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Abstract Plants deploy both primary and species-specific, specialized metabolites to communicate with other organisms and adapt to environmental challenges, including interactions with soil-dwelling microbial communities. However, the role of specialized metabolites in modulating plant-microbiome interactions often remains elusive. In this study, we report that maize ( Zea mays ) diterpenoid metabolites with known antifungal bioactivities also influence rhizosphere bacterial communities. Metabolite profiling showed that dolabralexins, antibiotic diterpenoids that are highly abundant in roots of some maize varieties, can be exuded from the roots. Comparative 16S rRNA gene sequencing determined the bacterial community composition of the maize mutant Zman2 ( anther ear 2 ), which is deficient in dolabralexins and closely related bioactive kauralexin diterpenoids. The Zman2 rhizosphere microbiome differed significantly from the wild-type sibling with the most significant changes observed for Alphaproteobacteria of the order Sphingomonadales. Metabolomics analyses support that these differences are attributed to the diterpenoid deficiency of the Zman2 mutant, rather than other large-scale metabolome alterations. Together, these findings support physiological functions of maize diterpenoids beyond known chemical defenses, including the assembly of the rhizosphere microbiome.
Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti–microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage (Leucophyllum frutescens) has demonstrated activity against multi–drug–resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L. frutescens and compare tissue–specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short–chain cis–prenyl transferase (LfCPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase (LfTPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short–chain cis–prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila. LfTPS1 represents neofunctionalization of a compartment–switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti–microbial therapeutics in heterologous systems.
Cyclic diterpenoid compounds have been found by various investigators in the geosphere (e.g., fossil resins, coals, soil, shale, and deep-sea sediments). These compounds occur in significant amounts only in higher plants and are therefore potential markers of terrigenous plant lipids. Diterpenoids with the abietane skeleton (mainly dehydroabietic acid) have been identified in the lipids of sediment samples from the northeast Pacific Ocean, Black Sea, and North Atlantic Ocean. The presence of these resin-derived compounds was correlated with the terrigenous clay components and with the presence of pollen. The presence of polycyclic diterpenoids was also correlated with the distribution patterns and inferred sources of other sediment lipid constituents. Potamic transport, followed by turbidite redistribution, is the probable input mechanism of these resin-derived compounds to the deep-sea sediments. These diterpenoids appear to be excellent biological markers of resinous higher plants.
The diterpenoid alkaloids are a group of specialized metabolites where the terpenoid and alkaloid classes intersect, and which are found primarily within the Aconitum (Wolf’s-Bane) and Delphinium (Larkspur) genera. While there is considerable interest in these compounds for their wide range of bioactivities, their structural complexity poses a significant challenge for chemical synthesis. Biosynthesis offers an appealing alternative for production, however, little progress has been made towards elucidation of their biosynthetic pathways. Here, we employ a comparative transcriptomics approach to identify six enzymatic steps in the biosynthesis of atisinium, conserved across both Delphinium grandiflorum and Aconitum plicatum . Key to this pathway is a reductase which selectively incorporates ethanolamine over ethylamine into the diterpenoid scaffold. While the majority of diterpenoid alkaloids contain an ethylamine moiety, we demonstrate through isotope labeling in Aconitum callus cultures and a computational metabolomics approach that ethanolamine is, unintuitively, the preferred source of nitrogen for these metabolites. Identification of these enzymes and production of a key intermediate in a heterologous host paves the way for biosynthetic production of this group of metabolites with promise for medicinal applications.
