eLife.44352.015

Additional files

Supplementary file 1

NMR data of discoidol recorded in C6D6.

https://doi.org/10.7554/eLife.44352.016
Supplementary file 2

Cytochrome p450 genes in Dictyostelium discoideum.

https://doi.org/10.7554/eLife.44352.017
Supplementary file 3

CYP genes of Dictyostelium discoideum that show significant coexpression with DdTPS8.

https://doi.org/10.7554/eLife.44352.018
Supplementary file 4

Cytochrome p450 genes in Dictyostelium purpureum.

https://doi.org/10.7554/eLife.44352.019
Supplementary file 5

Primers used in gene cloning and vector construction.

https://doi.org/10.7554/eLife.44352.020
Transparent reporting form
https://doi.org/10.7554/eLife.44352.021

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