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Terpenoids, also known as isoprenoids, are the largest and most diverse class of organic compounds in nature and are involved in many membrane-associated cellular processes, including membrane organization, electron transport chain, cell signaling, and phototrophy. Terpenoids are ancient compounds with their origin presumably before the last universal common ancestor. However, Bacteria and Archaea are known to possess two distinct terpenoid repertoires and utilize terpenoids differently. Most notably, archaea constitute their cellular membrane solely made of terpenoid-based phospholipids, contrary to the bacterial membrane that consists of fatty acid-based phospholipids. Thus, the composition of ancestral membranes at the beginning of cellular life and the diversification of terpenoids in early life remain enigmatic. This review addresses these key issues through comprehensive phylogenomic analyses of extant terpenoid biosynthesis enzymes in Bacteria and Archaea. We aim to infer the basal components of terpenoid biosynthesis machinery that have an ancient origin before the divergence of the two domains and shed light on the deep evolutionary connection between terpenoid biochemistry and early life.

Terpenoids are a group of hydrocarbons that are produced in large quantities by  plants and are mainly contained in the sap and cell vacuoles. In plants, terpene compounds and their modifications, terpenoids, are secondary metabolites. Terpenoids make up many of the essential oils produced by plants. The content of essential oils influences the use of spice products, both as seasonings, as fragrances, as well as ingredients for medicine, health, etc. The terpenoid-steroid test was carried out using the Lieberman-Buchard reagent which yielded positive results for terpenoids for all fractions. The results obtained for the n-hexane and ethyl acetate fractions on the addition of lieberman-buchard yielded positive results for steroids as indicated by the formation of a green color in the fractions. The purpose of this research article is to provide information to find out what methods are used in isolation in identifying terpenoid compounds and knowing the types of terpenoid compounds obtained from the results of previous research journals.

Strigolactones (SLs) are plant hormones that regulate shoot branching and diverse developmental processes. They are biosynthesized from carotenoid molecules via a key biosynthetic precursor called carlactone (CL) and its carboxylated analog, carlactonoic acid (CLA). We have previously identified the methyl esterified derivative of CLA, methyl carlactonoate (MeCLA), as an endogenous SL-like molecule in Arabidopsis. Neither CL nor CLA could interact with the receptor protein, Arabidopsis DWARF14 (AtD14), in vitro, while MeCLA could, suggesting that the methylation step of CLA is critical to convert a biologically inactive precursor to a bioactive compound in the shoot branching inhibition pathway. Here, we show that a member of the SABATH protein family (At4g36470) efficiently catalyzes methyl esterification of CLA using S-adenosyl-L-methionine (SAM) as a methyl donor. We named this enzyme CLAMT for CLA methyltransferase. The Arabidopsis loss-of-function clamt mutant accumulated CLA and had substantially reduced MeCLA content compared with wild type (WT), showing that CLAMT is the main enzyme that catalyzes CLA methylation in Arabidopsis. The clamt mutant displayed an increased branching phenotype, yet the branch number was less than that of severe SL biosynthetic mutants. Exogenously applied MeCLA, but not CLA, restored the branching phenotype of the clamt mutant. In addition, grafting experiments using the clamt and other SL biosynthetic mutants suggest that CL and CLA are transmissible from root to shoot. Taken together, our results demonstrate a significant role of CLAMT in the shoot branching inhibition pathway in Arabidopsis.

Several plant triterpenoids have valuable pharmaceutical properties, but their production and usage is limited since extraction from plants can burden natural resources, and result in low yields and purity. Here, we engineered oleaginous yeast Yarrowia lipolytica to produce three valuable plant triterpenoids (asiatic, madecassic, and arjunolic acids) by fermentation. First, we established the recombinant production of precursors, ursolic and oleanolic acids, by expressing plant enzymes in free or fused versions in a Y. lipolytica strain previously optimized for squalene production. Engineered strains produced up to 11.6 mg/g DCW ursolic acid or 10.2 mg/g DCW oleanolic acid. The biosynthetic pathway from ursolic acid was extended by expressing the Centella asiatica cytochrome P450 monoxygenases CaCYP716C11p, CaCYP714E19p, and CaCYP716E41p, resulting in the production of trace amounts of asiatic acid and 0.12 mg/g DCW madecassic acid. Expressing the same C. asiatica cytochromes P450 in oleanolic acid-producing strain resulted in the production of oleanane triterpenoids. Expression of CaCYP716C11p in the oleanolic acid-producing strain yielded 8.9 mg/g DCW maslinic acid. Further expression of a codon-optimized CaCYP714E19p resulted in 4.4 mg/g DCW arjunolic acid. Lastly, arjunolic acid production was increased to 9.1 mg/g DCW by swapping the N-terminal domain of CaCYP714E19p with the N-terminal domain from a Kalopanax septemlobus cytochrome P450. In summary, we have demonstrated the production of asiatic, madecassic, and arjunolic acids in a microbial cell factory. The strains and fermentation processes need to be further improved before the production of these molecules by fermentation can be industrialized.

