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
7 figures and 6 additional files


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.

Figure 1—figure supplement 1
Determination of the absolute configuration of discoidol.
Figure 1—figure supplement 2
Investigation of the reprotonation step in the cyclization mechanism of discoidol.
Figure 1—figure supplement 3
Investigation of the 1,2-hydride migration by incubation of (3–13C,2–2H)FDP with DdTPS8.
Figure 1—figure supplement 4
Incubation experiments with (12–13C)FDP and (13–13C)FDP and DdTPS8.
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.

Figure 2—figure supplement 1
Verification of DdTPS8 insertion mutant.
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.

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.

Figure 4—figure supplement 1
Activity of DdTPS8 and CYP521A1 in cell-free enzyme assays in the presence of (E,E)-FDP.
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.

Figure 5—figure supplement 1
Formation of discodiene by CYP521A1.
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.

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.


Additional files

Supplementary file 1

NMR data of discoidol recorded in C6D6.

Supplementary file 2

Cytochrome p450 genes in Dictyostelium discoideum.

Supplementary file 3

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

Supplementary file 4

Cytochrome p450 genes in Dictyostelium purpureum.

Supplementary file 5

Primers used in gene cloning and vector construction.

Transparent reporting form

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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
A terpene synthase-cytochrome P450 cluster in Dictyostelium discoideum produces a novel trisnorsesquiterpene
eLife 8:e44352.