Discovery of iridoid cyclase completes the iridoid pathway in asterids

Abstract

Iridoids are specialized monoterpenes ancestral to asterid flowering plants^1,2 that play key roles in defence and are also essential precursors for pharmacologically important alkaloids^3,4. The biosynthesis of all iridoids involves the cyclization of the reactive biosynthetic intermediate 8-oxocitronellyl enol. Here, using a variety of approaches including single-nuclei sequencing, we report the discovery of iridoid cyclases from a phylogenetically broad sample of asterid species that synthesize iridoids. We show that these enzymes catalyse formation of 7S-cis-trans and 7R-cis-cis nepetalactol, the two major iridoid stereoisomers found in plants. Our work uncovers a key missing step in the otherwise well-characterized early iridoid biosynthesis pathway in asterids. This discovery unlocks the possibility to generate previously inaccessible iridoid stereoisomers, which will enable metabolic engineering for the sustainable production of valuable iridoid and iridoid-derived compounds.

Fig. 1. Identification of ICYC.

a, Scheme showing iridoid biosynthesis including previously characterized iridoid pathway genes (grey). The 8-oxogeranial intermediate can be converted to either 7S-cis-trans nepetalactol or 7R-cis-cis nepetalactol by previously characterized species-specific stereoselective ISYs together with the newly identified ICYCs. For a complete scheme of the secoiridoid pathway including all intermediates, see Supplementary Fig. 1. b, C. ipecacuanha bulk tissue RNA-seq and snRNA-seq show tight co-expression of orthologues of previously characterized secoiridoid pathway genes from C. roseus (Supplementary Fig. 3a) and the newly identified CiICYC. Complete tissue-specific data are shown in Supplementary Fig. 3b. Cell clusters were grouped into cell types that were determined using marker genes (see Supplementary Fig. 4 for complete analysis of snRNA-seq dataset). c, Venn diagram showing overlay of co-expressed genes from both datasets. See Extended Data Fig. 1a for a detailed list of genes. MEP, 2-C-methyl-d-erythritol 4-phosphate. d, Identification of ICYC orthologues in various iridoid-producing orders. The tree is based on the latest angiosperm phylogeny. Circles depict reported presence of iridoids within at least one species of the respective order. An asterisk denotes that no sequencing data were publicly available from a reported iridoid-producing species from the respective clade, and thus ICYC presence could not be determined. Right: a scheme depicting the synteny of the ICYC and G8H gene cluster. The synteny is shown for representative species of each order: A. salviifolium (Cornales), V. corymbosum (Ericales), Escallonia rubra (Escalloniales), L. japonica (Dipsacales), Eucommia ulmoides (Garryales), A. majus (Lamiales) and C. ipecacuanha (Gentianales)^–. e, Representative ICYC orthologues from different orders all reconstituted the iridoid pathway up to loganic acid in N. benthamiana. Ci, C. ipecacuanha; IO, iridoid oxidase; 7DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid methyltransferase; SLS, secologanin synthase; As, A. salviifolium; Vc, V. corymbosum; Lj, L. japonica; Si, S. indicum; Am, A. majus; Ci, C. ipecacuanha; Cr, C. roseus.

Fig. 2. In vitro activity assays reveal stereoselectivity of ICYCs.

