Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants’ terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis

Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2–7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.

Functional kaurene-synthase-like diterpene synthases lacking a gamma domain are widely present in Oryza and related species

Various diterpene synthases have been functionally identified in cultivated rice (Oryza sativa). These are the homologs of ent-copalyl diphosphate (ent-CDP) synthase and ent-kaurene synthase (KS) that are responsible for the biosynthesis of gibberellins, diterpenoid phytohormones. We isolated a cDNA encoding full-length OsKSL12, a previously uncharacterized KS like (KSL) enzyme that consists of a β-domain and an α-domain with an active center, but lacks an N-terminal γ-domain. Functional analysis using a bacterial expression system showed that recombinant OsKSL12 converted ent-CDP into ent-manool or ent-13-epi-manool. Comparative genomics revealed that functional OsKSL12 homologs exist in diverse wild species in the Oryzeae- Oryza nivara (Oryza rufipogon), Oryza coarctata, Oryza granulata, Leersia perrieri and Leersia tisseranti. KSL12 homologs in O. granulata, L. perrieri and L. tisseranti preferentially reacted with GGDP rather than ent-CDP, resulting in geranyllinalool rather than ent-manool or ent-13-epi-manool as the main product, meaning that KSL12 functionally diversified during evolution in the Oryzeae.

Furanoditerpenoid biosynthesis in the bioenergy crop switchgrass is catalyzed by an alternate metabolic pathway

Specialized diterpenoid metabolites are important mediators of stress resilience in monocot crops. A deeper understanding of how species-specific diterpenoid-metabolic pathways and functions contribute to plant chemical defenses can enable crop improvement strategies. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop switchgrass (Panicum virgatum). Combinatorial pathway reconstruction showed that CYP71Z25-29 catalyze furan ring addition to diterpene alcohol intermediates derived from distinct class II diterpene synthases, thus bypassing the canonical role of class I diterpene synthases in plant diterpenoid metabolism. Transcriptional co-expression patterns and presence of select diterpenoids in droughted switchgrass roots support possible roles of CYP71Z25-29 in abiotic stress responses. Integrating molecular dynamics, structural analysis, and targeted mutagenesis, identified active site determinants controlling distinct CYP71Z25-29 catalytic specificities and, combined with broad substrate promiscuity for native and non-native diterpenoids, highlights the potential of these P450s for natural product engineering.Significance StatementDiterpenoids play important roles in stress resilience and chemically mediated interactions in many plant species, including major food and bioenergy crops. Enzymes of the cytochrome P450 monooxygenase family catalyze the various functional decorations of core diterpene scaffolds that determine the large diversity of biologically active diterpenoids. This study describes the identification and mechanistic analysis of an unusual group of cytochrome P450 monooxygenases, CYP71Z25-29, from the bioenergy crop switchgrass (Panicum virgatum). These enzymes catalyze the furan ring addition directly to class II diterpene synthase products, thus bypassing the conserved pairwise reaction of class II and class I diterpene synthases in labdane diterpenoid metabolism. Insight into the distinct substrate-specificity of CYP71Z25-29 offers opportunity for engineering of furanoditerpenoid bioproducts.

Evolution of Labdane-Related Diterpene Synthases in Cereals

Gibberellins (GAs) are labdane-related diterpenoid phytohormones that regulate various aspects of higher plant growth. A biosynthetic intermediate of GAs is ent-kaurene, a tetra-cyclic diterpene that is produced through successive cyclization of geranylgeranyl diphosphate catalyzed by the two distinct monofunctional diterpene synthases—ent-copalyl diphosphate synthase (ent-CPS) and ent-kaurene synthase (KS). Various homologous genes of the two diterpene synthases have been identified in cereals, including rice (Oryza sativa), wheat (Triticum aestivum) and maize (Zea mays), and are believed to have been derived from GA biosynthetic ent-CPS and KS genes through duplication and neofunctionalization. They play roles in specialized metabolism, giving rise to diverse labdane-related diterpenoids for defense because a variety of diterpene synthases generate diverse carbon-skeleton structures. This review mainly describes the diterpene synthase homologs that have been identified and characterized in rice, wheat and maize and shows the evolutionary history of various homologs in rice inferred by comparative genomics studies using wild rice species, such as Oryza rufipogon and Oryza brachyantha. In addition, we introduce labdane-related diterpene synthases in bryophytes and gymnosperms to illuminate the macroscopic evolutionary history of diterpene synthases in the plant kingdom—bifunctional enzymes possessing both CPS and KS activities are present in bryophytes; gymnosperms possess monofunctional CPS and KS responsible for GA biosynthesis and also possess bifunctional diterpene synthases facilitating specialized metabolism for defense.

Identification of Three Monofunctional Diterpene Synthases with Specific Enzyme Activities Expressed during Heartwood Formation in Western Redcedar (Thuja plicata) Trees

Upon harvest, Western redcedar (WRC; Thuja plicata) trees have a high incidence and extent of heartwood rot. While monoterpenoids and lignans have been linked to rot resistance in this species, other specialized metabolites, such as diterpenes, are likely to contribute to rot resistance. Here we report the cloning and functional assessment of three putative diterpene synthase (TpdiTPS) genes expressed during heartwood formation in WRC. The predicted proteins of the three genes lack either of the two catalytically independent active sites typical of most diTPS, indicating monofunctional rather than bifunctional activity. To identify potential catalytic activities of these proteins, we expressed them in genetically engineered Escherichia coli strains that produce four potential substrates, geranylgeranyl diphosphate (GGDP), ent, syn, and normal stereoisomers of copalyl diphosphate (CDP). We found that TpdiTPS3 used GGDP to produce CDP. TpdiTPS2 used normal CDP to produce levopimaradiene. TpdiTPS1 showed stereoselectivity as it used normal CDP to produce sandaracopimaradiene and syn-CDP to produce syn-stemod-13(17)-ene. These genes and protein enzymatic activities have not been previously reported in WRC and provide an opportunity to assess their potential roles in heartwood rot resistance in this economically important species.