Armillaria sinapina, a fungal pathogen of primary timber species of North American forests, causes white root rot disease that ultimately kills the trees. A more detailed understanding of the molecular mechanisms underlying this illness will support future developments on disease resistance and management, as well as in the decomposition of cellulosic material for further use. In this study, RNA-Seq technology was used to compare the transcriptome profiles of A. sinapina fungal culture grown in yeast malt broth medium supplemented or not with betulin, a natural compound of the terpenoid group found in abundance in white birch bark. This was done to identify enzyme transcripts involved in the metabolism (redox reaction) of betulin into betulinic acid, a potent anticancer drug. De novo assembly and characterization of A. sinapina transcriptome was performed using Illumina technology. A total of 170,592,464 reads were generated, then 273,561 transcripts were characterized. Approximately, 53% of transcripts could be identified using public databases with several metabolic pathways represented. A total of 11 transcripts involved in terpenoid biosynthesis were identified. In addition, 25 gene transcripts that could play a significant role in lignin degradation were uncovered, as well as several redox enzymes of the cytochromes P450 family. To our knowledge, this research is the first transcriptomic study carried out on A. sinapina.

Plants synthesise a vast range of secondary metabolites that are stored in specialised cells or organs. The presence of sub-dermal glands rich in volatile terpene essential oils is characteristic of the trees of the genus Eucalyptus (Myrtaceae). Recent studies showed that non-volatile compounds (NVCs), particularly monoterpene acid glucose esters (MAGEs), co-occur with volatile components in Eucalyptus foliar oil glands. The principal aim of this thesis is to characterise MAGEs and other non-volatiles localised to Eucalyptus oil glands and to explore their relationships to the co-housed oil components. Glandular extracts from a range of Eucalyptus species belonging to the two major subgenera, Symphyomyrtus and Eucalyptus, were investigated in Chapter 2. Non-volatiles were extracted from enzymatically isolated glands and analysed using high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS). MAGEs were identified based largely on diagnostic mass spectral fragmentation patterns. MAGEs were the dominant NVC in Symphyomyrtus species. In contrast, the sampled subgenus Eucalyptus species lacked MAGEs, but were rich in phenolics. Volatile oil components were also analysed using gas chromatography with flame ionisation detection and mass spectrometry (GC-FID and GC-MS). Monoterpenes and sesquiterpenes were identified and quantified for each species. In addition, cell suspensions of E. polybractea were successfully established from leaf-derived callus as a potential tool to investigate the biosynthesis of MAGEs. Glandular extracts from subgenus Eucalyptus species rich in non-volatiles containing phenolic moieties were further analysed in Chapter 3. A suite of unsubstituted B-ring flavanones was identified as the dominant glandular NVCs. In addition, flavones, flavanone-O-glucosides, flavanone-b-triketone conjugates, triketone heterodimers and chromone-C-glucosides were also identified. Flavanones were quantified and species-specific variations were observ

Structurally diverse natural products are valued for their targeted biological activity. The challenge of working with such metabolites is their low natural abundance and complex structure, often with multiple stereocenters, precludes large-scale or unsophisticated chemical synthesis. Since select plants contain the enzymatic machinery necessary to produce specialized compounds, tissue cultures can be used to achieve key transformations for large-scale chemical and/or pharmaceutical applications. In this context, plant tissue-culture bio-transformations have demonstrated great promise in the preparation of pharmaceutical products. This review describes the capacity of cultured plant cells to transform terpenoid natural products and the specific application of such transformations over the past three decades (1988-2019)., Felstavat författarnamn i publikationen: Hesham R. El Saedi

Current approaches for the production of high-value compounds in microorganisms mostly use the cytosol as a general reaction vessel. However, competing pathways and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Eukaryotic cells control the complexity of their metabolism by harnessing organelles to insulate biochemical pathways. Inspired by this concept, herein we transform yeast peroxisomes into microfactories for geranyl diphosphate-derived compounds, focusing on monoterpenoids, monoterpene indole alkaloids, and cannabinoids. We introduce a complete mevalonate pathway in the peroxisome to convert acetyl-CoA to several commercially important monoterpenes and achieve up to 125-fold increase over cytosolic production. Furthermore, peroxisomal production improves subsequent decoration by cytochrome P450s, supporting efficient conversion of (S)-(-)-limonene to the menthol precursor trans-isopiperitenol. We also establish synthesis of 8-hydroxygeraniol, the precursor of monoterpene indole alkaloids, and cannabigerolic acid, the cannabinoid precursor. Our findings establish peroxisomal engineering as an efficient strategy for the production of isoprenoids.

