18-Hydroxydolabella-3,7-diene synthase - a diterpene synthase from Chitinophaga pinensis

Abstract

The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products.

Keywords: Chitinophaga pinensis; Nicotiana benthamiana; biosynthesis; structure elucidation; terpenes.

Scheme 1

Germacrene A (1) and its Cope rearrangement to β-elemene (2).

Figure 1

In vitro terpene synthase activity of the investigated recombinant enzyme from C. pinensis, showing no formation of monoterpenes from GPP (A) and no formation of sesquiterpenes from FPP (B), but formation of a single diterpene alcohol 3 from GGPP (C) with the mass spectrum depicted in (D). Asterisks indicate non-terpenoid contaminants such as plasticisers.

Scheme 2

Product obtained from the diterpene synthase from C. pinensis. (A) Structure of (1R,3E,7E,11S,12S)-18-hydroxydolabella-3,7-diene (3), contiguous ¹H,¹H-COSY spin systems (bold), and diagnostic HMBC and NOESY correlations (single and double headed arrows). (B) Cyclisation mechanism for the conversion of GGPP into 3 by HdS. (C) Structure of the known stereoisomer 1,11-di-epi-3.

Figure 2

Determination of the absolute configuration of 3. (A) Partial HSQC spectrum of unlabelled 3 showing the region for C2, (B) cyclisation of GGPP to the two possible enantiomers of 3, (C) partial HSQC spectrum of the product obtained from (R)-(1-¹³C,1-²H)GGPP, and (D) partial HSQC spectrum of the product obtained from (S)-(1-¹³C,1-²H)GGPP. Purple dots indicate ¹³C-labelled carbons.

Figure 3

Determination of the absolute configuration of 3. (A) Partial HSQC spectrum of unlabelled 3 showing the region for C10, (B) elongation of GPP with IPP to GGPP and cyclisation to the two possible enantiomers of 3, (C) partial HSQC spectrum of the product obtained from (R)-(1-¹³C,1-²H)GPP, and (D) partial HSQC spectrum of the product obtained from (S)-(1-¹³C,1-²H)GPP. Purple dots indicate ¹³C-labelled carbons.

Figure 4

Assignment of H6α and H6β of 3. (A) Partial HSQC spectrum of unlabelled 3 showing the region for C6, (B) elongation of FPP with IPP to GGPP and cyclisation to 3, (C) partial HSQC spectrum of the product obtained from (R)-(1-¹³C,1-²H)FPP, and (D) partial HSQC spectrum of the product obtained from (S)-(1-¹³C,1-²H)FPP. Purple dots indicate ¹³C-labelled carbons.

Figure 5

Partial ¹³C NMR spectra of A) unlabeled 3, B) (¹³C₁)-3 arising from incubation of HdS and GGPPS with (12-¹³C)FPP + IPP, and C) (¹³C₁)-3 arising from incubation of HdS and GGPPS with (13-¹³C)FPP + IPP. Labelled carbons are indicated by purple dots.

Figure 6

Transient expression of 18-hydroxydolabella-3,7-diene synthase (HdS) in Nicotiana benthamiana. Total ion chromatograms of GC–MS analyses of N. benthamiana leaf extracts. A) HdS-mit (HdS expressed with mitochondrial targeting signal) showing the production of 3 in planta, B) HdS (expression without targeting signal) and C) empty vector.