Total Synthesis and Structure Confirmation of trans-Anhydromevalonate-5-phosphate, a Key Biosynthetic Intermediate of the Archaeal Mevalonate Pathway.

The mevalonate pathway is an upstream terpenoid biosynthetic route of terpenoids for providing the two five-carbon units, dimethylallyl diphosphate, and isopentenyl diphosphate. Recently, trans-anhydromevalonate-5-phosphate (tAHMP) was isolated as a new biosynthetic intermediate of the archaeal mevalonate pathway. In this study, we would like to report the first synthesis of tAHMP and its enzymatic transformation using one of the key enzymes, mevalonate-5-phosphate dehydratase from a hyperthermophilic archaeon, Aeropyrum pernix. Starting from methyl tetrolate, a Cu-catalyzed allylation provided an E-trisubstituted olefin in a stereoselective manner. The resulting E-olefin was transformed to tAHMP by cleavage of the olefin and phosphorylation. The structure of the synthetic tAHMP was unambiguously determined by NOESY analysis.

Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions.

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

The phosphorylation mechanism of mevalonate diphosphate decarboxylase: a QM/MM study.

Mevalonate diphosphate decarboxylase (MDD) catalyses a crucial step of the mevalonate pathway via Mg2+-ATP-dependent phosphorylation and decarboxylation reactions to ultimately produce isopentenyl diphosphate, the precursor of isoprenoids, which is essential to bacterial functions and provides ideal building blocks for the biosynthesis of isopentenols. However, the metal ion(s) in MDD has not been unambiguously resolved, which limits the understanding of the catalytic mechanism and the exploitation of enzymes for the development of antibacterial therapies or the mevalonate metabolic pathway for the biosynthesis of biofuels. Here by analogizing structurally related kinases and molecular dynamics simulations, we constructed a model of the MDD-substrate-ATP-Mg2+ complex and proposed that MDD requires two Mg2+ ions for maintaining a catalytically active conformation. Subsequent QM/MM studies indicate that MDD catalyses the phosphorylation of its substrate mevalonate diphosphate (MVAPP) via a direct phosphorylation reaction, instead of the previously assumed catalytic base mechanism. The results here would shed light on the active conformation of MDD-related enzymes and their catalytic mechanisms and therefore be useful for developing novel antimicrobial therapies or reconstructing mevalonate metabolic pathways for the biosynthesis of biofuels.

Catalytic mechanism of mevalonate kinase revisited, a QM/MM study.

Mevalonate Kinase (MVK) catalyses the ATP-Mg2+ mediated phosphate transfer of mevalonate to produce mevalonate 5-phosphate and is a key kinase in the mevalonate pathway in the biosynthesis of isopentenyl diphosphate, the precursor of isoprenoid-based biofuels. However, the crystal structure in complex with the native substrate mevalonate, ATP and Mg2+ has not been resolved, which has limited the understanding of its reaction mechanism and therefore its application in the production of isoprenoid-based biofuels. Here using molecular docking, molecular dynamics (MD) simulations and a hybrid QM/MM study, we revisited the location of Mg2+ resolved in the crystal structure of MVK and determined a catalytically competent MVK structure in complex with the native substrate mevalonate and ATP. We demonstrated that significant conformational change on a flexible loop connecting the α6 and α7 helix is induced by the substrate binding. Further, we found that Asp204 is coordinated to the Mg2+ ion. Arg241 plays a crucial role in organizing the triphosphoryl tail of ATP for in-line phosphate transfer and stabilizing the negative charge that accumulates at the ß,γ-bridging oxygen of ATP upon bond cleavage. Remarkably, we revealed that the phosphorylation of mevalonate catalyzed by MVK occurs via a direct phosphorylation mechanism, instead of the conventionally postulated catalytic base mechanism. The catalytically competent complex structure of MVK as well as the mechanism of reaction will pave the way for the rational engineering of MVK to exploit its applications in the production of biofuels.

Tracing the biosynthetic origin of limonoids and their functional groups through stable isotope labeling and inhibition in neem tree (Azadirachta indica) cell suspension.

