Influence of phosfon D and cycocel on growth and essential oil content of sage and peppermint

Foliar application of Phosfon D at 50–100 ppm stimulates the growth of Salvia officinalis (sage) and moderately retards the growth of Mentha piperita (peppermint), while increasing the essential oil yield of both species by 50–70 % Phosfon D increases the proportions of (−)-3-isothujone and (+)-3-thujone in sage oil and decreases the level of (−)-β-pinene and (+)-camphor, whereas this growth retardant increases the proportions of (+)-isomenthone and (+)-neoisomenthol in peppermint oil and decreases the level of(−)-menthone and (−)-menthoL Foliar application of Cycocel at 250–500 ppm slightly stimulates growth and essential oil formation in peppermint, and retards growth of sage with little effect on oil yield. The influence of Cycocel on sage oil composition was the opposite of that of Phosfon, with a tendency to increase the level of (−)-β-pinene and decrease the level of (−)-3-isothujone under severe stunting. The effect of Cycocel on the composition of peppermint varied with concentration. The influence of growth retardants on essential oil composition and yield are most readily explained by alterations in the levels or activities of the relevant enzymes.     

Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis)

(+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was detected at 144 ± 10 μg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases.     

Oxiranylphenyl esters from Pimpinella diversifolia

The major components of the essential oil from roots of Pimpinella diversifolia, gathered in the Kumaun Region of India, have been identified as the (+)-Z-2-methyl-2-butenoate (angelate) and (+)-isobutyrate esters of 4-methoxy-2-(E-3-methyloxiranyl)phenol. Aromatic ¹³C NMR resonances of these compounds and their synthetic acetate analog, as well as those of 2-methoxy-4-(E-3-methyloxiranyl)phenyl acetate prepared from isoeugenol, were found to be in excellent agreement with calculated values. Comparison of the EIMS of the natural and synthetic products with those reported for compounds previously identified as 2-methoxy-4-(E-3-methyloxiranyl)phenyl esters indicates that they also have the 4-methoxy-2-(E-3-methyloxiranyl)phenyl structure.     

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731 bp) and SasesquiTPS1 (1680 bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-α-terpineol and (−)-limonene, along with small quantities of linalool, myrcene, (−)-α-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were α-bulnesene, γ-muurolene, α- and β-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.     

A bioprocess for the production of high concentrations of R-(+)-α-terpineol from R-(+)-limonene

A bioprocess with a high conversion rate of limonene to α-terpineol was described. The enzyme hydratase involved in this process was found as being cofactor independent, non-inducible and able to perform the transformation of both R-(+) and S-(−)-limonene. The system used consisted of a biphasic medium in which the aqueous phase contained a concentrated resting cells of Sphingobium sp. and the organic phase was sunflower oil. After 30 h at 30 °C ca. 25 g of R-(+)-α-terpineol per liter of organic phase were obtained from R-(+)-limonene in Erlenmeyer flasks. Performance of the bioconversion in a bioreactor increased the production rate with no changes in yield and maximal R-(+)-α-terpineol concentration, which demonstrated that experiments in flasks were limited by liquid–liquid transport phenomena. A mathematical model able to explain the fact that the reaction always stopped before the precursor became exhausted has also been proposed and validated. Finally, the process reported was the most promising alternative for the biotechnological production of natural R-(+)-α-terpineol published so far and up to ca. 130 g L⁻¹ metabolite could finally be obtained.     

Nematicidal and Propagation Activities of Thyme Red and White Oil Compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae)

