Formation Of Terpenoids Research Articles

The Ion Formation and Quantitative Response of Isoprene, Monoterpenes and Terpenoids in Ion Mobility Spectrometry with Atmospheric-Pressure Chemical Ionization as a Function of Temperature.

Ion mobility spectrometry is successfully used as a sensor technology for different applications. A feature of this method is that characteristic ion mobility spectra are obtained for each measurement rather than a sum signal. The spectra result from the different drift velocities of ions in a drift tube at atmospheric pressure. In this study, we investigated the ion formation processes and the quantitative response of isoprene, monoterpenes and monoterpenoids as a function of the temperature of the spectrometer using a tritium ionization source. These substances are important target analytes in atmospheric monitoring and in the analysis of essential oils in different matrices. A drift tube temperature above 120 °C permitted the most sensitive detection of isoprene and monoterpenes, while 80 °C was sufficient for the sensitive detection of most terpenoids. Dimer ions were formed for isoprene over the whole temperature range. The ionization processes of monoterpenes and terpenoids were strongly influenced by the temperature. At temperatures of 40 °C, adduct ions were formed in addition to MH+ ions for monoterpenes. Enhanced temperatures provided a single peak with the same drift time for all monoterpenes. Structural differences influenced the ion formation of terpenoids, and much more complex spectra were obtained. The nature of the product ions changed depending on the temperature.

Differential Substrate Sensing in Terpene Synthases from Plants and Microorganisms: Insight from Structural, Bioinformatic, and EnzyDock Analyses.

Terpene synthases (TPSs) catalyze the first step in the formation of terpenoids, which comprise the largest class of natural products in nature. TPSs employ a family of universal natural substrates, composed of isoprenoid units bound to a diphosphate moiety. The intricate structures generated by TPSs are the result of substrate binding and folding in the active site, enzyme-controlled carbocation reaction cascades, and final reaction quenching. A key unaddressed question in class I TPSs is the asymmetric nature of the diphosphate-(Mg2+)3 cluster, which forms a critical part of the active site. In this asymmetric ion cluster, two diphosphate oxygen atoms protrude into the active site pocket. The substrate hydrocarbon tail, which is eventually molded into terpenes, can bind to either of these oxygen atoms, yet to which is unknown. Herein, we employ structural, bioinformatics, and EnzyDock docking tools to address this enigma. We bring initial data suggesting that this difference is rooted in evolutionary differences between TPSs. We hypothesize that this alteration in binding, and subsequent chemistry, is due to TPSs originating from plants or microorganisms. We further suggest that this difference can cast light on the frequent observation that the chiral products or intermediates of plant and bacterial terpene synthases represent opposite enantiomers.

Differential Substrate Sensing in Terpene Synthases from Plants and Microorganisms: Insight from Structural, Bioinformatic, and EnzyDock Analyses

AbstractTerpene synthases (TPSs) catalyze the first step in the formation of terpenoids, which comprise the largest class of natural products in nature. TPSs employ a family of universal natural substrates, composed of isoprenoid units bound to a diphosphate moiety. The intricate structures generated by TPSs are the result of substrate binding and folding in the active site, enzyme‐controlled carbocation reaction cascades, and final reaction quenching. A key unaddressed question in class I TPSs is the asymmetric nature of the diphosphate‐(Mg2+)3 cluster, which forms a critical part of the active site. In this asymmetric ion cluster, two diphosphate oxygen atoms protrude into the active site pocket. The substrate hydrocarbon tail, which is eventually molded into terpenes, can bind to either of these oxygen atoms, yet to which is unknown. Herein, we employ structural, bioinformatics, and EnzyDock docking tools to address this enigma. We bring initial data suggesting that this difference is rooted in evolutionary differences between TPSs. We hypothesize that this alteration in binding, and subsequent chemistry, is due to TPSs originating from plants or microorganisms. We further suggest that this difference can cast light on the frequent observation that the chiral products or intermediates of plant and bacterial terpene synthases represent opposite enantiomers.

