Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase.

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and ß-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

Evaluation of anti-oxidant and acetylcholinesterase activity and identification of polyphenolics of the invasive weed Dittrichia viscosa.

INTRODUCTION: The bioactive metabolites derived from weeds have attracted the interest of the food and pharmaceutical industries due to their health benefits. OBJECTIVE: To evaluate the anti-oxidant and acetylcholinesterase activity of Dittrichia viscosa extracts and characterise the polyphenolic metabolites using the LC coupled with diode-array detection (DAD) and positive mode electrospray ionisation (ESI) MS method with a view to evaluating the exploitation potential of this invasive weed. MATERIALS AND METHODS: Roots and aerial parts of D. viscosa were extracted with solvents of increasing polarity and their major polyphenolic metabolites were identified by LC - DAD/ESI(+)/MS. The total phenolic content of the extracts was determined using the Folin-Ciocalteu method, while their anti-oxidant activity was evaluated on the basis of their ability to scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide. Thin-layer chromatography was used to screen for acetylcholinesterase inhibitors. RESULTS: Stem extracts gave the highest phenolic content, whereas the roots showed the lowest content. Twenty-five polyphenolic constituents of the extracts were tentatively characterised according to their MS and UV spectroscopic data. Among the extracts studied, roots-ethyl acetate and flowers-diethyl ether revealed the highest activity according to the DPPH and chemiluminescence assays respectively. CONCLUSION: The metabolic profile of D. viscosa was studied and the structures of the major polyphenolic metabolites were tentatively assigned based on their MS and UV-vis spectra. The extracts exhibited high levels of anti-oxidant and acetylcholinesterase inhibitory activity and the inhibitors are probably localised mainly in flowers and roots.

Origanum species native to the island of Crete: in vitro antioxidant characteristics and liquid chromatography-mass spectrometry identification of major polyphenolic components.

Extracts from three Origanum species, including Origanum microphyllum, Origanum dictamnus and Origanum vulgare subsp. hirtum, native to the island of Crete (southern Greece), were partly fractionated through successive partition with ethyl acetate and n-butanol. All the fractions obtained were profiled with regard to their major polyphenolic constituents, using liquid chromatography-diode array-mass spectrometry. Furthermore, the antioxidant potency of each fraction was assessed by estimating the antiradical activity (A(AR)) and the hydroxyl free radical scavenging activity (SA(HFR)). The chromatographic analyses revealed a rich profile mainly for the ethyl acetate fractions, composed principally by flavones, which were accompanied by a limited number of phenylpropanoids, flavanones and dihydroflavonols. The highest values of antioxidant activity were displayed by the ethyl acetate extract of O. dictamnus, which also possessed the richest polyphenolic composition.

Phenolic profiles and antioxidant and anticarcinogenic activities of Greek herbal infusions; balancing delight and chemoprevention?

Total phenolic content, antioxidant activity and phenolic profiles of six herbal infusions - namely rosemary, Cretan dittany, St. John's Wort, sage, marjoram and thyme were assayed. Additionally, the infusion anticarcinogenic effect as to their ability to (a) scavenge free radicals, (b) inhibit cell growth, (c) decrease IL-8 levels and (d) regulate p65 subunit in epithelial colon cancer (HT29) and prostate (PC3) cancer cells was investigated. LC-DAD-MS and GC-MS analyses showed major qualitative and quantitative differences in phenolic profiles of the infusions. All herbal infusions exhibited antiradical activity which correlated strongly with their total phenolic content. Infusions exhibited the potential to inhibit cell growth and to reduce IL-8 levels in HT29 colon and PC3 prostate cancer cells. The regulation reported in p65 subunit in HT29 treated with St John's Wort and in PC3 treated with thyme might point to the NF-κB as the molecular target underlying the effect of these infusions.

Modulation of the antioxidant/pro-oxidant balance, cytotoxicity and antiviral actions of grape seed extracts.

Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system (regarding the cellular growth and growth recovery from H2O2 insult). GSEs antioxidant activity was detected in wild-type and mutant strains Δcta1, Δgsh1 and Δoye2glr1, while pro-oxidant activity in Δsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2 2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deficiencies and exhibit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV.

Herbal infusions; their phenolic profile, antioxidant and anti-inflammatory effects in HT29 and PC3 cells.

In this survey, we analyzed the phenolic profile of six herbal infusions namely Cretan marjoram, pink savory, oregano, mountain tea, pennyroyal and chamomile by LCDAD-MS and by GC-MS. Further, we investigated their anticarcinogenic effect as to their ability to (a) scavenge free radicals (b) inhibit proliferation (c) decrease IL-8 levels and (d) regulate nuclear factor-kappa B in epithelial colon cancer (HT29) and prostate (PC3) cancer cells. All herbal infusions exhibited antiradical activity correlated positevely with total phenolic content. Further, infusions exhibited the potential to inhibit cell proliferation and to reduce IL-8 levels in HT29 colon and PC3 prostate cancer cells. The molecular target for chamomile in HT29 seemed to be the NF-κB, while for the other herbal infusions needs to be identified. This study is the first to show the potential chemopreventive activity of infusions prepared from the examined herbs.

o-Quinone involvement in the prooxidant tendency of a mixture of quercetin and caffeic acid.

The oxidation products of a previously tested prooxidant mixture of quercetin (Q) and caffeic acid (CA) at 1:2 ratio were analysed by LC-MS. The UV-Vis and MS spectra of three chromatographic peaks eluting at t(R)=9.11min, t(R)=14.36min and t(R)=30.30min were studied further. The structures of the tentatively identified compounds indicate polymeric molecules. A pentamer formed by 3 units of quercetin and 2 units of caffeic acid was attributed in the case of peaks t(R)=9.11min and t(R)=14.36min. The quercetin quinone (QQ) - a polymerization intermediate - is a fragment of the compound identified in the MS spectrum with the m/z=323 coming from Q o-quinone m/z=300 plus 23 from Na(+). According to the UV-Vis spectrum, we suggest a different intermolecular arrangement which gives a more extended e-delocalisation. At t(R)=30.30min, the spectra helped us to tentatively identify this oxidation product as being a polymer of 4 CA units and 1 QQ.

Evaluation of anti-oxidant activity and identification of major polyphenolics of the invasive weed Oxalis pes-caprae.

INTRODUCTION: Phytochemical analyses of weeds, many of which have been used in traditional medicine worldwide, could lead to the identification of secondary metabolites with significant biological activity. OBJECTIVE: To perform an assessment of the chemical composition and exploitation potential of the invasive weed Oxalis pes-caprae. To evaluate the anti-oxidant activity of its extracts and isolate and characterise polyphenolic metabolites using LC-DAD-MS (ESI+) and NMR methods. METHODOLOGY: Aerial parts of the invasive weed O. pes-caprae were extracted with solvents of increasing polarity and their major polyphenolic metabolites were identified by LC-DAD-MS (ESI+). The total phenolic content of the extracts was determined using the Folin-Ciocalteu method, while their anti-oxidant activity was evaluated on the basis of their ability to scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl and hydrogen peroxide. RESULTS: The major polyphenolic constituents of the extracts were tentatively characterised as chlorogenic acid, quinic ferulate, luteolin glucoside and cernuoside according to their MS and UV spectroscopic data. Cernuoside, an aureusidin glucoside, was isolated from the methanolic extract of the weed's flowers and its structure was unambiguously identified by 1D- and 2D-NMR spectroscopy. The butanol extract of O. pes-caprae displayed the highest anti-oxidant activity. CONCLUSION: The metabolic profile of O. pes-caprae was studied and the structures of the major polyphenolic metabolites based on their MS and UV-vis spectra were tentatively assigned. The aureusidin glucoside cernuoside was isolated and characterised for the first time from O. pes-caprae. The extracts exhibited high levels of anti-oxidant activity.

Bioflavonoid profile of citrus juices from Greece.