The toxic and propagation effects on Bursaphelenchus xylophilus of 28 Thymus vulgaris red oil and white oil compounds were examined using direct contact and cotton ball bioassays. Results were compared with those of the trunk-injection nematicides emmamectin benzoate, levamisol hydrochloride and morantel tartrate. In direct contact bioassays, geraniol (LC₅₀, 0.47 mg/ml) was the most toxic compound, followed by thymol (1.08 mg/ml), carvacrol (1.23 mg/ml) and terpinen-4-ol (2.61 mg/ml). In cotton ball tests with 20 inactive compounds at 2 mg/cotton ball, p-cymene significantly inhibited propagation (propagation ratio [PR] 8), compared with the castor oil-ethanol-treated control (PR 56). Propagation stimulation was observed with (–)-caryophyllene oxide, (+)-ledene, (+)- and (–)-limonene, linalool oxide, β-myrcene, (–)-α-phellandrene, (+)-α-pinene and γ-terpinene (PR 63–100). The other 10 compounds exhibited low to moderate levels of propagation inhibition (PR 36–56). At 0.1 μg/cotton ball, emmamectin benzoate and morantel tartrate exhibited complete suppression of propagation, whereas a very low level of propagation inhibition was obtained from levamisol hydrochloride (PR 6). In conclusion, propagation-stimulating compounds can exist in plants in addition to nematicidal compounds, and careful use of plant preparations containing high quantities of these compounds is mandatory.     

Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B and evidence for their widespread occurrence in Eucalyptus

Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K₁ and K₂. Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C to be present in most, of the species examined. The widespread occurrence of these carbohydrate monoterpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to secretory cavity lumena.     

Variation in morphological characters,chemical composition,and anthocyanin content of different Chrysanthemum morifolium cultivars from Iran

The morphological traits, essential oil composition, and anthocyanin content of 17 Iranian C. morifolium cultivars were evaluated. Based on Shannon diversity index (I), such morphological characters as flower head diameter, ray floret, shape of apex, and main color of ray floret revealed more variability than the other traits. Significant genotypic variations were also observed in essential oil content (ranging from 0.1 to 0.56% (w/w)) and composition. Gas chromatography-mass spectrometry (GC-MS) analysis of the essential oil was conducted to identify 47 compounds in which camphor (0–44.6%), chrysanthenone (0–48.96%), chrysanthenyl acetate (0–16.22%), verbenol (0–11.78%), (+)-5-epi-neointermedeol (0–25.41%), and aromadendrene (0–14.98%) were the main constituents. Results of cluster analysis of essential oil compositions divided the cultivars into four groups. Hybridization among cultivars of divergent clusters led to heterotic effects for flower quality and phytochemical characters. The relationships established between the components revealed high correlations of anthocyanin with the two major aroma compounds of verbenol and chrysanthenyl acetate while they also provided a clue to the association between aroma compounds and color pigmentation. Moreover, a correlation was established between chrysanthenone and flower head diameter (r = −0.502). Based on the results obtained, it might be suggested that smaller flowers are capable of accumulating higher amounts of chrysanthenone. Clearly, the associations between morphological traits and essential oil components provide new insights for improved breeding programs in chrysanthemum.     

Investigation of monoterpenoids rich essential oils of two Ocimum basilicum L. varieties at different agro-climatic conditions in India

India is an emerging basil essential oil producer in South-east Asia. Two high essential oil yielding hybrids, namely one inter specific hybrid between of O. basilicum and O. kilimandscharicum Gürke (HYBL-1) and another intraspecific hybrid of O. basilicum × O. basilicum (OBL-1) of basil were analyzed using GC, enantiomeric GC, NMR, enantio-GC–MS and GC–MS methods. Inter specific hybrid HYBL-1 contained high essential oil-rich in linalool (68.5%), camphor (8%), and 1,8-cineole (4.6%) as characteristic constituents among monoterpenoids, whereas β-caryophyllene (1.9%), germacrene D (1.0%), and epi-α-cadinol (1.9%) were the sesquiterpenoids at the Lucknow (North Indian conditions) and linalool (71.8%), camphor (9.4%) and 1,8-cineole (4.3%) at Hyderabad (South Indian conditions) locations. Intraspecific hybrid (OBL-1) possessed linalool (66.1%), 1,8-cineole (5.4%) and geraniol (8.6%) with sesquiterpenoids in low proportions. Inter specific hybrid HYBL-1 showed superiority over OBL-1 in the multi-location trials conducted at Lucknow and Hyderabad. Average mean performance of inter specific hybrid over locations was: herb yield 44.80 t/ha, oil content 0.63%, oil yield 188.50 kg/ha, linalool content 67.65%, camphor content 8.90% v/s OBL-1 herb yield 21.32 t/ha, oil content 0.53%, oil yield 97.50 kg/ha, linalool content 65.55%, camphor content 0.00%, respectively. The essential oil of these two hybrids subjected to enantiomer differentiation revealed a high enantiomeric excess for (3R) -(?)-linalool, whereas (1R)- (+)-camphor was recorded exclusively in inter specific hybrid. The extensive NMR experiments were performed to confirm constituents in these hybrids and found that NMR spectroscopy could also be an ideal tool for the differentiation of essential oils from commercial samples declared as natural.     