Eucalypts (including Angophora, Corymbia, Eucalyptus) comprise a large and hyper-diverse group of ecologically important trees providing habitats for many forest dependent species. Eucalypts synthesize a plethora of specialized metabolites that moderate biotic and abiotic interactions. Until now, studies on eucalypt volatile organic compounds (VOCs) have primarily focused on isoprene and monoterpenoid emission from forest plantations. As a result, little is known about the volatile profile of adult eucalypt trees under natural conditions. Here we present an in-depth study of leaf and flower branch volatile emissions from mature trees of nine eucalypt species in south-eastern Australia. VOCs were captured using a non-invasive, dynamic flow-through enclosure technique enabling volatile collection into adsorbent cartridges, analyzed by GC–MS. For comparison, terpenoid extracts were also analyzed. Complex emission profiles composed of more than 100 different VOCs were recorded, including isoprene, mono- and sesquiterpenoids, and benzenoids. Emission profiles from eucalypts were highly species-specific, tissue dependent, diurnally regulated and affected by environmental conditions. The composition of stored terpenoids did not mirror the emission profiles, supporting differential regulation of formation, storage and emission of terpenoids. These results provide important knowledge about the entangled dynamics of eucalypt volatiles, and have wider implications for predictions of volatile emission from eucalypt forests and plantations.

Ferula assafoetida is a medicinal plant of the Apiaceae family that has traditionally been used for its therapeutic value. Particularly, terpenoid and phenylpropanoid metabolites, major components of the root-derived oleo-gum-resin, exhibit anti-inflammatory and cytotoxic activities, thus offering a resource for potential therapeutic lead compounds. However, genes and enzymes for terpenoid and coumarin-type phenylpropanoid metabolism have thus far remained uncharacterized in F. assafoetida Comparative de novo transcriptome analysis of roots, leaves, stems, and flowers was combined with computational annotation to identify candidate genes with probable roles in terpenoid and coumarin biosynthesis. Gene network analysis showed a high abundance of predicted terpenoid- and phenylpropanoid-metabolic pathway genes in flowers. These findings offer a deeper insight into natural product biosynthesis in F. assafoetida and provide genomic resources for exploiting the medicinal potential of this rare plant.

Guayule (Parthenium argentatum) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis-prenyltransferases (CPTs) and CPT-Binding Proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here, we identified four guayule genes - three PaCPTs (PaCPT1-3) and one PaCBP, whose protein products organize PaCPT/PaCBP complexes. Co-expression of both PaCBP and each of the PaCPTs could complemented the dolichol (a short cis-polyisoprene)-deficient yeast, whereas the individual expressions could not. Microsomes from the PaCPT/PaCBP-expressing yeast efficiently incorporated 14C-isopentenyl diphosphate into dehydrodolichyl diphosphates; however, NR with high molecular weight could not be synthesized in in vitro assays. Furthermore, co-immunoprecipitation and split-ubiquitin yeast 2-hybrid assays using PaCPTs and PaCBP confirmed the formation of protein complexes. Of the three PaCPTs, guayule transcriptomics analysis indicated that the PaCPT3 is predominantly expressed in stem and induced by cold-stress, suggesting its involvement in NR biosynthesis. The comprehensive analyses of these PaCPTs and PaCBP here provide the foundational knowledge to generate a high NR-yielding guayule.

Diatoms are among the most productive and ecologically important groups of microalgae in contemporary oceans. Due to their distinctive metabolic and physiological features, they offer exciting opportunities for a broad range of commercial and industrial applications. One such feature is their ability to synthesize a wide diversity of isoprenoid compounds. However, limited understanding of how these molecules are synthesized have until recently hindered their exploitation. Following comprehensive genomic and transcriptomic analysis of various diatom species, the biosynthetic mechanisms and regulation of the different branches of the pathway are now beginning to be elucidated. In this review, we provide a summary of the recent advances in understanding diatom isoprenoid synthesis and discuss the exploitation potential of diatoms as chassis for high-value isoprenoid synthesis.

Consumers are aware of diet causing health problems and therefore there is an increased demand for natural ingredients that are expected to be safe and health-promoting. Many of these compounds belong to the class of flavonoids and can be divided into these five groups: flavanones, flavones, flavonols, flavanols, isoflavones and anthocyanidins. Extracts from citrus fruits are usually used as functional ingredients for several products. The aim of this paper was to develop an UHPLC-UV-ESI-HRMS method to define the metabolite profile of different parts of citrus fruit, of a particular cultivar called ‘Ovale Calabrese’, and in its main by-products. The high resolution mass spectrometry analysis allowed the identification of 27 compounds belonging to the classes of flavonoids and terpenoids. The high contents of phytochemical compounds, reveal the potential use of the ‘Ovale Calabrese’ as a rich source of nutraceutical compounds.