Comprehensive Metabolomic and Transcriptomic Analysis of the Regulatory Network of Volatile Terpenoid Formation during the Growth and Development of Pears (Pyrus spp. ‘Panguxiang’)

Volatiles are essential substances that determine distinct fruit flavors and user preferences. However, the metabolic dynamic and molecular modulation models that regulate the overall flavor generation during fruit growth and ripening are still largely unclear for most fruit species. To comprehensively analyze the molecular mechanism and regulation mechanism of aroma accumulation and aroma component formation in Pyrus spp. ‘Panguxiang’ (‘Panguxiang’pear), this study compared pear phenotype, sugars, organic acid content, and the expression of related genes and metabolites amid pear growth and development in Pyrus spp. ‘Panguxiang’. A total of 417 VOCs (4 amines, 19 aromatics, 29 aldehydes, 31 alcohols, 38 ketones, 64 heterocyclic compounds, 89 terpenoids, 94 esters, and 49 others) were found. The potential gene expression patterns were explored by combining transcriptomics and metabolomics, and VOC-associated metabolism and transcriptome data from all samples were integrated during the growth and development period. On this basis, we constructed a colorful model depicting changes in the VOCs and genes throughout pear growth and development. Our findings reveal that terpenoid biosynthesis pathways are the main aroma production pathways during pear growth and development. In addition to providing novel insights into the metabolic control of fruit flavor during growth and development, this study also provides a new theoretical basis for studying aroma metabolites in pears.

Integrated transcriptomic and metabolomic analyses revealed the molecular mechanism of terpenoid formation for salicylic acid resistance in Pulsatilla chinensis callus.

As a kind of traditional Chinese medicine, Pulsatilla chinensis (Bunge) Regel is well known for its anti-inflammation and anti-cancer activities, which are attributed to its active components including total saponins and monomers. To clarify the synthesis and metabolism mechanisms of class components in callus terpenes of P. chinensis, a certain concentration of salicylic acid (SA) hormone elicitor was added to the callus before being analysed by transcriptomic and metabolomic techniques. Results showed that the content of Pulsatilla saponin B4 in the callus suspension culture was significantly increased up to 1.99% with the addition of SA. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the differentially expressed genes were mainly enriched in 122 metabolic pathways, such as terpenoid metabolism-related pathways: terpenoid skeleton synthesis pathway, monoterpenoid biosynthesis pathways, diterpenoid biosynthesis pathways, and ubiquinone and other terpenoid-quinone biosynthesis pathways. A total of 31 differentially accumulated metabolites were obtained from four differential groups. Amongst 21 kinds of known chemical components in P. chinensis, deoxyloganic acid was the only monoterpenoid; the others are triterpenoids. In summary, this study found that SA elicitors can affect the metabolic changes of terpenoids in P. chinensis callus, which provided a basis for analysing the genetic regulation of terpenoid components of leucons.

Another level of complex-ity: The role of metabolic channeling and metabolons in plant terpenoid metabolism.

Terpenoids constitute one of the largest and most diverse classes of plant metabolites. While some terpenoids are involved in essential plant processes such as photosynthesis, respiration, growth, and development, others are specialized metabolites playing roles in the interaction of plants with their biotic and abiotic environment. Due to the distinct functions and properties of specific terpenoid compounds, there is a growing interest to introduce or modify their production in plants by metabolic engineering for agricultural, pharmaceutical, or industrial applications. The MVA and MEP pathways and the prenyltransferases providing the general precursors for terpenoid formation, as well as the enzymes of the various downstream metabolic pathways leading to the formation of different groups of terpenoid compounds have been characterized in detail in plants. In contrast, the molecular mechanisms directing the metabolic flux of precursors specifically toward one of several potentially competing terpenoid biosynthetic pathways are still not well understood. The formation of metabolons, multi-protein complexes composed of enzymes catalyzing sequential reactions of a metabolic pathway, provides a promising concept to explain the metabolic channeling that appears to occur in the complex terpenoid biosynthetic network of plants. Here we provide an overview about examples of potential metabolons involved in plant terpenoid metabolism that have been recently characterized and the first attempts to utilize metabolic channeling in terpenoid metabolic engineering. In addition, we discuss the gaps in our current knowledge and in consequence the need for future basic and applied research.

