This review focuses on the monoterpene, sesquiterpene, and diterpene synthases of plant origin that use the corresponding C 10 , C 15 , and C 20 prenyl diphosphates as substrates to generate the enormous diversity of carbon skeletons characteristic of the terpenoid family of natural products. A description of the enzymology and mechanism of terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. Sequence relatedness and phylogenetic reconstruction, based on 33 members of the Tps gene family, are delineated, and comparison of important structural features of these enzymes is provided. The review concludes with an overview of the organization and regulation of terpenoid metabolism, and of the biotechnological applications of terpenoid synthase genes.The pathways of monoterpene, sesquiterpene, and diterpene biosynthesis are conveniently divided into several stages. The first encompasses the synthesis of isopentenyl diphosphate, isomerization to dimethylallyl diphosphate, prenyltransferase-catalyzed condensation of these two C 5 -units to geranyl diphosphate (GDP), and the subsequent 1Ј-4 additions of isopentenyl diphosphate to generate farnesyl (FDP) and geranylgeranyl (GGDP) diphosphate ( Fig. 1) (1). In the second stage, the prenyl diphosphates undergo a range of cyclizations based on variations on the same mechanistic theme to produce the parent skeletons of each class. Thus, GDP (C 10 ) gives rise to monoterpenes (2), FDP (C 15 ) to sesquiterpenes (3), and GGDP (C 20 ) to diterpenes (4). These transformations catalyzed by the terpenoid synthases (cyclases) may be followed by a variety of redox modifications of the parent skeletal types to produce the many thousands of different terpenoid metabolites of the essential oils, turpentines, and resins of plant origin (5).This review focuses on the synthases that use prenyl diphosphate substrates to generate the enormous diversity of carbon skeletons characteristic of terpenoids. Most of these natural products are cyclic, and many contain multiple ring systems, the basic structures of which are determined by the highly specific terpenoid synthases; examples of synthases that produce acyclic products are also known. The terpenoid synthases may be involved in the regulation of pathway flux because they operate at metabolic branch points and catalyze the first committed steps leading to the various terpene classes (6). The synthases responsible for generating the parent compounds of the various types are quite similar in properties (7), and all operate by electrophilic reaction mechanisms, as do the prenyltransferases (8, 9). Comprehensive treatment of the topic, especially enzymological and mechanistic aspects, has been provided recently (2-4), and the field is periodically surveyed (10, 11). After brief coverage of the three types of terpene synthases from higher plants, with emphasis on common features of structure and function, we focus here on molecular cloning and sequence analysis of these ...

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera ( Gc ), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc , MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc , and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

conifer defense ͉ gibberellic acid ͉ diterpene resin acids ͉ conifer genomics ͉ plant secondary metabolism D iterpene resin acids (DRAs) (Fig. 1A) are important defense compounds of conifers against potential herbivores and pathogens, such as bark beetles and their associated fungi (1-3). DRAs are formed and sequestered as major components of complex oleoresin blends in resin ducts, resin blisters, or resin cells in stems, needles, and roots of most conifers. Biosynthesis of DRAs involves formation of geranylgeranyl diphosphate (GGDP), cyclization of GGDP to a series of diterpene olefins by activity of diterpene synthases (diTPSs), and three subsequent oxidations at carbon 18 (Fig. 1B). A small family of singleproduct or multiproduct diTPSs of DRA biosynthesis has recently been cloned and characterized (4-6). Studies with grand fir (Abies grandis) and lodgepole pine (Pinus contorta) tissue extracts showed that stepwise oxidation of the diterpene olefin abietadiene (1a) to abietic acid (1d) can be achieved by membrane-bound cytochrome P450 monooxygenase (P450) and soluble aldehyde dehydrogenase enzyme activities (Fig. 1B) (7, 8). The general pathway scheme of oxidation of abietadiene to abietic acid in conifer secondary metabolism resembles that of oxidation of ent-kaurene to ent-kaurenoic acid in the biosynthesis of gibberellin phytohormones (9-11). A multifunctional P450 responsible for the three-step oxidation of ent-kaurene to kaurenoic acid has previously been identified by using genetic approaches in Arabidopsis thaliana (10-11). Similarly, a multifunctional P450 also catalyzes the subsequent three-step oxidation from kaurenoic acid to GA 12 in Arabidopsis (12). Cloning and identification of P450s of DRA secondary metabolism have been hampered by difficulties in purifying the corresponding enzymes and by lack of suitable forward genetic tools for conifers. However, large collections of ESTs for loblolly pine (Pinus taeda, Pt) (13) can enable gene discovery and biochemical identification of candidate P450 cDNAs of terpenoid secondary metabolism in conifers. Here, we describe cloning and functional characterization of abietadienol͞abietadienal oxidase (PtAO, CYP720B1), a P450 enzyme that is unusual in that it catalyzes an array of consecutive oxidations of multiple diterpene alcohol and aldehyde intermediates in DRA biosynthesis in loblolly pine. The methyl jasmonate (MJ)-inducible PtAO can account for much of the oxidative diversification of diterpenoid natural product defense compounds in loblolly pine. Materials and MethodsMaterials. Seedlings of loblolly pine and Sitka spruce (Picea sitchensis) were grown to 2-year-old trees as described in ref. 14. Yeast strain YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-⌬63, his3-⌬200, and leu2-⌬1) and yeast dual expression vectors (pESC-Leu and pESC-His) were from Stratagene. Diterpenoid substrates were prepared from the corresponding DRAs (Helix Biotech, Surrey, BC, Canada) as described in Supporting Materials and Methods, which is published as supporting informat...

Defense-related terpenoid biosynthesis in conifers is a dynamic process closely associated with specialized anatomical structures that allows conifers to cope with attack from many potential pests and pathogens. The constitutive and inducible terpenoid defense of conifers involves several hundred different monoterpenes, sesquiterpenes and diterpenes. Changing arrays of these many compounds are formed from the general isoprenoid pathway by activities of large gene families for two classes of enzymes, the terpene synthases and the cytochrome P450-dependent monooxygenases of the CYP720B group. Extensive studies have been conducted on the genomics, proteomics and molecular biochemical characterization of these enzymes. Many of the conifer terpene synthases are multi-product enzymes, and the P450 enzymes of the CYP720B group are promiscuous in catalyzing multiple oxidations, along homologous series of diterpenoids, from a broad spectrum of substrates. The terpene synthases and CYP720B genes respond to authentic or simulated insect attack with increased transcript levels, protein abundance and enzyme activity. The constitutive and induced oleoresin terpenoids for conifer defense accumulate in preformed cortical resin ducts and in xylem trauma-associated resin ducts. Formation of these resin ducts de novo in the cambium zone and developing xylem, following insect attack or treatment of trees with methyl jasmonate, is a unique feature of the induced defense of long-lived conifer trees.Zulak KG, Bohlmann J (2010) Terpenoid biosynthesis and specialized vascular cells of conifer defense.