The mechanism of neurodegeneration in Parkinson's disease (PD) remains unknown but it has been hypothesised that the intestinal tract could be an initiating and contributing factor to the neurodegenerative processes. In PD patients as well as in animal models for PD, alpha-synuclein-positive enteric neurons in the colon and evidence of colonic inflammation have been demonstrated. Moreover, several studies reported pro-inflammatory bacterial dysbiosis in PD patients. Here, we report for the first time significant changes in the composition of caecum mucosal associated and luminal microbiota and the associated metabolic pathways in a rotenone-induced mouse model for PD. The mouse model for PD, induced by the pesticide rotenone, is associated with an imbalance in the gut microbiota, characterised by a significant decrease in the relative abundance of the beneficial commensal bacteria genus Bifidobacterium. Overall, intestinal bacterial dysbiosis might play an important role in both the disruption of intestinal epithelial integrity and intestinal inflammation, which could lead or contribute to the observed alpha-synuclein aggregation and PD pathology in the intestine and central nervous system in the oral rotenone mouse model of PD.

Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain., Export Date: 22 October 2018; Article; CODEN: MEENF; Correspondence Address: Lindberg, P.; Department of Chemistry – Ångström, Uppsala University, Box 523, Sweden; email: pia.lindberg@kemi.uu.se; QC 20181106

Long-term, sustainable management of zebra chip disease of potato, caused by 'Candidatus Liberibacter solanacearum' (Lso) and vectored by potato psyllids (Bactericera cockerelli Sulc [Hemiptera: Triozidae]), requires development of cultivars resistant or tolerant to infection or capable of reducing spread or both. We examined the influence that five experimental breeding clones of potato had on potato psyllids and their ability to vector Lso. The ability of these potato clones to resist aphids (green peach aphids, Myzus persicae Sulzer [Hemiptera: Aphididae]) also was examined. Due to the importance of host chemistry on plant-insect interactions, levels of primary metabolites of amino acids and sugars, as well as secondary metabolites including polyphenolics, terpenoids, and alkaloids were compared between breeding clones and a commercial cultivar. Findings for compound levels then were associated with observed changes in host susceptibility to psyllids or aphids. Psyllids oviposited less on three breeding clones than Atlantic, but no significant effects of breeding clones on psyllid feeding or choice were observed. Aphid reproduction was reduced on two clones relative to Atlantic. A05379-211 had greater sugar levels and postpsyllid amino acid levels than Atlantic. Total alkaloid and phenolic levels were greater in all breeding clones than Atlantic. Total terpenoid levels were greater in PALB03016-3 and PALB03016-6 than Atlantic, which might explain, in part, the observed resistance to psyllid oviposition and aphid reproduction. Overall, these results suggest that increased levels of certain metabolites in breeding clones could affect psyllid and aphid reproduction.

In the first chapter of this dissertation, a three-step total synthesis of the antimalarial (+)-cardamom peroxide, an endoperoxide-containing monoterpene dimer, is reported. This concise synthesis utilized (–)-myrtenal as an inexpensive terpene building block, and applied a novel oxygen stitching strategy to install all the oxygen atoms in the natural product from molecular oxygen. This strategy has enabled the preparation of large quantities of (+)-cardamom peroxide, and subsequent studies have determined its absolute configuration, and this molecule’s reactivity in iron(II)-induced reductive cleavage. Additionally, the antimalarial activities of cardamom peroxide against the clinical isolates of Plasmodium falciparum from several Cambodia provinces have been evaluated.In the second chapter, a double allylation strategy for the construction of the [5,7,5]-fused carbocyclic system found in guaianolide sesquiterpenoids is disclosed. This strategy features a robust intramolecular allylation mediated by tin(II) chloride to assemble the seven-membered core. An efficient and scalable total synthesis of the trans-fused 8,12-guaianolide (+)-mikanokryptin via this strategy is reported in nine to ten steps from the abundant monoterpene (+)-carvone, which constitutes the first example of gram-scale total synthesis of any guaianolide natural product. Subsequently, the oxygen stitching strategy has been expanded from the synthesis of endoperoxides to the strategic installation of multiple oxygen atoms on complex guaianolides in a stereocontrolled manner. A concise total synthesis of (–)-nortrilobolide and a formal synthesis of the anticancer agent (–)-thapsigargin are achieved by merging the oxygen stitching strategy and double allylation strategy.Preliminary studies on a modular synthesis of guaianolides from the Apiaceae family of plants are also disclosed via an intramolecular allylation strategy. These studies include concise total syntheses of sinodielide A and grilactone

