Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory

Abstract

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

Fig. 1. Species and genome assemblies used in this study.

The cladogram shows evolutionary relationships between the species studied. Numbers at the nodes represent estimations of clade age in millions of years (MYA)^–. Ploidy level of species not assumed to be diploid are shown in parenthesis next to their genome size (Supplementary Table 2).

Fig. 2. Syntenic relationships of a miltiradiene biosynthetic gene cluster present across the Lamiaceae.

Genes are represented with arrows and pseudogenes are represented with boxes. A core set of genes are common to many species examined, including a diTPS class II (+)-CPP synthase, a diTPS class I miltiradiene synthase, and CYP450s in the 76AH and 71D subfamilies. Notably, there is divergence in gene number, cluster length, and unique genes, indicating lineage-specific evolution. Synteny between each species is shown here with colored curves. Species tree adapted from Mint Evolutionary Genomics Consortium 2018. Figure created using BioRender.com. Source data are provided as a Source Data file.

Fig. 3. Phylogenetic evidence shows the relatedness of each gene class in the clusters.

Enzymes present in each cluster with syntenic support from MCScanX and sequence identity from BLASTp are highlighted in red (TPS-e), orange (TPS-c), light blue (CYP76AH), and dark blue (CYP71D). DiTPSs characterized in previous reports are highlighted in pink and periwinkle ((+)-CPP synthases for TPS-c and miltiradiene synthases for TPS-e, respectively). Reference enzymes are bolded. Black solid dots at the base of the nodes represent 80% bootstrap confidence. Gray circles around clade nodes represent hypothetical expansion points for syntelogs and share approximately 70% or more sequence similarity. DiTPS trees are rooted to Physcometrium patens ent-kaurene synthase (PpEKS), and CYP trees are rooted to Arabidopsis thaliana AtCYP701A3. Source data are provided as a Source Data file.

Fig. 4. Predicted Lamiaceae minimal ancestral BGC and species-specific expansion of each.

Based on maximum parsimony, we suggest that a cluster containing a class II diTPS, class I diTPS, CYP76AH, and CYP71D gene was formed in an early Lamiaceae ancestor. Lineage-specific expansion and refinement are evident from the number and composition of genes in each gene family present in the extant species studied. Source data are provided as a Source Data file.

Fig. 5. Tissue specific expression of a miltiradiene BGC in C. americana obtained from RNA sequencing.

Functional characterization of these enzymes refers to this study. This figure represents Chr10:21.92-22.33 Mb. Approximate location on the chromosome is indicated. Two differentially expressed metabolic clusters are boxed to highlight similar expression patterns. Colors indicate diTPS, CYP, or unrelated gene family, including pseudogenes (unnamed). Data obtained from Hamilton et al..

Fig. 6. GC-MS analysis of C. americana BGC diTPS products.

CamTPS9 was confirmed to be a miltiradiene synthase by comparison with the retention time and mass spectra of PbTPS3^– products when both were expressed with the (+)-CPP synthase CamTPS6, forming miltiradiene (1) and abietatriene (2). CamTPS10 was found to make 4 from (+)-CPP but not ent-CPP (CamTPS1). This product has a different retention time but similar mass spectrum to ent-kaurene (3), made by the combination of CamTPS1 and CamTPS12 (Supplementary Fig. 11). All chromatograms shown are total ion chromatograms. Red and black traces correspond to combinations yielding 1, 2, 3, and 4 respectively, as indicated in the mass spectra. Each combination includes P. barbatus 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and GGPP synthase (GGPPS), shown as a control in gray. Source data are provided as a Source Data file.

Fig. 7. GC-MS chromatograms showing oxidation products of C. americana BGC CYPs.

a Oxidation products of the CamCYP76AHs from 1 and 2, assigned based on analysis of mass spectra (Supplementary Fig. 12). b CamCYP71D717 catalyzes the production of (+)-manool (6), likely from (+)-copalol (5) (Supplementary Fig. 16,) and the addition of CamCYP71D716 results in 3(S)-hydroxy-(+)-manool (7). Each combination includes P. barbatus 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and GGPP synthase (GGPPS), shown as a control in gray. CamTPS6 and CamTPS6 + CamTPS9 controls given in red. Source data are provided as a Source Data file.

Fig. 8. Pathway schematic for CYP oxidations in C. americana.

a Proposed mechanism for enzyme-assisted conversion of 1 to 2, followed by an additional oxidation of 2 to form 2c. Mass spectra supports assignment of the hydroxy group in 2c to the c-ring (Supplementary Fig. 12). b Proposed conversion of 5 to 6 by CamCYP71D717, and oxidation of 6 by CamCYP71D716. This occurs in the same position as a keto group on calliterpenone, which is derived from 4. c Structures of abietane diterpenoids found in two other species of Callicarpa.