The Lamiaceae (mint family) is the largest known source of furanoclerodanes, a subset of clerodane diterpenoids with broad bioactivities including insect antifeedant properties. The Ajugoideae subfamily, in particular, accumulates significant numbers of structurally related furanoclerodanes. The biosynthetic capacity for formation of these diterpenoids is retained across most Lamiaceae subfamilies, including the early-diverging Callicarpoideae which forms a sister clade to the rest of Lamiaceae. VacCYP76BK1, a cytochrome P450 monooxygenase from Vitex agnus-castus, was previously found to catalyze the formation of the proposed precursor to furan and lactone-containing labdane diterpenoids. Through transcriptome-guided pathway exploration, we identified orthologs of VacCYP76BK1 in Ajuga reptans and Callicarpa americana. Functional characterization demonstrated that both could catalyze the oxidative cyclization of clerodane backbones to yield a furan ring. Subsequent investigation revealed a total of 10 CYP76BK1 orthologs across six Lamiaceae subfamilies. Through analysis of available chromosome-scale genomes, we identified four CYP76BK1 members as syntelogs within a conserved syntenic block across divergent subfamilies. This suggests an evolutionary lineage that predates the speciation of the Lamiaceae. Functional characterization of the CYP76BK1 orthologs affirmed conservation of function, as all catalyzed furan ring formation. Additionally, some orthologs yielded two novel lactone ring moieties. The presence of the CYP76BK1 orthologs across Lamiaceae subfamilies closely overlaps with the distribution of reported furanoclerodanes. Together, the activities and distribution of the CYP76BK1 orthologs identified here support their central role in furanoclerodane biosynthesis within the Lamiaceae family. Our findings lay the groundwork for biotechnological applications to harness the economic potential of this promising class of compounds.
Abstract Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g −1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐ d ‐erythritol 4‐phosphate pathway, Coleus forskohlii 1‐deoxy‐ d ‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g −1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.
Teucrium is well known for making clerodane-type diterpenoids that are produced from the backbone kolavanyl diphosphate. In order to begin to elucidate some of the complex biosynthetic pathways of these medicinal compounds, we identified and functionally characterized several kolavanyl diphosphate synthases from T. chamaedrys . Along the way, we discovered the genome of this species to be one of the largest genomes published from the Lamiaceae family, to which it belongs. This tetraploid, 3 Gbp genome is especially rich in diterpene synthase genes, with 74 putative sequences identified.
Diterpenoid alkaloids are known for their range of potential medicinal applications and are found primarily in the Delphinium and Aconitum genera. We sequenced RNA isolated from different tissue types of Delphinium grandiflorum, Aconitum plicatum, and Aconitum lycoctonum to identify six entry steps in their biosynthesis.
CYP76BK1 orthologs catalyze furan and lactone ring formation in clerodane diterpenoids across the mint family
Transcriptome assemblies, open reading frames in nucleotide and peptide sequences, clustered transcriptomes and corresponding amino acid files, and expression matrices in TPM and raw counts for RNA-seq datasets from Delphinium grandiflorum, Aconitum plicatum, Aconitum lycoctonum, Aconitum carmichaelii, Aconitum japonicum, Aconitum kusnezoffii, and Aconitum vilmorinianum. Also included are phylogenetic trees for terpene synthases and cytochromes P450 mined from these assemblies.
Abstract The diverse class of plant diterpenoid metabolites serves important functions in mediating growth, chemical defence, and ecological adaptation. In major monocot crops, such as maize (Zea mays), rice (Oryza sativa), and barley (Hordeum vulgare), diterpenoids function as core components of biotic and abiotic stress resilience. Switchgrass (Panicum virgatum) is a perennial grass valued as a stress‐resilient biofuel model crop. Previously we identified an unusually large diterpene synthase family that produces both common and species‐specific diterpenoids, several of which accumulate in response to abiotic stress. Here, we report discovery and functional characterization of a previously unrecognized monofunctional class I diterpene synthase (PvKSL1) viain vivoco‐expression assays with different copalyl pyrophosphate (CPP) isomers, structural and mutagenesis studies, as well as genomic and transcriptomic analyses. In particular, PvKSL1 convertsent‐CPP intoent‐abietadiene,ent‐palustradiene,ent‐levopimaradiene, andent‐neoabietadiene via a 13‐hydroxy‐8(14)‐ent‐abietene intermediate. Notably, although featuring a distinctent‐stereochemistry, this product profile is near‐identical to bifunctional (+)‐levopimaradiene/abietadiene synthases occurring in conifer trees. PvKSL1 has three of four active site residues previously shown to control (+)‐levopimaradiene/abietadiene synthase catalytic specificity. However, mutagenesis studies suggest a distinct catalytic mechanism in PvKSL1. Genome localization ofPvKSL1distant from other diterpene synthases, and its phylogenetic distinctiveness from known abietane‐forming diterpene synthases, support an independent evolution of PvKSL1 activity. Albeit at low levels,PvKSL1gene expression predominantly in roots suggests a role of diterpenoid formation in belowground tissue. Together, these findings expand the known chemical and functional space of diterpenoid metabolism in monocot crops.