A library of bidentate diols, as well as tridentate triols and aminodiols, derived from (+)-sabinol, was synthesized in a stereoselective manner. Sabinol was transformed into allylic trichloroacetamide via Overman rearrangement of the corresponding trichloroacetimidate. After changing the protecting group to Boc, the enamine was subjected to stereospecific dihydroxylation with OsO4/NMO, resulting in the (1R,2R,3R,5R)-aminodiol diastereomer. The obtained primary aminodiol was transformed to a secondary analogue. The ring closure of the N-benzyl-substituted aminodiol with formaldehyde was investigated and regioselective formation of the spiro-oxazolidine ring was observed. Hydroboration or dihydroxylation of sabinol or its benzyl ether with OsO4/NMO resulted in the formation of sabinane-based diols and triols following a highly stereospecific reaction. Treatment of sabinol with m-CPBA afforded O-benzoyl triol as a diastereoisomer of the directly dihydroxylated product, instead of the expected epoxy alcohol. The resulting aminodiols, diol, and triols were applied as chiral catalysts in the reaction of diethylzinc and benzaldehyde from moderate to good selectivity.

Six new compounds, omaezol, intricatriol, hachijojimallenes A and B, debromoaplysinal, and 11, 12-dihydro-3-hydroxyretinol have been isolated from four collections of Laurencia sp. These structures were determined by MS and NMR analyses. Their antifouling activities were evaluated together with eight previously known compounds isolated from the same samples. In particular, omaezol and hachijojimallene A showed potent activities (EC50 = 0.15–0.23 µg/mL) against larvae of the barnacle Amphibalanus amphitrite.

Several kalihinol natural products, members of the broader isocyanoterpene family of antimalarial agents, are potent inhibitors of Plasmodium falciparum, the agent of the most severe form of human malaria. Our previous total synthesis of kalihinol B provided a blueprint to generate many analogues within this family, some as complex as the natural product and some much simplified and easier to access. Each analogue was tested for blood-stage antimalarial activity using both drug-sensitive and -resistant P. falciparum strains. Many considerably simpler analogues of the kalihinols retained potent activity, as did a compound with a different decalin scaffold made in only three steps from sclareolide. Finally, one representative compound showed reasonable stability toward microsomal metabolism, suggesting that the isonitrile functional group that is critical for activity is not an inherent liability in these compounds.

The marine soft corals Sarcophyton trocheliophorum crude extracts possessed antimicrobial activity towards pathogenic bacterial strains, i.e. Bacillus cereus, Salmonella typhi, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Bioassay-guided fractionation indicated that the antimicrobial effect was due to the presence of terpenoid bioactive derivatives. Further biological assays of the n-hexane fractions were carried out using turbidity assay, inhibition zone assay and minimum inhibitory concentration for investigating the growth-inhibition effect towards the Gram-positive and Gram-negative bacteria. The fractions were screened and the structure of the isolated compound was justified by interpretation of the spectroscopic data, mainly mass spectrometry (GC-MS). The structure was assigned as (5S)-3-[(3E,5S)-5-hydroxy-3-hepten-6-yn-1-yl]-5-methyl-2(5H)-furanone and was effective at concentrations as low as 0.20mg/mL. The above findings, in the course of our ongoing research on marine products, may implicate that the profound anti-microbial activity of the S. trocheliophorum soft corals, inhabiting the red sea reefs, is attributed to the presence of growth-inhibiting secondary metabolites mainly terpenoids.

Further chemical investigation of two Ligularia virgaurea samples collected in China resulted in the isolation of 21 new compounds, two of which were bakkane-type sesquiterpenoids bearing an anhydride-type ring C, which was a previously unknown partial structure. These samples belonged to the V-type (the major component was virgaurenone) among the five chemotypes found in this species., Tetrahedron, 71(44), pp.8428-8435; 2015