Effects of elevated growth temperature and enhanced atmospheric vapour pressure deficit on needle and root terpenoid contents of two Douglas fir provenances

In the present work, we studied the effects of elevated air temperatures, which were above the optimum for photosynthesis, in combination with enhanced atmospheric VPD on two Douglas fir provenances grown under controlled conditions in a climate chamber. Provenance Monte Creek (MC) from the menziesii-glauca transition zone, Southern British Columbia, Canada, was derived from a dry environment receiving ca. half of the precipitation at its natural site than the interior provenance Pend Oreille (PO) from a mesic site in Northeast Washington State, US. We determined the terpenoid contents in needles and roots of the trees as well as terpene emission from needles and terpenoid synthase activities observing clear provenance-specific patterns. Whereas total terpenoid contents in needles dropped significantly in provenance PO in response to thermal stress, they remained unaffected in MC. The drop in terpenoid content in PO was due to decreased abundance of almost all identified terpenoids with exception of five compounds. Terpene emission was significantly enhanced in thermal-stressed provenance MC but it was unaffected in provenance PO. Oppositely, root terpenoid contents were rather stable in both provenances upon high temperature and enhanced atmospheric VPD. Similarly, we did not observe stress effects on terpenoid synthase activity, which was used as a proxy for the formation of terpenoids. The results indicate that features of the original habitat of the trees determine plant chemotypic properties, for example, thermal stress related responses. The observed decrease of terpenoid levels in needles of PO after long-term exposure to elevated temperature/enhanced atmospheric VPD, might weaken stress-exposed trees. Since terpenoids are essential components of the conifers’ defense arsenal against herbivores, decreased terpenoid levels might increase susceptibility of stressed trees to above- and belowground herbivore challenges.

Integration of Metabolite Profiling and Transcriptome Analysis Reveals Genes Related to Volatile Terpenoid Metabolism in Finger Citron (C. medica var. sarcodactylis).

Finger citron (Citrus medica var. sarcodactylis) is a popular ornamental tree and an important source of essential oils rich in terpenoids, but the mechanisms behind volatile formation are poorly understood. We investigated gene expression changes combined with volatile profiling of ten samples from three developing organs: flower, leaf, and fruit. A total of 62 volatiles were identified with limonene and γ-terpinene being the most abundant ones. Six volatiles were identified using partial least squares discriminant analysis (PLS-DA) that could be used as markers for distinguishing finger citron from other citrus species. RNA-Seq revealed 1,611,966,118 high quality clean reads that were assembled into 32,579 unigenes. From these a total of 58 terpene synthase (TPS) gene family members were identified and the spatial and temporal distribution of their transcripts was measured in developing organs. Transcript levels of transcription factor genes AP2/ERF (251), bHLH (169), bZIP (76), MYB (155), NAC (184), and WRKY (66) during finger citron development were also analyzed. From extracted subnetworks of three modules constructed by weighted gene co-expression network analysis (WGCNA), thirteen TPS genes and fifteen transcription factors were suggested to be related to volatile terpenoid formation. These results provide a framework for future investigations into the identification and regulatory network of terpenoids in finger citron.

Traumatic Resin Duct Development, Terpenoid Formation, and Related Synthase Gene Expression in Pinus massoniana Under Feeding Pressure of Monochamus alternatus

Conifer-produced oleoresin is mainly composed of monoterpenoids, sesquiterpenoids, and diterpenoids that can protect conifers against herbivores and pathogens. Pinus massoniana Lamb. is an important economic species in southern China. However, the traumatic resin duct (TD) formation and terpenoid accumulation induced by adult Monochamus alternatus (Coleoptera: Cerambycidae) of P. massoniana have not been clearly reported. Here, we infested P. massoniana with M. alternatus and characterized the induced terpene defenses by microscopy, gas chromatography-mass spectrometry (GC-MS) and GCflame ionization detector (GC-FID), and quantitative reverse transcriptase PCR (qRT-PCR). The induced responses involve TD formation, tissue-specific terpenoid accumulation differentiation, and temporal related gene changes. Formation of TDs was observed in secondary xylem 7 days after feeding. GC-MS and GC-FID results showed terpenoid composition varied in bark and xylem tissue in response to infestation. Following feeding damage, most of the terpenoid synthase genes were up-regulated, which is consistent with the changes in terpenoid constitution. The genes were activated before terpenoid changes were detected indicating that this complex physiological and biochemical process requires time. This study provides evidence of the temporal sequence of changes in terpenoid defenses against herbivory in P. massoniana.