a, Scheme depicting reaction products of 7S and 7R stereo-selective ISY together with ICYC. b,c, Assays were performed as indicated and analysed by GC-MS. BSA was used as a negative control. Displayed chromatograms are total ion chromatograms of one representative replicate. See Supplementary Fig. 9 for detailed descriptions of chromatograms including side products and Supplementary Fig. 10 for fragmentation patterns of standards and enzymatic products. Bar graphs depict peak areas from N = 3 replicates of nepetalactol (shaded in grey); error bars are standard error of the mean (s.e.m.). b, Assays of ICYC orthologues with the 7S selective C. roseus ISY (CrISY) show a dramatic increase in 7S-cis-trans nepetalactol in the presence of ICYC orthologues. c, Assays of ICYC orthologues with the 7R selective A. majus ISY (AmISY) reveal appearance of a peak consistent with 7R-cis-cis nepetalactol in the presence of ICYC. The asterisk indicates that these side products are the 7R enantiomers that coelute with the 7S series when using an achiral stationary phase as used here (Supplementary Fig. 9). d, To confirm the identity of the AmISY–AmICYC enzymatic product, five enzymatic reactions were pooled and chemically oxidized to 7R-cis-cis nepetalactone (Methods). e, Chiral GC-MS analysis of the chemically oxidized product showed that it had the same retention time and the identical mass (extracted ion chromatogram for nepetalactone mass m/z 166.1) as the authentic standard on a chiral GC column. f, The MS fragmentation pattern of the oxidized enzymatic product was identical to the standard confirming that the enzymatic product is indeed 7R-cis-cis nepetalactol. Ctrl, control. Source data

Fig. 3. ICYC is a member of the MES family.

a, A condensed phylogenetic tree showing that ICYC is related to MESs that esterify substrates in plant hormone activation pathways and in alkaloid biosynthesis. Each clade is named after a member found within the respective clade. See Supplementary Fig. 12 for an extended version of this tree. Sequence data highlight the conservation of the catalytic triad and other amino acid residues that were subjected to mutation in CiICYC. The asterisk indicates that AtMES10 was a single-member clade, and thus no sequence logos were generated. At, Arabidopsis thaliana; CXE, carboxylesterases; CiDE, C. ipecacuanha deacetyl(iso)ipecoside esterase; CpDCE, Cinchona pubescens dihydrocorynantheine aldehyde esterase; RsPNAE, Rauvolfia serpentina polyneuridine aldehyde esterase. b, Putative active site of an alphafold3 structural model of CiICYC with the docked (AutoDock Vina) substrate S-8-oxocitronellyl enol substrate. Indicated amino acids were chosen for mutagenesis studies because they are part of the catalytic triad (in bold) or are in proximity to the docked substrate and are conserved in ICYC orthologues. c, In vitro assays show that D210A and H238A ICYC mutants yield a reaction profile that is identical to the negative control. S83A, V15G and W133A mutants show reduced levels of nepetalactol compared to wild-type ICYC, whereas H17A and G84A mutants behaved similarly to wild-type (WT) ICYC. The asterisk indicates that the D210A mutant exhibited poor solubility, and thus the assays with this mutant should be interpreted cautiously. Bar graphs depict normalized peak areas from N = 3 replicates of nepetalactol (shaded in grey); error bars are s.e.m. d, Reconstitution of loganic acid biosynthesis in N. benthamiana with CiICYC and CiICYC mutants yielded results that are consistent with results from in vitro assays shown in c. Bar graphs depict normalized peak areas from N = 3 biological replicates; error bars are s.e.m. EV, empty vector. Source data

Extended Data Fig. 1. Combination of bulk and single nuclei RNA-seq co-expression analysis.

Because it was unclear which type of enzyme would catalyze this cyclization reaction, we attempted to narrow down the list of candidates through co-expression analyses rather than using functional annotation. To achieve the most highly resolved co-expression list, candidate genes from tissue specific co-expression analysis (Pearson correlation to CiISY expression > 0.8) and cell type specific co-expression analysis (co-expression module 16) were overlayed. a, In addition to co-expression, only genes with high absolute expression in bulk RNA-seq (>50 CPM in young leaves from plant 1) and snRNA-seq (normalized average expression > 1 in cell cluster 30) were included. The overlay contained the 13 genes listed with functional annotations based on sequence homology. ICYC is shown in bold. Blue indicates orthologs of known iridoid pathway genes, green indicates orthologs of known MEP pathway genes. CYPADH has been previously associated with iridoid biosynthesis but its exact function could not be determined (Brown et al, 2015). b, Without a cutoff for absolute expression in bulk RNA-seq, the list of genes common to both datasets contained 9 additional genes, including orthologs of the C. roseus bHLH iridoid synthesis (BIS) transcription factors that are known to induce expression of IPAP specific iridoid pathway genes^–. Blue indicates orthologs of C. roseus BIS. CPM, counts per million;; GES, geraniol synthase; G8H, geraniol 8-hydroxylase; 8HGO, 8-hydroxygeraniol oxidase; ISY, iridoid synthase; ICYC, iridoid cyclase; IO, iridoid oxidase; 7DLGT, 7-deoxyloganetic acid glucosidase; 7DLH, 7-deoxyloganic acid hydroxylase.