Plants produce an extensive array of specialised metabolites, chemicals that are in addition to those that are essential for the basic functioning of the plant. These metabolites have evolved in response to the many selective pressures experienced by plants. Terpenoids form a large class of specialised metabolites and are important mediators of many ecological interactions. They can protect plants from pests and diseases, suppress competitors and attract pollinators. The work presented in this thesis focuses on the terpenoid chemistry of Eucalyptus, a genus with significant ecological and economic importance. Species in this genus produce an essential oil rich in mono- and sesquiterpenes (collectively termed terpenes). The oil is synthesised and stored in glands that are present throughout the plant but are particularly abundant in leaves. Until recently this was considered to be the only function of oil glands but new evidence suggests they may have a much broader role to play in terpenoid metabolism. The overall aim of this thesis is to explore this idea further with a focus on a specific group of terpenoids, the monoterpene-acid sugar esters (MSEs). Firstly, the prevalence and diversity of MSEs in eucalypt oil glands was investigated using a high resolution LC-FTMS approach. Tandem MS experiments with three purified MSEs showed a characteristic fragmentation pattern. Along with accurate mass measurements, this was used to identify known and novel MSEs in the oil gland extracts of 18 species. Eight known and 10 novel MSEs were identified using this method. The use of the fragmentation pattern was validated with the structural elucidation by NMR of one of these novel compounds, named here as eucaglobulin B. The results of this study indicate that MSEs are common constituents of oil glands supporting the idea that these structures are a more general site of terpenoid accumulation and possibly biosynthesis in eucalypts. Interestingly, the non-volatile extract from oi

Long-lived tree species are subject to attack by various pests and pathogens during their lifetime. This problem is exacerbated by climate change, which may increase the host range for pathogens and extend the period of infestation by pests. Plant defences may involve preformed barriers or induced resistance mechanisms based on recognition of the invader, complex signalling cascades, hormone signalling, activation of transcription factors and production of pathogenesis-related (PR) proteins with direct antimicrobial or anti-insect activity. Trees have evolved some unique defence mechanisms compared with well-studied model plants, which are mostly herbaceous annuals. The genome sequence of Eucalyptus grandis W. Hill ex Maiden has recently become available and provides a resource to extend our understanding of defence in large woody perennials. This review synthesizes existing knowledge of defence mechanisms in model plants and tree species and features mechanisms that may be important for defence in Eucalyptus, such as anatomical variants and the role of chemicals and proteins. Based on the E. grandis genome sequence, we have identified putative PR proteins based on sequence identity to the previously described plant PR proteins. Putative orthologues for PR-1, PR-2, PR-4, PR-5, PR-6, PR-7, PR-8, PR-9, PR-10, PR-12, PR-14, PR-15 and PR-17 have been identified and compared with their orthologues in Populus trichocarpa Torr. & A. Gray ex Hook and Arabidopsis thaliana (L.) Heynh. The survey of PR genes in Eucalyptus provides a first step in identifying defence gene targets that may be employed for protection of the species in future. Genomic resources available for Eucalyptus are discussed and approaches for improving resistance in these hardwood trees, earmarked as a bioenergy source in future, are considered.

The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects.

Citral is a linear monoterpene which is present, as a volatile component, in the essential oil of several different aromatic plants. Previous studies have demonstrated the ability of citral to alter the mitotic microtubules of plant cells, especially at low concentrations. The changes to the microtubules may be due to the compound acting directly on the treated root and coleoptile cells or to indirect action through certain phytohormones. This study, performed in Arabidopsis thaliana, analysed the short-term effects of citral on the auxin content and mitotic cells, and the long-term effects of these alterations on root development and ethylene levels. The results of this study show that citral alters auxin content and cell division and has a strong long-term disorganising effect on cell ultra-structure in A. thaliana seedlings. Its effects on cell division, the thickening of the cell wall, the reduction in intercellular communication, and the absence of root hairs confirm that citral is a strong phytotoxic compound, which has persistent effects on root development. © 2013 Springer Science+Business Media New York.

[EN]: The volatile organic compound (VOC) a-pinene, one of the most abundant component emitted during the composting of urban waste (MSW) and pruning waste (P), was lreated in biofilters filled up with a MSW compost or a MSW+P compost. A photoionization detector was used to carry out the monitoring of the biofilters efficiency but GC-MS and an electronic nose were also used for the process study. Moisture content in biofilters below 66% (dw, MSW) or 51% (dw, MSWP) made efficiency decrease to less than 90%. GC-MS spectra indicated no intermediate products from 0- pi nene degradation appeared but e-nose data indicates a "smell" corresponding to Scompounds produced in MSW biofilter. These results show that electronic noses could become a powerful tool for the monitoring of VOC compounds in biofiltering and composting processes., [ES]: Las plantas de compostaje causan frecuentemente problemas de malos olores por los compuestos orgánicos volátiles (COV) emitidos, entre ellos el pineno. En el presente trabajo se estudia la eficacia de dos biofiltros rellenos con diferentes compost de RSU y restos de poda (P) en la biofiltración de a-pineno y la incidencia de diferentes condiciones operativas, en particular de la humedad del lecho de compost, haciendo uso de una combinación de técnicas: medidor de COV con detector de fotoionización, nariz electrónica y GC-MS. Ambos tipos de biofiltros mostraron gran eficacia en la biodegradación de a-pineno, con eficiencia del 100% para una concentración inicial de hasta 50 ppmv. La eficiencia de biodegradación resultó muy dependiente de la humedad del lecho, con una importante reducción al disminuir la humedad por debajo de 66% sms en el caso de RSU o del 51% sms en el caso de RSU+P. Tanto con la nariz electrónica como mediante GC-MS se comprobó que la degradación de a-pineno fue completa, sin que aparecieran compuestos intermedios de degradación. Sin embargo, la nariz mostró una emisión de fondo propia de cada biofiltro por debajo del límite de detección de las otras técnicas. La nariz electrónica demostró ser una herramienta muy útil, simplificando el muestreo y permitiendo realizar un seguimiento casi en continuo. Los biofiltros basados en RSU y restos de poda son eficaces para la biofiltración de pineno aunque la humedad del sistema debe ser cuidadosamente controlada.

