The diverse sesquiterpene profile of patchouli, Pogostemon cablin, is correlated with a limited number of sesquiterpene synthases

Pogostemon cablin (patchouli), like many plants within the Lamiaceae, accumulates large amounts of essential oil. Patchouli oil is unique because it consists of over 24 different sesquiterpenes, rather than a blend of different mono-, sesqui- and di-terpene compounds. To determine if this complex mixture of sesquiterpenes arises from an equal number of unique sesquiterpene synthases, we developed a RT-PCR strategy to isolate and functionally characterize the respective patchouli oil synthase genes. Unexpectedly, only five terpene synthase cDNA genes were isolated. Four of the cDNAs encode for synthases catalyzing the biosynthesis of one major sesquiterpene, including a gamma-curcumene synthase, two germacrene D synthases, and a germacrene A synthase. The fifth cDNA encodes for a patchoulol synthase, which catalyzes the conversion of FPP to patchoulol plus at least 13 additional sesquiterpene products. Equally intriguing, the yield of the different in vitro reaction products resembles quantitatively and qualitatively the profile of sesquiterpenes found in patchouli oil extracted from plants, suggesting that a single terpene synthase is responsible for the bulk and diversity of terpene products produced in planta.     

The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes

The mature leaves and husks of Zea mays release a complex blend of terpene volatiles after anthesis consisting predominantly of bisabolane-, sesquithujane-, and bergamotane-type sesquiterpenes. The varieties B73 and Delprim release the same volatile constituents but in significantly different proportions. To study the molecular genetic and biochemical mechanisms controlling terpene diversity and distribution in these varieties, we isolated the closely related terpene synthase genes terpene synthase4 (tps4) and tps5 from both varieties. The encoded enzymes, TPS4 and TPS5, each formed the same complex mixture of sesquiterpenes from the precursor farnesyl diphosphate but with different proportions of products. These mixtures correspond to the sesquiterpene blends observed in the varieties B73 and Delprim, respectively. The differences in the stereoselectivity of TPS4 and TPS5 are determined by four amino acid substitutions with the most important being a Gly instead of an Ala residue at position 409 at the catalytic site of the enzyme. Although both varieties contain tps4 and tps5 alleles, their differences in terpene composition result from the fact that B73 has only a single functional allele of tps4 and no functional alleles of tps5, whereas Delprim has only a functional allele of tps5 and no functional alleles of tps4. Lack of functionality was shown to be attributable to frame-shift mutations or amino acid substitutions that greatly reduce the activity of their encoded proteins. Therefore, the diversity of sesquiterpenes in these two maize cultivars is strongly influenced by single nucleotide changes in the alleles of two terpene synthase genes.     

Four terpene synthases produce major compounds of the gypsy moth feeding-induced volatile blend of Populus trichocarpa

After herbivore damage, many plants increase their emission of volatile compounds, with terpenes usually comprising the major group of induced volatiles. Populus trichocarpa is the first woody species with a fully sequenced genome, enabling rapid molecular approaches towards characterization of volatile terpene biosynthesis in this and other poplar species. We identified and characterized four terpene synthases (PtTPS1-4) from P. trichocarpa which form major terpene compounds of the volatile blend induced by gypsy moth (Lymantria dispar) feeding. The enzymes were heterologously expressed and assayed with potential prenyl diphosphate substrates. PtTPS1 and PtTPS2 accepted only farnesyl diphosphate and produced (−)-germacrene D and (E,E)-α-farnesene as their major products, respectively. In contrast, PtTPS3 and PtTPS4 showed both mono- and sesquiterpene synthase activity. They produce the acyclic terpene alcohols linalool and nerolidol but exhibited opposite stereospecificity. qRT-PCR analysis revealed that the expression of the respective terpene synthase genes was induced after feeding of gypsy moth caterpillars. The TPS enzyme products may play important roles in indirect defense of poplar to herbivores and in mediating intra- and inter-plant signaling.     