High-performance liquid chromatography with confirmation by UV-visible photodiode array detector-positive electrospray ionization-mass spectrometry [HPLC-UV-vis-DAD-(+ESI)-MS] with enhanced fragmentation by appropriate adjustment of the cone voltage was used to determine bioflavonoid content of five citrus species (tangerine, sanguine, sour orange, lemon and grapefruit) cultivated in Greece which come from citrus varieties analyzed for the first time. The main groups of bioflavonoids found in the juice of the citrus species according to HPLC retention times, spectral data and literature references were O-glycosylated flavanones and flavones, C-glucosylated flavones, O-glucosylated flavones, O-C-glucosylated flavones like saponarin and a phenolic derivative.

Aurones: a promising heterocyclic scaffold for the development of potent antileishmanial agents.

A series of (Z)-2-benzylidenebenzofuran-3-(2H)-ones (aurones) bearing a variety of substituents on rings A and B were synthesized and evaluated for their antiparasitic activity against the intracellular amastigote form of Leishmania infantum and their cytotoxicity against human THP1-differentiated macrophages. In general, aurones bearing no substituents on ring A (compounds 4a-4f) exhibit higher toxicity than aurones with 4,6-dimethoxy substitution (compounds 4g-4l). Among the latter, two aurones possessing a 2'-methoxy or a 2'-methyl group (compounds 4i and 4j) exhibit potent antileishmanial activity (IC50 = 1.3 ± 0.1 µM and IC50 = 1.6 ± 0.2 µM, resp.), comparable to the activity of the reference drug Amphotericin B, whereas they present significantly lower cytotoxicity than Amphotericin B as deduced by the higher selectivity index.

Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids.

BACKGROUND: Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. RESULTS: S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. CONCLUSIONS: Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields.

Polyphenolic composition and antioxidant characteristics of kumquat (Fortunella margarita) peel fractions.

The polyphenolic composition of two Fortunella margarita (Nagami kumquat) specimens from Greece and Egypt was investigated employing fractionation by solvent partition and liquid chromatography-mass spectrometry. The main groups of phenolics identified in the different fractions generated were C-glycosylated flavones, O-glycosylated flavones, C-glycosylated flavanones, O-glycosylated flavanones, flavonols, chalcones, phenolic acids and derivatives thereof. The antioxidant potency of the fractions was assessed using two representative in vitro tests, including antiradical activity and hydroxyl free radical scavenging activity. It was revealed that the ethyl acetate fractions from both specimens contained the higher polyphenol content and exhibited the better antioxidant characteristics. The results indicated that F. margarita peels may be regarded as a rich source of potentially bioactive polyphenols.

Natural and synthetic 2'-hydroxy-chalcones and aurones: synthesis, characterization and evaluation of the antioxidant and soybean lipoxygenase inhibitory activity.

A series of 2'-hydroxy-chalcones and their oxidative cyclization products, aurones, have been synthesized and tested for their antioxidant and lipoxygenase inhibitory activity. The natural product aureusidin (31) was synthesized in high yield by a new approach. An extensive structure-relationship study was performed and revealed that several chalcones and aurones possess an appealing pharmacological profile combining high antioxidant and lipid peroxidation activity with potent soybean LOX inhibition.

Biocatalytic properties of a peroxidase-active cell-free extract from onion solid wastes: caffeic acid oxidation.

The exploitation of food residual sources consists of a major factor in reducing the polluting load of food industry wastes and developing novel added-value products. Plant food residues including trimmings and peels might contain a range of enzymes capable of transforming bio-organic molecules with potential phytotoxicity, including hydrolases, peroxidases and polyphenoloxidases. Although the use of bacterial and fungal enzymes has gained interest in studies pertaining to bioremediation applications, plant enzymes have been given less attention or even disregarded. In this view, this study aimed at the investigating the use of a crude peroxidase preparation from onion solid by-products for oxidising caffeic acid, a widespread o-diphenol, whose various derivatives may occur in food industry wastes, such as olive mill waste waters. Increased enzyme activity was observed at a pH value of 5, but considerable activity was also retained for pH up to 7. Favourable temperatures for increased activity varied between 20 degrees C and 40 degrees C, 30 degrees C being the optimal. Liquid chromatography-mass spectrometry analysis of a homogenate/H(2)O(2)-treated caffeic acid solution revealed the existence of a tetramer as major oxidation product. Based on the data generated, a putative pathway for the formation of the peroxidase-mediated caffeic acid tetramer was proposed.