A C16 norsesterterpenoid (euplectellodiol, 1) and a norditerpenoid (2) have been isolated from the marine sponges Mycale euplectelloides and Diacarnus megaspinorhabdosa, respectively. Their structures have been determined by spectroscopic methods. Compounds 1 and 2 are new natural products., Journal Article, info:eu-repo/semantics/published

During attempted enzyme-catalysed resolution of sterically hindered secondary alcohols, hemiacetals and their esters were unexpectedly detected. Hemiacetal esters are reactive compounds that decompose to alcohol, aldehyde and acid under ordinary work-up conditions i.e. in contact with water, acid, or silica gel. Thus, the presence of these side products might decrease the enantiomeric excess of the residual alcohol after workup of a lipase-catalysed resolution. The formation of these hemiacetal esters were further studied using both terpenoid and non-terpenoid substrate alcohols, various acyl donors, and lipases. The prerequisite for their formation is the presence of a sterically hindered substrate alcohol, an aldehyde or an aldehyde releasing acyl donor, and a lipase (PCL-L6, PCL-PS and CAL-B). Enantioselective synthesis of (S)- and (R)-cryptone was performed via a ring closing metathesis (RCM) of (S)- and (R)-6-isopropyl-1,7-octadien-3-one. The stereochemistry was induced by using pseudoephedrine as chiral auxiliary in an alkylation reaction which provided a chiral octadienone. Problems with removal of the RCM-catalyst resulted in low yields and low enantiomeric purity. In an alternative approach, racemic cryptone was subjected to conjugate addition with thiophenol followed by reduction to the corresponding alcohol. Lipase-catalysed resolution of this alcohol yielded, after oxidation and elimination, (R)- and (S)- cryptone with 76% and 98% ee, respectively. Marine fouling of immersed objects is a serious problem. Many coatings contain effective antifouling compounds having the drawback of being toxic to the marine environment. The marine natural product 10-isocyano-4-cadinene is a potentially non-toxic antifouling agent against the barnacle Balanus amphitrite and therefore an interesting target for organic synthesis. Cryptone was used as a starting material in attempted syntheses of this compound and other similar model compounds., QC 20100901

平成15年度～16年度科学研究費補助金(基盤研究(C)(2))研究成果報告書, 研究概要：マングローブのテルペノイド画分が塩分負荷により増加することをTLC分析で確認しているので、マングローブのテルペノイド組成について詳細な分析を行った。また、マングローブの生合成酵素の一種であるOxidesqualene cyclase(OSC)の分子構造を明らかにした。方法:7種類の沖縄産マングローブの葉と根から調製した総脂質抽出液をケン化後、不ケン化物をガスクロマトグラフにより分析した。テルペノイドの同定には^<13>C-NMRと3C-MSを用いた。OSC遺伝子はメヒルギ(Kandelia candel)根のcDNAを鋳型にしたPCR法によりクローニングした。結果:マングローブには11種類のトリテルペノイドと6種類の植物ステロールが検出され、主要成分はLupeol,β-Amyrin, Taraxerol、β-Sitosterol, Stigmasterolであった。テルペノイド組成は各マングローブ間で異なり、同一マングローブにおいても葉と根では異なる場合があった。根のテルペノイド濃度は、内側より外側で高かった。単離したOSCはβ-Amirin synthaseと高い相同性を示した。また、クローン間で塩基配列が異なっていたことからマングローブ根には複数個のOSC遺伝子が存在することが示唆された。本遺伝子の機能解析を行うため、OSC欠損酵母であるGIL 77に導入発現させ、Oxidosqualene代謝産物の解析をおこなったが、特定のテルペノイドの合成は確認できなかった。発現系の誘導に問題がある可能性が考えられ、引き続き機能解析を行う予定である。, 要約（欧文）:Our previous study showed that the terpenoid concentration of mangrove root increased with salinity. For this reason, nonsaponifiable lipids(NSL) from Okinawan mangroves were analyzed with a special reference to terpenoids. Identifications of the triterpenoids and phytosterols were based on the analysis of ^<13>C-NMR and GC-MS spectra. Triterpenoids and phytosterols comprised the major proportion of NSL. The triterpenoids and phytosterols mainly consisted of 11 and 6 compounds, respectively. The major components were lupeol, β-amyrin and taraxerol for triterpenoids and were β-sitosterol and stigmasterol for phytosterols. The diversity in the NSL composition was noted with mangrove species for both leaves and roots, implying the occurrence of divergent enzyme systems for biosynthesis. Terpenoid compositions of the root are not always similar to that of leaf, suggesting that terpenoids in the root are produced by biosynthesis in situ, not a translocation of the synthate from the leaf. Terpenoids existed in greater proportion in the outer parts of the root, suggesting the protective roles of triterpenoids in mangrove. Oxidosqualene cyclase(OSC) gene was isolated from mangrove root by PCR, introduced into osc-defective yeast mutant GIL 77. Transformation of GIL 77 with OSC gene resulted in no accumulation of the OSC related terpenoids. Expression and functional analysis of the isolated OSC is in progress.