Summary Switchgrass ( Panicum virgatum ) is a bioenergy model crop valued for its energy efficiency and drought tolerance. The related monocot species rice ( Oryza sativa ) and maize ( Zea mays ) deploy species‐specific, specialized metabolites as core stress defenses. By contrast, specialized chemical defenses in switchgrass are largely unknown. To investigate specialized metabolic drought responses in switchgrass, we integrated tissue‐specific transcriptome and metabolite analyses of the genotypes Alamo and Cave‐in‐Rock that feature different drought tolerance. The more drought‐susceptible Cave‐in‐Rock featured an earlier onset of transcriptomic changes and significantly more differentially expressed genes in response to drought compared to Alamo. Specialized pathways showed moderate differential expression compared to pronounced transcriptomic alterations in carbohydrate and amino acid metabolism. However, diterpenoid‐biosynthetic genes showed drought‐inducible expression in Alamo roots, contrasting largely unaltered triterpenoid and phenylpropanoid pathways. Metabolomic analyses identified common and genotype‐specific flavonoids and terpenoids. Consistent with transcriptomic alterations, several root diterpenoids showed significant drought‐induced accumulation, whereas triterpenoid abundance remained predominantly unchanged. Structural analysis verified select drought‐responsive diterpenoids as oxygenated furanoditerpenoids. Drought‐dependent transcriptome and metabolite profiles provide the foundation to understand the molecular mechanisms underlying switchgrass drought responses. Accumulation of specialized root diterpenoids and corresponding pathway transcripts supports a role in drought stress tolerance.
Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generate the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial α-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.
Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Furthermore, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450-derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25-CYP71Z29 for native and non-native diterpenoids.
Abstract Plant derived bioactive small molecules have attracted attention of scientists across fundamental and applied scientific disciplines. We seek to understand the influence of these phytochemicals on rhizosphere and root-associated fungi. We hypothesize that – consistent with accumulating evidence that switchgrass genotype impacts microbiome assembly – differential terpenoid accumulation contributes to switchgrass ecotype-specific microbiome composition. An initial in vitro Petri plate-based disc diffusion screen of 18 switchgrass root derived fungal isolates revealed differential responses to upland- and lowland-isolated metabolites. To identify specific fungal growth-modulating metabolites, we tested fractions from root extracts on three ecologically important fungal isolates – Linnemania elongata, Trichoderma sp. and Fusarium sp. Saponins and diterpenoids were identified as the most prominent antifungal metabolites. Finally, analysis of liquid chromatography-purified terpenoids revealed fungal inhibition structure – activity relationships (SAR). Saponin antifungal activity was primarily determined by the number of sugar moieties – saponins glycosylated at a single core position were inhibitory whereas saponins glycosylated at two core positions were inactive. Saponin core hydroxylation and acetylation were also associated with reduced activity. Diterpenoid activity required the presence of an intact furan ring for strong fungal growth inhibition. These results inform future breeding and biotechnology strategies for crop protection with reduced pesticide application.
Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent-, (+)- and syn-copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien-19-ol, syn-pimara-7,15-dien-19-ol and germacrene-D-4-ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth-inhibiting activity of abietadien-19-ol and syn-pimara-7,15-dien-19-ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid-mediated stress resilience in these agriculturally important species.