(+)-2,3-Trans-pubeschin,the first catechin analogue of peltogynoids from Peltogyne pubescens and P. venosa

The heartwoods of Peltogyne pubescens and P. venosa contain the predominant pair (+)-peltogynol and (+)-mopanol, their 4-epimers, (+)-peltogynol B and (+)-mopanol B, together with the first catechin analogue of peltogynol, (+)-2,3- trans-pubeschin. These are accompanied by ±-2,3-cis- and ±-2,3-trans-3-O-methylfustins, and by α, 2′,3,4,4′-pentahydroxychalcone. Other minor metabolises are 4′,7-dihydroxy- and 3′,4′,7-trihydroxy-flavanones and 5,6-dihydroxyphthalide. (+)-2,3-Trans-pubeschin trimethyl ether was synthesized by reduction of the corresponding (+)-2,3-trans-peltogynone analogue with NaBH₄/BF₃ in diglyme, and its absolute configuration shown to be 2R: 3S.     

Fumariaceae alkaloids including the biogenetic precursor of cularine

Corydalis claviculata has yielded (+)-crassifoline, the first 7,8,3′,4′-oxygenated benzylisoquinoline and biogenetic precursor of cularine, as well as the new cularine alkaloids (+)-sarcocapnidine, (+)-claviculine and (+)-O-methylcularicine.     

Mathematical Modeling-Guided Evaluation of Biochemical,Developmental, Environmental,and Genotypic Determinants of Essential Oil Composition and Yield in Peppermint Leaves

