The seco-iridoid pathway from Catharanthus roseus

Abstract

The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.

Figure 1. The secologanin–strictosidine pathway.

Genes indicated in boxes were published before (black background) or during (white background) the present study, or are reported here (yellow background). Frames indicate mRNA localization in the leaf IPAP (pink) or epidermis (blue). Numbers indicate predicted enzyme classes in the initial gene discovery strategy. 1: oxidoreductase, 2: cytochrome P450, 3: UGT. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; Glc, glucose; GPPS, geranyl diphosphate synthase, GES, geraniol synthase; G8O, geraniol 8-oxidase; 8-HGO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7-DLGT, 7-deoxyloganetic acid glucosyl transferase; 7-DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; SLS, secologanin synthase; STR, strictosidine synthase; TDC, tryptophan decarboxylase. Iridotrial indicated in brackets was a previously proposed intermediate that we did not detect in vitro or in vivo.

Figure 2. Gene discovery strategy.

(a,b) Complete-linkage hierarchical clustering of early MIA pathway gene expression in C. roseus based on our data (a) or the Medicinal Plant Genomics Resource consortium (http://medicinalplantgenomics.msu.edu) (b). Colours indicate transcriptional activation (blue) or repression (yellow) relative to untreated samples. Tissues: Fl, flower; mL, mature leaves; iL, immature leaves; St, stem; Ro, root; Sdlg, seedling. Suspension cells (CellSus): Wt, wild-type; O2, ORCA2; O3, ORCA3. Hairy roots (HairRt): Wt, wild-type; Td, TDCi; RebH, RebH_F. Treatments: Not, no treatment; MeJA, methyl jasmonate (6, 12 or 24 h); Con, mock; YE, yeast extract. (c) Candidate P450 protein hits in the epidermis and mesophyll samples from proteomics analysis±s.d. (n=3). GES, geraniol synthase; G8O, geraniol 8-oxidase; 8-HGO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7-DLGT, 7-deoxyloganetic acid glucosyl transferase; 7-DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferase; SGD, strictosidine β-D-glucosidase; SLS, secologanin synthase; STR, strictosidine synthase (1–3: three related genes); TDC, tryptophan decarboxylase.

Figure 3. Functional characterization of recombinant 8-HGO.

Affinity-purified enzyme expressed in E. coli was incubated with 8-OH-geraniol and NAD⁺. (a) GC–MS profile of the reaction extract compared with authentic standards. (b) The identity of peaks 1–4 was confirmed by comparison of MS spectra with authentic standards. 8-HGO catalyses the stepwise conversion of 8-OH-geraniol into 8-oxogeraniol or 8-OH-geranial and then into 8-oxogeranial.

Figure 4. Functional characterization of IO.

The cytochrome P450 enzyme was expressed in yeast and assayed as a microsomal preparation by incubation with cis-trans-nepetalactol and NADPH. (a) GC–MS profile of the reaction extract compared with the negative control and authentic standards. (b) Product identity was confirmed by comparison of the MS spectrum with the authentic standard. Cis-trans-nepetalactol is the peak 3 (ref. 13), in the substrate standard, peaks 1 and 2 are its open dialdehyde forms (cis and trans iridodial). These compounds are in equilibrium in water. Peak 3 is the first converted by IO, but eventually, possibly after interconversion, all three compounds are metabolized (not shown).

Figure 5. Functional characterization of 7-DLGT.

Affinity-purified enzyme expressed in E. coli was incubated with 7-deoxyloganetic acid and UDP-glucose. (a) HPLC-DAD profile of the reaction extract compared with authentic standards. (b) Product identity was confirmed by comparison of the mass spectrum with the authentic standard.

Figure 6. Functional characterization of 7-DLH.

The cytochrome P450 enzyme was expressed in yeast and assayed as a microsomal preparation by incubation with 7-deoxyloganic acid and NADPH. (a) HPLC-DAD profile of the reaction extract compared with the negative control and authentic standards. (b) Product identity was confirmed by comparison of the mass spectrum with the authentic standard.

Figure 7. Expression of the transcriptional regulon required for loganic acid biosynthesis in the IPAP cells.

In situ hybridization on serial longitudinal sections of young developing leaves was carried out with antisense (AS) probes and sense (S) probes as controls. G8O and strictosidine β-D-glucosidase (SGD) AS probes were used as IPAP and epidermis markers, respectively. Sense probe controls gave no signals (not shown). 8-HGO, 8-hydroxygeraniol oxidoreductase; IO, iridoid oxidase; 7-DLGT, 7-deoxyloganetic acid glucosyl transferase; 7-DLH, 7-deoxyloganic acid hydroxylase; IPAP, internal phloem-associated parenchyma; ep, epidermis. Scale bar=100 μm.

Figure 8. Reconstitution of the strictosidine pathway in N. benthamiana.

(a) Gene combinations infiltrated in leaves in triplicate. (b) Principal component analysis. PC1 and PC2 describe 36.2 and 31.1% of the total mass variation, respectively. (c) LC-MS analysis showing selected masses 401 and 359 representing (acetylated) 7-deoxyloganic acid (7-DLA) from infiltrations with 8-carboxygeranic acid (CGA), 7-DLA or gene combinations 6 or 7 (negative control). The two peaks likely represent 7-DLA acetylated at two different positions in the glucose moiety. (d) LC-MS analysis showing selected masses 433 (formic acid adduct of secologanin) and 575 (formic acid adduct of strictosidine) from infiltrations with secologanin or strictosidine, or with gene combinations 9 or 10, with or without iridodial. *Identical profiles with iridotrial or 7-DLH. Hex=hexosyl; CPS=counts per second.