Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase

Figures & data

Figure 1. Structures of (−)-conidiogenone (1), conidiogenol, and deoxyconidiogenol (2).

Figure 2. GC-MS and LC-MS profiles of the extracts from enzymatic reaction and heterologous expression.

(a). GC-MS profile of PchDS reaction. Upper panel: chromatogram showing total ion current (TIC). Lower panel: MS spectrum of 2. (b). GC-MS profile of metabolites extracted from AO-PrDS and A. oryzae NSAR1. Upper panel: chromatograms extracted at m/z 290. Lower panel: MS spectrum of 2. (c). LC-MS profile of AO-PrDS and AO-PrDS/PrP450. Chromatograms are extracted at m/z 305.

Table 1. ¹³C chemical shifts and multiplicity of [¹³C₂₀]-2 (CDCl₃, 150 MHz).

	[^13C20]-2	Multiplicity^b
No.	δC^a	[^13C5]-2	[^13C10]-2	[^13C15]-2	[^13C20]-2
1	43.9	*	*	*	*
2	60.6	t	q	quint	quint
3	35.1	*	*	*	*
4	27.2	d	t	t	t
5	28.9	-	*	*	*
6	74.2	-	t	t	t
7	49.1	-	quint	quint	quint
8	37.6	-	br,d	br,t	br,t
9	31.0	-	-	*	*
10	53.8	-	-	*	*
11	53.9	-	-	q	quint
12	40.9	-	-	d	t
13	40.4	-	-	-	t
14	73.7	-	-	-	q
15	42.4	-	-	-	quint
16	27.1	-	-	-	d
17	31.7	-	-	-	d
18	17.6	d	d	d	d
19	22.0	-	*	*	*
20	31.6	-	-	d	d

^aMeasured in CDCl₃.

^bCoupling patterns were directly observed in one-dimensional ¹³C NMR.

*Coupling pattern could not be assigned due to broadening of the peaks.

Figure 3. ¹³C NMR spectra of ¹³C-labeled 2 enzymatically synthesized by PchDS (CDCl₃, 150 MHz).

The solvent signal at δ_C 77.0 was used as a reference.

Scheme 1. Proposed cyclization mechanism catalyzed by PchDS/PrDS.

The numbers of intermediates are consistent with those in ref. 22.

Figure 4. ¹³C NMR spectra of deoxyconidiogenol (2) measured at room temperature (a, toluene-d₈, 125 MHz) and at −80 °C (b, toluene-d₈, 150 MHz).

Chemical shifts are referenced to the methyl signal of toluene-d₈ (δ_C 20.4).

Figure 5. Stereostructures of the two conformers, I_ax and II_eq.

M0001-M0006 are superposed for I_ax (81% distribution) and M0007 and M0012 are superposed for II_eq (18% distribution).