We have previously reported the use of a combination of computational simulations and targeted experiments to build a first generation mathematical model of peppermint (Mentha × piperita) essential oil biosynthesis. Here, we report on the expansion of this approach to identify the key factors controlling monoterpenoid essential oil biosynthesis under adverse environmental conditions. We also investigated determinants of essential oil biosynthesis in transgenic peppermint lines with modulated essential oil profiles. A computational perturbation analysis, which was implemented to identify the variables that exert prominent control over the outputs of the model, indicated that the essential oil composition should be highly dependent on certain biosynthetic enzyme concentrations [(+)-pulegone reductase and (+)-menthofuran synthase], whereas oil yield should be particularly sensitive to the density and/or distribution of leaf glandular trichomes, the specialized anatomical structures responsible for the synthesis and storage of essential oils. A microscopic evaluation of leaf surfaces demonstrated that the final mature size of glandular trichomes was the same across all experiments. However, as predicted by the perturbation analysis, differences in the size distribution and the total number of glandular trichomes strongly correlated with differences in monoterpenoid essential oil yield. Building on various experimental data sets, appropriate mathematical functions were selected to approximate the dynamics of glandular trichome distribution/density and enzyme concentrations in our kinetic model. Based on a χ² statistical analysis, simulated and measured essential oil profiles were in very good agreement, indicating that modeling is a valuable tool for guiding metabolic engineering efforts aimed at improving essential oil quality and quantity.The essential oil distilled from peppermint (Mentha × piperita) leaves is used in numerous consumer products (e.g. chewing gum, toothpaste, and mouthwash), as a flavor in the confectionary and pharmaceutical industries, and as a source of active ingredients for aromatherapy. Peppermint oil consists primarily of p-menthane-type monoterpenes, with smaller amounts of other monoterpenes and very minor quantities of sesquiterpenes (Rohloff, 1999). The essential oil is synthesized and accumulated in specialized anatomical structures called peltate glandular trichomes (Gershenzon et al., 1989; McCaskill et al., 1992). These trichomes contain secretory cells, arranged in an eight-celled disc, which are responsible for the synthesis of the oil. Nascent essential oil is secreted into an emerging cavity formed by the separation of a preformed layer of cuticular material (Amelunxen, 1965). Over the last two decades, the entire complement of genes and enzymes involved in the peppermint monoterpenoid essential oil biosynthetic pathway has been characterized (for review, see Croteau et al., 2005).Transgenic peppermint plants have been generated in efforts aimed at modulating essential oil yield and composition. Mahmoud and Croteau (2001) reported that, by overexpressing the gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), oil yield increases (compared with wild-type plants) of up to 50% were observed. Antisense suppression of the (+)-menthofuran synthase (MFS) gene led to a dramatic decrease in the amounts of the undesirable side product (+)-menthofuran (elite transgenic line designated MFS7a; Mahmoud and Croteau, 2001). A slight increase in overall monoterpene yields was reported for transgenic plants with increased expression levels of the gene encoding (−)-limonene synthase (LS; Diemer et al., 2001), whereas only negligible effects on yield were detected in an independent study (Krasnyansky et al., 1999). Transgenic plants overexpressing the gene coding for (−)-limonene 3-hydroxylase (L3H) did not accumulate increased levels of the recombinant protein, and the composition and yield of the essential oils were the same as in wild-type controls; however, cosuppression of the L3H gene resulted in a vastly increased accumulation of the intermediate (−)-limonene, without notable effects on oil yield (elite transgenic line designed L3H20; Mahmoud et al., 2004).Mathematical modeling can be a powerful tool to support metabolic engineering efforts, including those performed with peppermint. Stoichiometric modeling only requires knowledge of the topology of reactions in the pathway and inputs/outputs. This is a particularly useful approach to determine flux distributions and the systemic characteristics of metabolic networks (for review, see Llaneras and Picó, 2008). When experimental designs supporting metabolic and isotopic steady state are employed, isotope labeling data can be utilized for the development of quantitative flux maps of metabolic pathways (for review, see Libourel and Shachar-Hill, 2008). For dynamic systems, kinetic modeling is regarded as the generally most suitable method (McNeil et al., 2000; Poolman et al., 2004; Bruggeman and Westerhoff, 2006; Rios-Estepa and Lange, 2007; Mendes et al., 2009). Building on the rich body of published data on the enzymology and physiology of the peppermint monoterpene pathway (for review, see Croteau et al., 2005), we recently developed a first generation kinetic model to simulate the dynamics of peppermint monoterpene composition (Rios-Estepa et al., 2008). Modeling indicated that the monoterpene profiles observed in leaves of plants grown under low-light conditions could be explained if one assumed that (+)-menthofuran, a dead-end side product, acted as a heretofore unknown competitive inhibitor against (+)-pulegone, the primary substrate of the branch point enzyme (+)-pulegone reductase (PR; Fig. 1). Follow-up biochemical studies established that this prediction was correct (Rios-Estepa et al., 2008), thus illustrating the utility of an approach that integrates mathematical modeling with experimental testing.Open in a separate windowFigure 1.Outline of p-menthane monoterpene biosynthesis in peppermint glandular trichomes. The following enzymes are involved in this pathway: 1, 1-deoxy-d-xylulose 5-phosphate synthase; 2, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; 3, 2C-methyl-d-erythritol 4-phosphate cytidyltransferase; 4, 4-(cytidine 5′-diphospho)-2C-methyl-d-erythritol kinase; 5, 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase; 6, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate synthase; 7, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase; 8, isopentenyl diphosphate isomerase; 9, geranyl diphosphate synthase; 10, (−)-limonene synthase; 11, (−)-limonene 3-hydroxylase; 12, (−)-trans-isopiperitenol dehydrogenase; 13, (−)-trans-isopiperitenone reductase; 14, (+)-cis-isopulegone isomerase; 15, (+)-menthofuran synthase; 16a, (+)-pulegone reductase [(−)-menthone-forming activity]; 16b, (+)-pulegone reductase [(+)-isomenthone-forming activity]; 17a, (−)-menthone:(−)-menthol reductase [(−)-menthol-forming activity]; 17b, (−)-menthone:(−)-menthol reductase [(+)-neoisomenthol-forming activity]; 18a, (−)-menthone:(+)-neomenthol reductase [(+)-neomenthol-forming activity]; 18b, (−)-menthone:(+)-neomenthol reductase [(+)-isomenthol-forming activity]. The subcellular compartmentation of p-menthane metabolic enzymes is color coded as follows: Cyt (blue), cytosol; ER (orange), endoplasmic reticulum; Lpl (green), leucoplasts; Mit (red), mitochondria. The inhibitory effects of (+)-menthofuran on (+)-pulegone reductase and geranyl diphosphate on isopentenyl diphosphate isomerase are indicated by red arcs with orthogonal red lines. Names of selected metabolites are shown in the colors that are used to indicate the corresponding profiles in Figures 2 to 5​5​​.As part of this study, a computational perturbation analysis was used to predict factors with the potentially greatest impacts on peppermint essential oil yield and composition (specific biosynthetic enzymes and the density of oil-synthesizing trichomes). To test these modeling predictions experimentally, we first acquired biometric data with peppermint plants grown under several environmental conditions known to adversely affect oil accumulation (Burbott and Loomis, 1967; Clark and Menary, 1980) and the transgenic line MFS7a, for which an altered essential oil profile had been reported earlier (Mahmoud and Croteau, 2001). Building on these experimental data sets, we then developed a second generation model that accounts for biochemical, developmental, environmental, and genotypic factors of essential oil formation. This updated model was then used to simulate monoterpenoid essential oil profiles for the transgenic line MFS7a grown under low-light environmental stress conditions and the transgenic line L3H20, which had previously been shown to have vastly reduced expression levels of the gene encoding L3H. In both cases, simulated and measured monoterpene patterns were very similar, indicating that mathematical modeling has great potential for guiding efforts aimed at developing peppermint lines with high oil yields and favorable composition, even under adverse environmental conditions.     