Antioxidant small molecules confer variable protection against oxidative damage in yeast mutants.

To assess the capacity of small molecules to function as antioxidants in pathologic conditions, a set of yeast assays utilizing strains deficient in the antioxidant machinery was applied with measurements of reactive oxygen species (ROS), glutathione (GSH/GSSG), and induction of the stress responsive proteins oye2 and oye3. Yeast strains deficient in superoxide dismutase (Delta sod1), catalase A (Delta cta1), and double-deficient in Old Yellow enzyme 2 and glutathione reductase 1 (Delta oye2 glr1) were supplemented with ascorbic acid, beta-carotene, caffeic acid, or quercetin, subjected to pro-oxidant insult, and monitored for growth recovery. Ascorbic acid and caffeic acid protected cells under most circumstances, whereas beta-carotene and quercetin protection was highly context dependent, exhibiting protection in some cases and inhibition in others. Beta-carotene and quercetin elevated substantially endogenous levels of ROS in some yeast mutants. Quercetin supplementation increased significantly GSH and GSSG levels but could not maintain GSH levels in H(2)O(2)-exposed cells. Induction of the stress response machinery was manifested by the strong up-regulation of a chromosomally encoded OYE2-GFP fusion. In the case of quercetin, there was simultaneous induction of OYE3-GFP, which was previously shown to sensitize cells to H(2)O(2)-induced programmed cell death (PCD). Taken together, the results show that mutations in the antioxidant machinery affect significantly the capacity of dietary antioxidants to protect cells.

Peroxidase-active cell free extract from onion solid wastes: biocatalytic properties and putative pathway of ferulic acid oxidation.

The exploitation of food residuals can be a major contribution in reducing the polluting load of food industry waste and in developing novel added-value products. Plant food residues including trimmings and peels might contain a range of enzymes capable of transforming bioorganic molecules, and thus they may have potential uses in several biocatalytic processes, including green organic synthesis, modification of food physicochemical properties, bioremediation, etc. Although the use of bacterial and fungal enzymes has gained attention in studies pertaining to biocatalytic applications, plant enzymes have been given less consideration or even disregarded. Therefore, we investigated the use of a crude peroxidase preparation from solid onion by-products for oxidizing ferulic acid, a widespread phenolic acid, various derivatives of which may occur in food wastes. The highest enzyme activity was observed at a pH value of 4, but considerable activity was retained up to a pH value of 6. Favorable temperatures for increased activity varied between 20-40 degrees C, 30 degrees C being the optimal. Liquid chromatography-mass spectrometry analysis of a homogenate/H(2)O(2)-treated ferulic acid solution showed the formation of a dimer as a major oxidation product.

Optimisation of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems and response surface methodology.