BACKGROUND: Neem tree serves as a cornucopia for triterpenoids called limonoids that are of profound interest to humans due to their diverse biological activities. However, the biosynthetic pathway that plant employs for the production of limonoids remains unexplored for this wonder tree. RESULTS: Herein, we report the tracing of limonoid biosynthetic pathway through feeding experiments using 13C isotopologues of glucose in neem cell suspension. Growth and development specific limonoid spectrum of neem seedling and time dependent limonoid biosynthetic characteristics of cell lines were established. Further to understand the role of mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways in limonoid biosynthesis, Ultra Performance Liquid Chromatography (UPLC)- tandem mass spectrometry based structure-fragment relationship developed for limonoids and their isotopologues have been utilized. Analyses of labeled limonoid extract lead to the identification of signature isoprenoid units involved in azadirachtin and other limonoid biosynthesis, which are found to be formed through mevalonate pathway. This was further confirmed by treatment of cell suspension with mevinolin, a specific inhibitor for MVA pathway, which resulted in drastic decrease in limonoid levels whereas their biosynthesis was unaffected with fosmidomycin mediated plastidial methylerythritol 4-phosphate (MEP) pathway inhibition. This was also conspicuous, as the expression level of genes encoding for the rate-limiting enzyme of MVA pathway, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) was comparatively higher to that of deoxyxylulose-phosphate synthase (DXS) of MEP pathway in different tissues and also in the in vitro grown cells. Thus, this study will give a comprehensive understanding of limonoid biosynthetic pathway with differential contribution of MVA and MEP pathways. CONCLUSIONS: Limonoid biosynthesis of neem tree and cell lines have been unraveled through comparative quantification of limonoids with that of neem tree and through 13C limonoid isotopologues analysis. The undifferentiated cell lines of neem suspension produced a spectrum of C-seco limonoids, similar to parental tissue, kernel. Azadirachtin, a C-seco limonoid is produced in young tender leaves of plant whereas in the hard mature leaves of tree, ring intact limonoid nimocinol accumulates in high level. Furthermore, mevalonate pathway exclusively contributes for isoprene units of limonoids as evidenced through stable isotope labeling and no complementation of MEP pathway was observed with mevalonate pathway dysfunction, using chemical inhibitors.

Modeling of Dolichol Mass Spectra Isotopic Envelopes as a Tool to Monitor Isoprenoid Biosynthesis.

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials.

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells' global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell-material interactions and suggest alternative research routes for evaluating biomaterials to improve their design.

Harzianone Biosynthesis by the Biocontrol Fungus Trichoderma.

Analysis of the volatile terpenes produced by seven fungal strains of the genus Trichoderma by use of a closed-loop stripping apparatus (CLSA) revealed a common production of harzianone, a bioactive, structurally unique diterpenoid consisting of a fused tetracyclic 4,7,5,6-membered ring system. The terpene cyclization mechanism was studied by feeding experiments using selectively 13 C- and 2 H-labeled synthetic mevalonolactone isotopologues, followed by analysis of the incorporation patterns by 13 C NMR spectroscopy and GC/MS. The structure of harzianone was further supported from a 13 C,13 C COSY experiment of the in-vivo-generated fully 13 C-labeled diterpene.

Scalable production of mechanically tunable block polymers from sugar.

Development of sustainable and biodegradable materials is essential for future growth of the chemical industry. For a renewable product to be commercially competitive, it must be economically viable on an industrial scale and possess properties akin or superior to existing petroleum-derived analogs. Few biobased polymers have met this formidable challenge. To address this challenge, we describe an efficient biobased route to the branched lactone, ß-methyl-δ-valerolactone (ßMδVL), which can be transformed into a rubbery (i.e., low glass transition temperature) polymer. We further demonstrate that block copolymerization of ßMδVL and lactide leads to a new class of high-performance polyesters with tunable mechanical properties. Key features of this work include the creation of a total biosynthetic route to produce ßMδVL, an efficient semisynthetic approach that employs high-yielding chemical reactions to transform mevalonate to ßMδVL, and the use of controlled polymerization techniques to produce well-defined PLA-PßMδVL-PLA triblock polymers, where PLA stands for poly(lactide). This comprehensive strategy offers an economically viable approach to sustainable plastics and elastomers for a broad range of applications.

Simvastatin inhibits CD44 fragmentation in chondrocytes.

In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1ß + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1ß + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix.

Production of the forskolin precursor 11ß-hydroxy-manoyl oxide in yeast using surrogate enzymatic activities.