Expression of MEP Pathway Genes and Non-volatile Sequestration Are Associated with Circadian Rhythm of Dominant Terpenoids Emission in Osmanthus fragrans Lour. Flowers.

Osmanthus fragrans Lour. is one of the top 10 traditional ornamental flowers in China famous for its unique fragrance. Preliminary study proved that the terpenoids including ionone, linalool, and ocimene and their derivatives are the dominant aroma-active compounds that contribute greatly to the scent bouquet. Pollination observation implies the emission of aromatic terpenoids may follow a circadian rhythm. In this study, we investigated the variation of volatile terpenoids and its potential regulators. The results showed that both volatile and non-volatile terpenoids presented circadian oscillation with high emission or accumulation during the day and low emission or accumulation during the night. The volatile terpenoids always increased to reach their maximum values at 12:00 h, while free and glycosylated compounds continued increasing throughout the day. The depletion of non-volatile pool might provide the substrates for volatile emission at 0:00–6:00, suggesting the sequestration of non-volatile compounds acted like a buffer regulating emission of terpenoids. Further detection of MEP pathway genes demonstrated that their expressions increased significantly in parallel with the evident increase of both volatile and non-volatile terpenoids during the day, indicating that the gene expressions were also closely associated with terpenoid formation. Thus, the expression of MEP pathway genes and internal sequestration both played crucial roles in modulating circadian rhythm of terpenoid emission in O. fragrans.

Biological role for synthesis and release of isoprene by photosynthesizing cells in view of the entropy phenomenon

In this review, the issues of photobiological synthesis and release of isoprene by chlorophyll-containing cells are considered from the viewpoint of thermodynamics of open nonequilibrium systems, with an emphasis on fundamental significance of the entropy phenomenon. The excretory function of the living cell is envisioned as a result of the total release of energy by dissipative structures. The living cell metabolism represents a continuous transformation of a huge number of biologically significant chemical substances. The complex of these transformations results in maintenance of cell homeostasis. The cell functioning can be viewed as energy flows and matter conversions occurring on biological matrices. The flows of irreversible metabolic reactions proceed under steady-state condition of the system and ensure its balanced disequilibrium. The hypotheses considered in this review are based on the principles of energy dynamics; they permit the description of cell metabolism from the laws of nonequilibrium thermodynamics of open systems. It is concluded that the biogenic release of isoprene ensures entropy dissipation, which is required for regulation of fluxes leading to the formation of terpenoids and allowing the maintenance of cell homeostasis.

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants.

Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.

Terpenoids and their biosynthesis in cyanobacteria.

Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids.

Defining the Potassium Binding Region in an Apple Terpene Synthase

Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M(2+)) metal ion-assisted electrophilic attack. In addition to M(2+), monovalent cations (M(+)) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple alpha-farnesene synthase (MdAFS1), which has a dependence upon potassium (K(+)), was used to identify active site regions that may be important for M(+) binding. Protein homology modeling revealed a surface-exposed loop (H-alphal loop) in MdAFS1 that fulfilled the necessary requirements for a K(+) binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K(+) response and strongly implicated specific residues in direct K(+) binding. The role of the H-alphal loop in terpene synthase K(+) coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M(+) binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K(+) activation in other terpene synthases and establish a new role for the H-alphal loop region in terpene synthase catalysis.