Within-plant distribution and emission of sesquiterpenes from Copaifera officinalis

Copaifera officinalis, the diesel tree, is known for massive production of oleoresin, mainly composed of sesquiterpene hydrocarbons. In this study, composition of these sesquiterpenes and their concentrations in leaves, stems and roots of C. officinalis at two developmental stages, including the three-week old (TW) seedlings and two-year old (TY) trees, were determined. The leaves of TW seedlings and TY trees contained similar number of sesquiterpenes, which also had comparable concentrations. The stems of TW seedlings had higher concentrations of sesquiterpenes than those of TY trees. In contrast, the number of sesquiterpene species and their concentrations in the roots of TW seedlings were much lower than those in the roots of TY trees. Cluster analysis of sesquiterpenes estimated that there are at least four terpene synthase genes involved in the production of sesquiterpenes in C. officinalis. Because sesquiterpenes are highly volatile, emissions of sesquiterpenes from healthy and wounded TW seedlings were examined using headspace analysis. Whereas very low emission of sesquiterpenes was detected from undamaged plants, the physically injured seedlings emitted a large number of sesquiterpenes, the quality and the relative quantity of which were similar to those in leaves determined using organic extraction. The implications of our findings to the biosynthetic pathways leading to the production of sesquiterpenes as well as their biological roles in C. officinalis are discussed.     

Intensive sampling identifies previously unknown chemotypes,population divergence and biosynthetic connections among terpenoids in Eucalyptus tricarpa

Australian members of the Myrtaceae produce large quantities of ecologically and economically important terpenes and display abundant diversity in both yield and composition of their oils. In a survey of the concentrations of leaf terpenes in Eucalyptus tricarpa (L.A.S. Johnson) L.A.S. Johnson & K.D. Hill, which were previously known from few samples, exceptional variability was found in composition. The aim was to characterize the patterns of variation and covariation among terpene components in this species and to use this information to enhance our understanding of their biosynthesis. There were marked discontinuities in the distributions of numerous compounds, including the overall proportions of mono- and sesquiterpenes, leading us to delineate three distinct chemotypes. Overall, positive covariation predominated, but negative covariation suggested competitive interactions involved in monoterpene synthesis. Two groups of covarying monoterpenes were found, each of which was positively correlated with a group of sesquiterpenes and negatively correlated with the alternate sesquiterpene group. These results imply substantial cross-talk between mono- and sesquiterpene biosynthesis pathways. However, only those compounds hypothesized to share final carbocation intermediates or post-processing steps were strongly positively correlated within chemotypes. This suggests that the broader patterns of covariation among groups of compounds may result from co-regulation of multiple biosynthetic genes, controlling the complex terpene profiles of the chemotypes of Eucalyptus.     

Metabolic engineering of sesquiterpene metabolism in yeast

Terpenes are structurally diverse compounds that are of interest because of their biological activities and industrial value. These compounds consist of chirally rich hydrocarbon backbones derived from terpene synthases, which are subsequently decorated with hydroxyl substituents catalyzed by terpene hydroxylases. Availability of these compounds is, however, limited by intractable synthetic means and because they are produced in low amounts and as complex mixtures by natural sources. We engineered yeast for sesquiterpene accumulation by introducing genetic modifications that enable the yeast to accumulate high levels of the key intermediate farnesyl diphosphate (FPP). Co-expression of terpene synthase genes diverted the enlarged FPP pool to greater than 80 mg/L of sesquiterpene. Efficient coupling of terpene production with hydroxylation was also demonstrated by coordinate expression of terpene hydroxylase activity, yielding 50 mg/L each of hydrocarbon and hydroxylated products. These yeast now provide a convenient format for investigating catalytic coupling between terpene synthases and hydroxylases, as well as a platform for the industrial production of high value, single-entity and stereochemically unique terpenes.     

