Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana

Abstract

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.

Figure 1. Biosynthetic pathway of costunolide in Asteraceae.

GAS, germacrene A synthase.

Figure 2. Germacrene A production in yeast.

A yeast culture transformed by either CiGAS-long, CiGAS-short , or TpGAS . Induced yeast culture medium was extracted and analysed by GC-MS.

Figure 3. Headspace analysis of volatiles emitted from agro-infiltrated Nicotiana benthamiana leaves.

A, GC-MS chromatograms are shown for the volatiles emitted from N.benthamiana leaves infiltrated with the indicated genes. Line a is a negative control, line b and c display the different amount of compound 1 (germacrene A) produced by N. benthamiana leaves infiltrated with TpGAS with different targeting signals: mTpGAS, mitochondrial targeting; cTpGAS, cytosolic targeting. Line d shows that compound 1 which is produced upon mTpGAS agro-infiltration disappears upon agro-infiltration with CiGAO. Agro-infiltration with CiGAO alone does not induce any volatile formation (Line e). B, the mass fragmentation patterns of compound 1 (a) and a β-elemene from the Wiley library (b). C, cope rearrangement of germacrene A to β-elemene by heat.

Figure 4. Costunolide production in yeast.

A, GC-MS chromatograms are shown for the metabolites from yeast transformed with the indicated genes. Line a is a negative control, line b displays the metabolites in the yeast transformed with two genes (TpGAS, CiGAO), and line c displays the metabolites in the yeast transformed with three genes (TpGAS, CiGAO, and CiCOS). B, the mass spectra of compound 1 (a) and compound 2 (b) produced by yeast and elematrien-12-oic acid (c) and costunolide standards (d) are shown. C, cope rearrangement of germacrene acid to elematrien-12-oic acid by heat is shown. 1 = germacra-1(10),4,11(13)-trien-12-oic acid; 2 = costunolide.

Figure 5. Phylogenetic analysis of Asteraceae GAO genes and five chicory CYP71 P450 ESTs.

Chicory candidate 3368 was later identified as Cichorium intybus costunolide synthase (CiCOS). Amino acid seuqences of GAOs were obtained from cDNAs deposited at the NCBI. LsGAO germacrene A oxidase from Lactuca sativa (GU198171) or from Cichorium intybus (Ci; GU256644), Helianthus annuus (Ha; GU256646), Saussurea costus (Sc; GU256645) and Barnadesia. spinosa (Bs; GU256647). Bootstrap values are shown in frequency values from 1000 replicates.

Figure 6. LC-MS/MS analysis of non-volatile metabolites in N. benthamiana leaves agro-infiltrated with empty vector, TpGAS, TpGAS+CiGAO and TpGAS+CiGAO+CiCOS.

The two new peaks in TpGAS+CiGAO+CiCOS agro-infiltrated leaves were further fragmented by MS/MS. The [m/z]⁻ for the parent ion of peak 22.30 is 352.1615 The [m/z]⁻ for the parent ion of peak 22.50 is 538.2206. Inserted figures show MS/MS spectrum of peak 22.30 and peak 22.50 at 25 eV. Arrows indicate characteristic Cys and GSH MS/MS fragments, respectively. The Y-axis scale is identical in all chromatograms.

Figure 7. Costunolide-glutathione and costunolide-cysteine conjugate identification by GST enzyme assay and LC-MS analysis.

A. LC-MS chromatograms of [m/z]⁻ = 538 of extracts of N. benthamiana leaves agro-infiltrated with TpGAS+CiGAO+CiCOS, costunolide-GSH conjugate formed in an enzyme assay of costunolide and GSH with GST, costunolide-GSH conjugate formed by non-enzymatic conjugation of costunolide and GSH. B. LC-MS chromatograms of [m/z]⁻ = 352 of extracts of N. benthamiana leaves agro-infiltrated with TpGAS+CiGAO+CiCOS, costunolide-cysteine (Cys) conjugate formed in an enzyme assay of costunolide and Cys with GST, costunolide-Cys conjugate formed non enzymatically from costunolide and Cys. C. [m/z]⁻ spectrum of peak 22.48 and costunolide-GSH conjugate (RT = 22.52). Arrows indicate parent ions of GSH-glutathione. D. [m/z]⁻ spectrum of peak 22.30 and costunolide-Cys conjugate (RT = 22.30). Arrows indicate parent ions of GSH-cysteine. E. Presumed molecular structure of costunolide-GSH (a) and costunolide-Cys (b) conjugates. GSH, glutathione; Cys, cysteine; GST, glutathione S-transferase. RT, retention time. Y-axis scale is identical in all chromatograms.