Developmental Control of Monoterpene Content and Composition in Micromeria fruticosa(L.) Druce

White micromeria [ Micromeria fruticosa(L.) Druce, Lamiaceae]is a dwarf evergreen shrub endemic to Israel and the easternMediterranean. The essential oil of M. fruticosa largely comprisesthe monoterpenes (+)-pulegone and isomenthol. Seasonal variationsin the levels and composition of the monoterpene componentsof the essential oil of M. fruticosa were noted. During thesummer months, when growth rates are maximal, (+)-pulegone constitutedup to 80% of the essential oil, while in early winter, a periodof growth-rest in Mediterranean climates, (+)-pulegone levelsdropped dramatically to a few percent, while isomenthol constitutedup to 80% of the essential oil. Experiments in which plantswere grown under controlled temperature and photoperiodic regimesindicated that the variation was not directly associated withenvironmental conditions, but the composition of the monoterpenesobtained from mature flowering branches was strikingly differentto that obtained from young vegetative branches. Additionally,there were marked differences in the extracts obtained fromindividual leaf pairs from the same plant. In young upper leaves,the main component was (+)-pulegone, constituting up to 70%of the total essential oil extracted. During maturation, levelsof this component dropped steadily, becoming negligible in olderleaves. Reciprocally, levels of isomenthol increased steadilywith leaf position, from 0% in young leaves to more than 60%in older leaves. Less pronounced but significant decreases inthe levels of limonene, isopulegone, piperitenone oxide, germacreneD and bicyclogermacrene, accompanied by increases in neoiso-isopulegol,isopulegol, neoisomenthol and pulegol were noted. This studyindicates that the strong seasonal variation observed in thechemical composition of M. fruticosa is primarily due to leafmaturation. Copyright 2001 Annals of Botany Company Micromeria fruticosa, Lamiaceae, essential oil, leaf age, monoterpenes, (+)-pulegone, isomenthol, pulegol     

Variabilin,a 6a-hydroxypterocarpan from Dalbergia variabilis

Bark and wood of the creeper Dalbergia variabilis contain the previously described friedelin, O-acetyl-oleanolic acid, formononetin, 8-O-methylretusin, (+)-vestitol, (±)-mucronulatol, (+)- and (±)-medicarpin, besides (+)-variabilin [(6aR,11aR)-6a-hydroxy-3,9-dimethoxypterocarpan]. This structure was confirmed by the conversion of (+)-variabilin into di-O-methylcoumestrol.     