Ambers from Peñacerrada (Basque Country, North Spain) have been analysed by IR spectroscopy and GC-MS in order to study the polymeric and solvent-soluble GC-amenable fractions, respectively. All samples showed branched monoalkybenzenes, bicyclic sesquiterpenoids and tricyclic diterpenoids related to pimaric acids precursors, suggesting a paleobotanic origin from araucarian species. Molecules containing oxygenated polar functionalities were not found, which is in agreement with the Cretaceous origin of the samples and in good correspondence with the IR spectra and with the low oxygen content estimated from elemental composition analysis. In addition, no IR bands for exocyclic double bonds and very few GC-amenable monounsaturated molecules were found, which provides further evidence of the loss of olefinic groups. Theses features show that the age-dependent loss of oxygenated and olefinic functions is a general process affecting the whole amber structure, not only the solvent soluble fraction.

Filamentous fungi produce asexual dispersive spores (conidia), when conditions are not suitable for apical extension growth. Conidiation (the process by which conidia produced) in genus Penicillium has attracted considerable interest because of its wide usage in different areas. In addition, several Penicillium species are pathogenic toward humans and plants, causing crop spoilage as well as decay processes. It is consequently important to understand this process since it could provide tools to inhibit or to induce conidiation in the case when fungal conidia are harmful or are useful. However, the precise mechanism by which conidiation induction is triggered has proved to be elusive. Recently, two fungal metabolites conidiagenol (1) and conidiagenone (2) were isolated from the fermentation broth of Penicillium cyclopium. They are related diterpenes, and show potent and selective conidiation inducing activity in liquid culture. 2 is believed to be the true inducer and 1 a biosynthetic precursor of 2, and its effect depends on its oxidation to 2 by the fungus. In order to initiate a structure-activity relationship study, and to obtain analogues of the natural product with similar or more potent biological activity but more readily available, three types of analogues of conidiogenone 2 were prepared. Starting from the ring A which bears all the functional groups, the simplified ring A, ring AB and tricyclic analogues were designed and synthesized. They share the same functional groups as the parent molecular but differ in size. In addition, a reasonable route towards the total synthesis of conidiogenone (2) is proposed. In the course of synthesis of the analogues, a mild method for hydrolysis of acetals in solvent-free conditions under microwave irradiation was developed, to meet the need for the selective deprotection of acetals.

Cyanobacteria are photosynthetic microorganisms that can be engineered to convert carbon dioxide into a myriad of useful chemicals. Terpenoids are the largest class of natural products and have applications in a wide variety of industries including fuels, pharmaceuticals, materials, cosmetics, and flavors and fragrances. Many of these compounds can be chemically synthesized or extracted from plants, however this is often either unsustainable or economically prohibitive. An alternative method of production relies on the heterologous expression of terpene synthase enzymes in cyanobacteria, producing the desired terpenoids directly from carbon dioxide. That strategy was explored in this work, whereby a heterologous mevalonate pathway, an enhanced methylerythritol phosphate (MEP) pathway, and a heterologous artemisinin biosynthetic pathway were all designed for expression in both Synechocystis and Synechococcus. Additionally, a heterologous patchoulol synthase from Pogostemon cablin (patchouli) was expressed in a cyanobacterium for the first time. A highly sensitive GC-MS quantification method was developed for the detection of patchoulol, and patchoulol production was characterized under four different growth conditions.