Extended Data Fig. 2. Virus Induced Gene Silencing (VIGS) of ICYC and ISY in C. roseus.

Magnesium Chelatase (MgChel) was co-silenced in all cases to visualize silenced tissues. In the negative control (in orange) only MgChel was silenced. As a positive control CrISY was silenced. a- b, qPCR confirming the downregulation CrICYC (a) or CrISY (b) compared to MgChel negative control. Expression values are shown as fold-changes relative to expression in MgChel negative control. Bar graphs depict mean N = 6 biological replicates, error bars are standard error of the mean. Black dot symbols depict values for individual replicates. P-value of unpaired two-tailed ttest with Welch’s test correction: **P = 0.0061, ****P < 0.0001. c, Secologanin levels in silenced plants compared to control. LC-MS peak areas normalized to internal standard are shown as individual dots of each replicate, the line depicts the mean of N = 6 biological replicates. P-value of unpaired two-tailed ttest with Welch’s test correction: **P = 0.0047, ***P = 0.0002.

Extended Data Fig. 3. ISY and ICYC must be simultaneously present for nepetalactol formation and may interact.

a, Enzyme assays under the conditions shown in Fig. 2a (main text) but with ICYC, NmMLPL or BSA added simultaneously for 3 hours or after 1.5 hours reaction with CrISY alone (sequential incubation). Interestingly, nepetalactol is only formed to higher amounts in simultaneous incubations. This could indicate that the formed ISY product 8-oxocitronellyl enol, which is the substrate for the cyclases, is unstable und must be immediately taken up by the cyclases. Bar graphs depict peak areas from N = 3 replicates of nepetalactol, error bars are standard error of the mean. b, Split-Luciferase assay with CiICYC and CiISY in N. benthamiana. The C-terminal part of luciferase (CLuc) and the N-terminal part of luciferase (NLuc) are always fused N-terminally or C-terminally, respectively, to the target protein. Empty vectors (EV) contained non-fused CLuc or NLuc, respectively, and served as negative controls. Leaf disks were cut from N = 4 biological replicates of agroinfiltrated N. benthamiana plants, transferred to a well plate, where luciferin was added, and imaged using a Nightshade camera (see methods for details). Images are pictures of the same leaf disks with different indicated exposure times. The assays indicate that CiICYC interacts with itself and that CiICYC interacts with CiISY. Note that when NLuc is fused to CiISY interaction is weaker and only revealed at longer exposure times.

Extended Data Fig. 4. Esterase activity of ICYC.

a, Reaction scheme of the assay. Esterase activity of the proteins was assessed with 4-nitrophenyl acetate as a substrate. The formation of product is assessed by measuring absorbance of the 4-nitrophenol (yellow) at 405 nm. b, Esterase activity assays of 0.5 µM ICYC orthologs compared to NmMLPL and the negative control BSA. Esterase activity greatly varies among ICYC proteins but is detectable for all ICYC orthologs. c, Esterase activity assays of 0.5 µM CiICYC wild-type and mutant proteins (see Fig. 3, main text). Mutations in the catalytic triad led to complete abolishment of esterase activity (the asterisk indicates that D210A mutant was poorly soluble, thus activity could not be assessed reliably). Esterase activity appeared to be poorly correlated with cyclase activity (Fig. 3, main text). Each curve was created by curve fitting of absorbance values of N = 3 replicates. Absorbance was measured every minute on a microplate reader (see methods for details).