BACKGROUND: Several plant diterpenes have important biological properties. Among them, forskolin is a complex labdane-type diterpene whose biological activity stems from its ability to activate adenylyl cyclase and to elevate intracellular cAMP levels. As such, it is used in the control of blood pressure, in the protection from congestive heart failure, and in weight-loss supplements. Chemical synthesis of forskolin is challenging, and production of forskolin in engineered microbes could provide a sustainable source. To this end, we set out to establish a platform for the production of forskolin and related epoxy-labdanes in yeast. RESULTS: Since the forskolin biosynthetic pathway has only been partially elucidated, and enzymes involved in terpene biosynthesis frequently exhibit relaxed substrate specificity, we explored the possibility of reconstructing missing steps of this pathway employing surrogate enzymes. Using CYP76AH24, a Salvia pomifera cytochrome P450 responsible for the oxidation of C-12 and C-11 of the abietane skeleton en route to carnosic acid, we were able to produce the forskolin precursor 11ß-hydroxy-manoyl oxide in yeast. To improve 11ß-hydroxy-manoyl oxide production, we undertook a chassis engineering effort involving the combination of three heterozygous yeast gene deletions (mct1/MCT1, whi2/WHI2, gdh1/GDH1) and obtained a 9.5-fold increase in 11ß-hydroxy-manoyl oxide titers, reaching 21.2 mg L(-1). CONCLUSIONS: In this study, we identify a surrogate enzyme for the specific and efficient hydroxylation of manoyl oxide at position C-11ß and establish a platform that will facilitate the synthesis of a broad range of tricyclic (8,13)-epoxy-labdanes in yeast. This platform forms a basis for the heterologous production of forskolin and will facilitate the elucidation of subsequent steps of forskolin biosynthesis. In addition, this study highlights the usefulness of using surrogate enzymes for the production of intermediates of complex biosynthetic pathways. The combination of heterozygous deletions and the improved yeast strain reported here will provide a useful tool for the production of numerous other isoprenoids.

Lethality of cytochalasin B and other compounds isolated from fungus Aspergillus sp. (Trichocomaceae) endophyte of Bauhinia guianensis (Fabaceae).

Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis.

Inhibition of bacterial mevalonate diphosphate decarboxylase by eriochrome compounds.

Mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33) catalyzes the irreversible decarboxylation of mevalonate diphosphate in the mevalonate pathway to form isopentenyl diphosphate, which is a precursor in the biosynthesis of many essential polyisoprenoid natural products, including sterols. In low G/C Gram-positive bacteria, which utilize the mevalonate pathway, MDD is required for cell viability and thus is a potential target for development of antibiotic drugs. To identify potential inhibitors of the enzyme, the National Cancer Institute's Mechanistic Diversity Set library of compounds was screened for inhibitors of Staphylococcus epidermidis MDD. From this screen, the compound Eriochrome Black A (EBA), an azo dye, was found to inhibit the enzyme with an IC50 value<5µM. Molecular docking of EBA into a crystal structure of S. epidermidis MDD suggested binding at the active site. EBA, along with the related Eriochrome B and T compounds, was evaluated for its ability to not only inhibit enzymatic activity but to inhibit bacterial growth as well. These compounds exhibited competitive inhibition towards the substrate mevalonate diphosphate, with Ki values ranging from 0.6 to 2.7µM. Non-competitive inhibition was observed versus ATP indicating binding of the inhibitor in the mevalonate diphosphate binding site, consistent with molecular docking predictions. Fluorescence quenching analyses also supported active site binding of EBA. These eriochrome compounds are effective at inhibiting S. epidermidis cell growth on both solid media and in liquid culture (MIC50 from 31 to 350µM) raising the possibility that they could be developed into antibiotic leads targeting pathogenic low-G/C Gram-positive cocci.

Structural Revision and Elucidation of the Biosynthesis of Hypodoratoxide by (13) C,(13) C COSY NMR Spectroscopy.

Feeding of (2,3,4,5,6-(13) C5 )mevalonolactone to the fungus Hypomyces odoratus resulted in a completely labeled sesquiterpene ether. The connectivity of the carbon atoms was easily deduced from a (13) C,(13) C COSY spectrum, revealing a structure that was different from the previously reported structure of hypodoratoxide, even though the reported (13) C NMR data matched. A structural revision of hypodoratoxide is thus presented. Its absolute configuration was tentatively assigned from its co-metabolite cis-dihydroagarofuran. Its biosynthesis was investigated by feeding of (3-(13) C)- and (4,6-(13) C2 )mevalonolactone, which gave insights into the complex rearrangement of the carbon skeleton during terpene cyclization by analysis of the (13) C,(13) C couplings.

Identification in Haloferax volcanii of phosphomevalonate decarboxylase and isopentenyl phosphate kinase as catalysts of the terminal enzyme reactions in an archaeal alternate mevalonate pathway.

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.

De novo sequencing and comparative analysis of holy and sweet basil transcriptomes.

