A Cytochrome P450-Mediated Intramolecular Carbon-Carbon Ring Closure in the Biosynthesis of Multidrug-Resistance-Reversing Lathyrane Diterpenoids

Abstract

The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer. We report here a gene cluster from Jatropha curcas that contains the genes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids, including the lathyranes jolkinol C and epi-jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon-carbon ring closure. This requires the activity of two cytochrome P450s that we propose form a 6-hydroxy-5,9-diketocasbene intermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologous production of these potential anticancer drugs, which can often only be sourced in limited quantities from their native plant.

Keywords: casbene; cytochromes; diterpenoids; gene clusters; lathyranes.

Scheme 1

The lathyranes as proposed intermediates in the biosynthesis of diterpenoids with tigliane, daphnane, ingenane, rhamnofolane and jatropholane carbon skeletons.

Scheme 2

Structures of Euphorbia factor L1 (a lathyrane), ingenol mebutate (an ingenane), prostratin (a tigliane) and resiniferatoxin (a daphnane). The red and blue oxygen atoms highlighted on each of the molecules correspond to the 5 and 9 positions of casbene, respectively. The green carbon–carbon bond corresponds to the 6 and10 positions of casbene.

Figure 1

A diterpenoid biosynthesis gene cluster. The diagram corresponds to a 300 kb region present on scaffold 123 of the J. curcas genome (Genbank accession no. NW_012124159). Different classes of enzymes have been colour coded, for example, cytochrome P450 genes are shown in blue.

Figure 2

GC and LC chromatograms of casbene and casbene metabolites produced by transient expression of casbene synthase and casbene synthase with a single cytochrome P450 from the J. curcas gene cluster in N. benthamiana. The structures of the numbered metabolites are shown in Scheme 3. The corresponding mass and NMR spectra are provided in the Supporting Information and Figure S6.

Scheme 3

Roles of CYP726A35, CYP726A20 and CYP71D495 in diterpenoid biosynthesis. The blue numbering is based on the casbene numbering system.

Figure 3

LC chromatograms obtained from co‐expression of casbene synthase with two cytochrome P450s from the J. curcas diterpenoid gene cluster. The lower panels show the results from co‐expression of the J. curcas genes with 1‐deoxy‐d‐xylulose 5‐phosphate synthase (DXS) and a plastidial geranylgeranyl pyrophosphate synthase (GGPPS) from Arabidopsis thaliana. The structures of the numbered metabolites are shown in Scheme 3. The corresponding mass and NMR spectra are provided in the Supporting Information and Figure S6.

Scheme 4

Presumed facile enolisation at the 5‐keto group is the key step for the Δ^7,8 to Δ^6,7 double bond isomerisation in 6‐hydroxy‐5,9‐diketocasbene, which leads to a triketo precursor that spontaneously converts to jolkinol C through an intramolecular aldol reaction.