Characterization of four terpene synthase cDNAs from methyl jasmonate-induced Douglas-fir, Pseudotsuga menziesii

Numerous terpenoid compounds are present in copious amounts in the oleoresin produced by conifers, especially following exposure to insect or fungal pests. CDNA clones for many terpene synthases responsible for the biosynthesis of these defense compounds have been recovered from several conifer species. Here, the use of three terpene synthase sequences as heterologous probes for the discovery of related terpene synthase genes in Douglas-fir, Pseudotsuga menziesii (Mirbel) Franco (Pinaceae), is reported. Four full-length terpene synthase cDNAs were recovered from a methyl jasmonate-induced Douglas-fir bark and shoot cDNA library. These clones encode two multi-product monoterpene synthases [a (-)-alpha-pinene/(-)-camphene synthase and a terpinolene synthase] and two single-product sesquiterpene synthases [an (E)-beta-farnesene synthase and a (E)-gamma-bisabolene synthase].     

Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif

The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant-insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D,E), Sc11 is unusual in that this motif occurs as (303)NDTYD. Mutagenesis of this motif to (303)DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.     

Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases

5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol. To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined. Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis. The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate. The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.     

Population divergence in the ontogenetic trajectories of foliar terpenes of a Eucalyptus species

Background and Aims The development of plant secondary metabolites during early life stages can have significant ecological and evolutionary implications for plant–herbivore interactions. Foliar terpenes influence a broad range of ecological interactions, including plant defence, and their expression may be influenced by ontogenetic and genetic factors. This study investigates the role of these factors in the expression of foliar terpene compounds in Eucalyptus globulus seedlings.Methods Seedlings were sourced from ten families each from three genetically distinct populations, representing relatively high and low chemical resistance to mammalian herbivory. Cotyledon-stage seedlings and consecutive leaf pairs of true leaves were harvested separately across an 8-month period, and analysed for eight monoterpene compounds and six sesquiterpene compounds.Key Results Foliar terpenes showed a series of dynamic changes with ontogenetic trajectories differing between populations and families, as well as between and within the two major terpene classes. Sesquiterpenes changed rapidly through ontogeny and expressed opposing trajectories between compounds, but showed consistency in pattern between populations. Conversely, changed expression in monoterpene trajectories was population- and compound-specific.Conclusions The results suggest that adaptive opportunities exist for changing levels of terpene content through ontogeny, and evolution may exploit the ontogenetic patterns of change in these compounds to create a diverse ontogenetic chemical mosaic with which to defend the plant. It is hypothesized that the observed genetically based patterns in terpene ontogenetic trajectories reflect multiple changes in the regulation of genes throughout different terpene biosynthetic pathways.     

Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450_cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450_cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.     

A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.     

Plant coexistence alters terpene emission and content of Mediterranean species

There is evidence that secondary metabolism may modulate plant interactions and is modified by different biotic stress agents, such as herbivores or pathogens. However, it is poorly understood whether secondary metabolism is altered during competition among plants. The intraspecific and interspecific coexistence of some Mediterranean potted seedlings, namely Rosmarinus officinalis, Pinus halepensis, Cistus albidus and Quercus coccifera was investigated through their terpene accumulation within leaves (except for Q. coccifera, a non-storing species) and terpene emissions (for all species). Competition had both positive and negative effects for both terpene emissions and content, depending on the species a seedling coexisted with. For R. officinalis, terpene concentrations (1.8-cineole and camphor) and terpene emissions (camphene, camphor and overall monoterpenes) were lower when the neighbour species was P. halepensis. For C. albidus, no changes were observed in its content, while the overall sesquiterpene emissions (70% of total emissions) were reduced in all competition conditions, except in intraspecific competition. In the case of P. halepensis, the highest terpene content occurred when it grew with C. albidus, and in intraspecific competition, while its emissions were reduced under these conditions. Only emissions of Q. coccifera showed no significant changes in the different competition treatments.     

Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis)

Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (−)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (−)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (−)-drimenol, from which useful drimane-type terpenes can be synthesized.     

Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum)

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography–mass spectrometry (GC–MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.     

Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the first committed step in the synthesis of antimicrobial melleolides

Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6-7 double bond into the 7-8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns.     

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants

The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.