BACKGROUND: Ocimum L. of family Lamiaceae is a well known genus for its ethnobotanical, medicinal and aromatic properties, which are attributed to innumerable phenylpropanoid and terpenoid compounds produced by the plant. To enrich genomic resources for understanding various pathways, de novo transcriptome sequencing of two important species, O. sanctum and O. basilicum, was carried out by Illumina paired-end sequencing. RESULTS: The sequence assembly resulted in 69117 and 130043 transcripts with an average length of 1646 ± 1210.1 bp and 1363 ± 1139.3 bp for O. sanctum and O. basilicum, respectively. Out of the total transcripts, 59648 (86.30%) and 105470 (81.10%) from O. sanctum and O. basilicum, and respectively were annotated by uniprot blastx against Arabidopsis, rice and lamiaceae. KEGG analysis identified 501 and 952 transcripts from O. sanctum and O. basilicum, respectively, related to secondary metabolism with higher percentage of transcripts for biosynthesis of terpenoids in O. sanctum and phenylpropanoids in O. basilicum. Higher digital gene expression in O. basilicum was validated through qPCR and correlated to higher essential oil content and chromosome number (O. sanctum, 2n = 16; and O. basilicum, 2n = 48). Several CYP450 (26) and TF (40) families were identified having probable roles in primary and secondary metabolism. Also SSR and SNP markers were identified in the transcriptomes of both species with many SSRs linked to phenylpropanoid and terpenoid pathway genes. CONCLUSION: This is the first report of a comparative transcriptome analysis of Ocimum species and can be utilized to characterize genes related to secondary metabolism, their regulation, and breeding special chemotypes with unique essential oil composition in Ocimum.

Pogostol biosynthesis by the endophytic fungus Geniculosporium.

Six (13)C-labelled isotopomers of mevalonolactone were synthesised and used in feeding experiments with the endophytic fungus Geniculosporium. The high incorporation rates of (13)C-label into a sesquiterpene that was found in headspace extracts of the fungus enabled unambiguous identification of this volatile as pogostol without the need for compound purification, simply by collecting the volatile fraction with a closed-loop stripping apparatus followed by direct (13)C NMR analysis (CLSA-NMR). The feeding experiments also gave insights into the biosynthesis of pogostol, including stereochemical aspects of the terpene cyclisation reaction. The possible biological function of pogostol is discussed.

Tracking the sterol biosynthesis pathway of the diatom Phaeodactylum tricornutum.

Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the pathway leading to sterol biosynthesis in diatoms has remained hitherto unidentified. Through the DiatomCyc database we reconstructed the mevalonate and sterol biosynthetic pathways of the model diatom Phaeodactylum tricornutum in silico. We experimentally verified the predicted pathways using enzyme inhibitor, gene silencing and heterologous gene expression approaches. Our analysis revealed a peculiar, chimeric organization of the diatom sterol biosynthesis pathway, which possesses features of both plant and fungal pathways. Strikingly, it lacks a conventional squalene epoxidase and utilizes an extended oxidosqualene cyclase and a multifunctional isopentenyl diphosphate isomerase/squalene synthase enzyme. The reconstruction of the P. tricornutum sterol pathway underscores the metabolic plasticity of diatoms and offers important insights for the engineering of diatoms for sustainable production of biofuels and high-value chemicals.

Sterol partitioning by HMGR and DXR for routing intermediates toward withanolide biosynthesis.

Withanolides biosynthesis in the plant Withania somnifera (L.) Dunal is hypothesized to be diverged from sterol pathway at the level of 24-methylene cholesterol. The conversion and translocation of intermediates for sterols and withanolides are yet to be characterized in this plant. To understand the influence of mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways on sterols and withanolides biosynthesis in planta, we overexpressed the WsHMGR2 and WsDXR2 in tobacco, analyzed the effect of transient suppression through RNAi, inhibited MVA and MEP pathways and fed the leaf tissue with different sterols. Overexpression of WsHMGR2 increased cycloartenol, sitosterol, stigmasterol and campesterol compared to WsDXR2 transgene lines. Increase in cholesterol was, however, marginally higher in WsDXR2 transgenic lines. This was further validated through transient suppression analysis, and pathway inhibition where cholesterol reduction was found higher due to WsDXR2 suppression and all other sterols were affected predominantly by WsHMGR2 suppression in leaf. The transcript abundance and enzyme analysis data also correlate with sterol accumulation. Cholesterol feeding did not increase the withanolide content compared to cycloartenol, sitosterol, stigmasterol and campesterol. Hence, a preferential translocation of carbon from MVA and MEP pathways was found differentiating the sterols types. Overall results suggested that MVA pathway was predominant in contributing intermediates for withanolides synthesis mainly through the campesterol/stigmasterol route in planta.

(-)-R-mevalonolactone studied by ROA and SERS spectroscopy.

Here we present for the first time the experimental and theoretical (DFT/B3LYP/6-311++G**) Raman optical activity (ROA) spectra of (-)-R-mevalonic acid as the δ-lactone form in neat liquid and in the aqueous solution. Quantum chemical calculations show the conformational diversity of (-)-R-mevalonolactone originated from small energy differences between the various conformation of the six-membered ring and the arrangement of the hydroxyl group. According to calculations, the investigated compound takes predominantly the chair conformation with the hydroxyl group in axial position, but the contribution of the other chair and boat conformers in the equilibrium at room temperature is not negligible. Additionally, we present normal Raman and the surface enhanced Raman (SERS) spectra of (-)-R-mevalonolactone adsorbed on the colloidal silver.