An experimental setup based on a 2(3) full-factorial, central-composite design was implemented with the aim of optimising the recovery of polyphenols from olive leaves by employing reusable and nontoxic solutions composed of water/ethanol/citric acid as extracting media. The factors considered were (i) the pH of the medium, (ii) the extraction time and (iii) the ethanol concentration. The model obtained produced a satisfactory fit to the data with regard to total polyphenol extraction (R(2) = 0.91, p = 0.0139), but not for the antiradical activity of the extracts (R(2) = 0.67, p = 0.3734). The second-order polynomial equation obtained after analysing the experimental data indicated that ethanol concentration and time mostly affected the extraction yield, but that increased pH values were unfavourable in this regard. The maximum theoretical yield was calculated to be 250.2 +/- 76.8 mg gallic acid equivalent per g of dry, chlorophyll-free tissue under optimal conditions (60% EtOH, pH 2 and 5 h). Liquid chromatography-electrospray ionisation mass spectrometry of the optimally obtained extract revealed that the principal phytochemicals recovered were luteolin 7-O-glucoside, apigenin 7-O-rutinoside and oleuropein, accompanied by smaller amounts of luteolin 3',7-O-diglucoside, quercetin 3-O-rutinoside (rutin), luteolin 7-O-rutinoside and luteolin 3'-O-glucoside. Simple linear regression analysis between the total polyphenol and antiradical activity values gave a low and statistically insignificant correlation (R(2) = 0.273, p > 0.05), suggesting that it is not the sheer amount of polyphenols that provides high antioxidant potency; instead, this potency is probably achieved through interactions among the various phenolic constituents.

Extraction of phenolics in liquid model matrices containing oak chips: Kinetics, liquid chromatography-mass spectroscopy characterisation and association with in vitro antiradical activity.

Four different liquid model matrices were utilised to study the leaching of polyphenols from oak chips. The matrices included distilled water, 12% (v/v) ethanol, 12% (v/v) ethanol adjusted to pH 3.4, and 55% (v/v) ethanol. Extraction of phenolics into the liquid systems was monitored by the estimation of the total polyphenol concentration, using the Folin-Ciocalteu method. The in vitro antiradical activity was also recorded using the stable DPPH radical, to ascertain enrichment of the solutions with potentially antioxidant compounds. As a final step, the polyphenolic composition of each matrix was characterised by means of liquid chromatography-electrospray ionisation mass spectrometry. The kinetics of polyphenol leaching into the liquid phase was found to obey a 2nd parameter power equation of the type y=ax(b), which produced a good fit of the data (p<0.0001). Kinetics was faster in distilled water up to a point, where after polyphenol extraction occurred at higher rate in the 55% ethanolic solution. The antiradical activity in all cases was highly correlated with total polyphenol concentration (p<0.001), providing that the amount of polyphenols extracted into the liquid media exerted a proportional antioxidant effect. The analytical examination by liquid chromatography-mass spectrometry revealed that the compounds implicated are hydrolysable tannins and hydrolysis products thereof.

Beta-orcinol metabolites from the lichen Hypotrachyna revoluta.

Four new beta-orcinol metabolites, hypotrachynic acid (1), deoxystictic acid (2), cryptostictinolide (3) and 8'-methylconstictic acid (4) along with the metabolites 8'-methylstictic acid (5), 8'-methylmenegazziaic acid (6), stictic acid (7), 8'-ethylstictic acid (8) and atranorin (9), that have been previously described, were isolated for the first time from the tissue extracts of the lichen Hypotrachyna revoluta (Flörke) Hale. The structures of the new metabolites were elucidated on the basis of extensive spectroscopic analyses. Radical scavenging activity (RSA) of the metabolites isolated in adequate amounts, was evaluated using luminol chemiluminescence and comparison with Trolox.

Storage of olives (Olea europaea L.) under CO2 atmosphere: liquid chromatography-mass spectrometry characterization of indices related to changes in polyphenolic metabolism.

Olives (Olea europaea cv. Chondrolia) were stored under a CO2 atmosphere immediately after harvesting for a period of 12 days. Samples obtained at 24-h intervals were analyzed by HPLC to identify components that may reflect changes in the biochemical behavior of the tissue. Four substances were shown to undergo significant fluctuations during storage, while their evolution was found to be different in olives stored under CO2 from those stored under regular atmospheric conditions (control). On the basis of data provided by liquid chromatography-electrospray ionization mass spectrometry, these substances were tentatively identified as hydroxytyrosol glucoside, demethylated ligstroside aglycone, ibotalactone A methyl ester, and verbascoside. The data are discussed in relation to the effect of postharvest treatments of olives for purposes of manipulating their polyphenolic content and plausible development of novel debittering processes.