Early herbivore-elicited events in terpenoid biosynthesis

Volatile terpenoids, the major products among the herbivore-induced plant volatiles in the legume, mediate interactions that attract herbivores’ natural enemies and serve as signals to neighboring plants. We recently demonstrated cross-talk among the signaling components involving Ca2+, jasmonic acid, and ethylene, which are altogether responsible for volatile terpenoid formation in Medicago truncatula. Herbivore-stimulated Ca2+ transients are an additional element that has an impact on the composition of the blend of terpenoids, whose biosynthesis depends on the jasmonic acid/ethylene pathway. The molecular diversity of the blend is expanded and modulated by the transcriptional regulation of terpene synthases, some of which are multi-functional enzymes producing a large set of sesqui- and monotepenes or precursors of C11 and C16 homoterpenes from different prenyl diphosphates. In this addendum, we discuss a new perspective on early events leading to terpenoid biosynthesis.

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.

Recent advances in the molecular genetics of resin biosynthesis and genetic engineering strategies to improve defenses in conifers

Since the first terpenoid synthase cDNA was obtained by the reverse genetic approach from grand fir, great progress in the molecular genetics of terpenoid formation has been made with angiosperms and genes encoding a monoterpene synthase, a sesquiterpene synthase, and a diterpene synthase. Tree killing bark beetles and their vectored fungal pathogens are the most destructive agents of conifer forests worldwide. Conifers defend against attack by the constitutive and inducible production of oleoresin that accumulates at the wound site to kill invaders and both flush and seal the injury. Although toxic to the bark beetle and fungal pathogen, oleoresin also plays a central role in the chemical ecology of these boring insects. Recent advances in the molecular genetics of terpenoid biosynthesis provide evidence for the evolutionary origins of oleoresin and permit consideration of genetic engineering strategies to improve conifer defenses as a component of modern forest biotechnology. This review described enzymes of resin biosynthesis, structural feathers of genes genomic intron and exon organization, pathway organization and evolution, resin production and accumulation, interactions between conifer and bark beetle, and engineering strategies to improve conifer defenses.

(E)-4-Hydroxy-3-methylbut-2-enyl Diphosphate: An Intermediate in the Formation of Terpenoids in Plant Chromoplasts

The missing link in the new deoxyxylulose phosphate metabolic pathway leading to the biosynthesis of plant terpenoids has been identified. The intermediate between the cyclic diphosphate 1 and the basic isoprenoid building blocks dimethylallyl diphosphate and isopentenyl diphosphate has been shown for the first time to be (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (2) by incorporation of tritium-labeled 2 into phytoene.

Biotransformation of monoterpene alcohols by Saccharomyces cerevisiae, Torulaspora delbrueckii and Kluyveromyces lactis.

Monoterpenoids are important flavour compounds produced by many plant species, including grapes (Vitis vinifera) and hops (Humulus lupulus). Biotransformation reactions involving monoterpenoids have been characterized in filamentous fungi, but few examples have been observed in yeasts. As monoterpenoids are in contact with yeasts during beer and wine production, biotransformation reactions may occur during the fermentation of these beverages. This paper describes the biotransformation of monoterpene alcohols, of significance in the alcoholic beverage industries, by three yeast species. All three species analysed had the ability to convert monoterpenoids. Saccharomyces cerevisiae and Kluyveromyces lactis reduced geraniol into citronellol, whilst all three yeasts produced linalool from both geraniol and nerol. Monocyclic alpha-terpineol was formed from both linalool and nerol, by all three yeasts. alpha-Terpineol was then converted into the diol cis-terpin hydrate. K. lactis and Torulaspora delbrueckii also had the ability to form geraniol from nerol. Finally, the stereospecificity of terpenoid formation was analysed. Both (+) and (-) enantiomers of linalool and alpha-terpineol were formed in roughly equal quantities, from either geraniol or nerol.

Cell-free conversion of 1-deoxy-D-xylulose 5-phosphate and 2- C-methyl-D-erythritol 4-phosphate into β-carotene in higher plants and its inhibition by fosmidomycin

The presence of the deoxyxylulose pathway in chromo- and chloroplasts of various higher plants was demonstrated by the conversion of labelled 1-deoxy-D-xylulose 5-phosphate into β-carotene and geranylgeraniol, in the presence of ATP. The antibiotic fosmidomycin inhibited the formation of terpenoids from 1-deoxy-D-xylulose 5-phosphate, whereas it did not affect the conversion of 2- C-methyl-D-erythritol 4-phosphate or isopentenyl diphosphate into terpenoids.