Effects of dietary n-3 fatty acids on contractility, Na+ and K+ currents in a rat cardiomyocyte model of arrhythmia

The n-3 polyunsaturated fatty acids (PUFAs) have been reported to prevent ventricular fibrillation in human clinical studies and in studies involving experimental animals and isolated cardiomyocytes. This study aimed to determine whether dietary n-3 PUFAs could prevent isoproterenol and free radical-induced arrhythmic (asynchronous) contractile activity in adult rat cardiomyocytes and whether whole-cell Na(+) and K(+) currents measured by patch-clamp techniques were affected. Dietary supplementation with fish oil for 3 weeks significantly increased the proportion of total n-3 PUFAs in ventricular membrane phospholipids compared with saturated fat supplementation (18.8 +/- 0.6% vs. 8.1 +/- 1.0%, respectively). Cardiomyocytes from the fish oil group were less susceptible to isoproterenol-induced asynchronous contractile activity than were those from the saturated fat group [EC(50) values: 892 +/- 130 nM, n = 6 and 347 +/- 91 nM, n = 6 (P < 0.05), respectively]. Fish oil supplementation also prolonged the time taken to develop asynchronous contractile activity induced by superoxide and hydrogen peroxide. The voltage dependence of inactivation of Na(+) currents were significantly altered (-73.5 +/- 1.2 mV, n = 5 vs. -76.7 +/- 0.7 mV, n = 5, P < 0.05, for saturated fat and fish oil treated groups, respectively). The voltage dependence of activation of Na(+) and K(+) currents was not significantly affected by the dietary fish oil treatment. These results demonstrate the antiarrhythmic effects of dietary fish oil in a cardiomyocyte model of arrhythmia.     

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208 mg L⁻¹ cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.     

Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis

(+)-Menthofuran is an undesirable monoterpenoid component of peppermint (Mentha x piperita) essential oil that is derived from the alpha,beta-unsaturated ketone (+)-pulegone. Microsomal preparations, from the oil gland secretory cells of a high (+)-menthofuran-producing chemotype of Mentha pulegium, transform (+)-pulegone to (+)-menthofuran in the presence of NADPH and molecular oxygen, implying that menthofuran is synthesized by a mechanism analogous to that of mammalian liver cytochrome P450s involving the hydroxylation of the syn-methyl group of (+)-pulegone, spontaneous intramolecular cyclization to the hemiketal, and dehydration to the furan. An abundant cytochrome P450 clone from a peppermint oil gland cell cDNA library was functionally expressed in Saccharomyces cerevisiae and Escherichia coli and shown to encode the (+)-menthofuran synthase (i.e., (+)-pulegone-9-hydroxylase). The full-length cDNA contains 1479 nucleotides, and encodes a protein of 493 amino acid residues of molecular weight 55,360, which bears all of the anticipated primary structural elements of a cytochrome P450 and most closely resembles (35% identity) a cytochrome P450 monoterpene hydroxylase, (+)-limonene-3-hydroxylase, from the same source. The availability of this gene permits transgenic manipulation of peppermint to improve the quality of the derived essential oil.     

(+)-Pinpollitol: A di-O-methyl d-(+)-chiro-inositol from Pinus radiata

(+)-Pinpollitol, a new cyclitol recently isolated from the pollen of Pinus radiata, was found in the needles of this species. (+)-Pinpollitol was found to be a di-O-methyl ether Of d-(+)-chiro-inositol, and tentative isomeric structures have been proposed for the cyclitol. (+)-Pinpollitol is the first di-O-methyl inositol to be found in a gymnosperm and is one of only three di-O-methyl inositols yet found in nature.     

(+)-5,17-dehydromatrine N-oxide,a new alkaloid in Euchresta japonica

A new lupin alkaloid, (+)-5,17-dehydromatrine N-oxide, was isolated from the fresh aerial parts of Euchresta japonica. Its structure was confirmed by spectrometric data and by direct comparison with a synthetic sample, prepared from (+)-sophoranol ((+)-5-hydroxymatrine). It was also concluded that (+)-5,17-dehydromatrine N-oxide and (+)-matrine N-oxide possess the same configuration with respect to the asymmetric nitrogen by NMR spectra.     

Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300

Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through ¹H and ¹³C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated.