Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids

The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

Carbohydrate and elicitor enhanced withanolide (withaferin A and withanolide A) accumulation in hairy root cultures of Withania somnifera (L.)

Leaves of Withania somnifera contained more withaferin A and withanolide A than roots indicating that these compounds mainly accumulate in leaves. With an increase in age of the plant, withaferin A was enhanced with a corresponding decrease in withanolide A. Hairy root cultures were induced from leaf explants using Agrobacterium rhizogenes and the transgenic nature of hairy roots was confirmed by partial isolation and sequencing of rolB gene, which could not be amplified in untransformed plant parts. In hairy roots, withaferin A accumulated at 2, 3 and 4% but not at 6% sucrose, the highest amount being 1733 microg/g dry weight at 4% level. High and equal amounts of withaferin A and withanolide A accumulated (890 and 886 microg/g dry tissue respectively) only at 3% sucrose. Increasing concentrations of glucose enhanced withaferin A and it peaked at 5% level (3866 microg/g dry tissue). This amount is 2842 and 34% higher compared to untransformed roots and leaves (collected from 210-day-old plants) respectively. Withanolide A was detected at 5% glucose but not at other concentrations. While chitosan and nitric oxide increased withaferin A, jasmonic acid decreased it. Acetyl salicylic acid stimulated accumulation of both withaferin A and withanolide A at higher concentrations. Triadimefon, a fungicide, enhanced withaferin A by 1626 and 3061% (not detected earlier) compared to hairy and intact roots respectively.     

Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal

Withania somnifera Dunal is an important and extensively studied medicinal plant; however, there is no report available that relates withanolide content and its profile in relation to the expression of pathway genes during different morphogenic stages. In this study, withanolide A, withaferin A, and withanone, the major withanolides of W. somnifera, were measured in different in vitro stages during organogenesis, viz., shoot to root (direct rhizogenesis)/root to shoot (indirect via callus phase) transition vis-à-vis expression levels of key pathway genes involved in withanolide biosynthetic pathways. The morphogenic transitions were found to be tightly linked to the pattern of accumulation of withanolides. The high expression levels of most of the pathway genes in in vitro shoots in comparison to in vitro root and callus tissues exhibited a direct co-relation with the maximum withanolide content (>2.7 mg/gDW). The biogenesis of withaferin A, a major constituent of the leaves, was however found to be tightly linked to shoots/green tissue. In addition, we were also able to establish an efficient regeneration system from roots for their further utilization in biotechnological applications.     

Biosynthetic pathway of insect juvenile hormone III in cell suspension cultures of the sedge Cyperus iria

In most insect species, juvenile hormones regulate critical physiological processes such as metamorphosis and reproduction. In insects, these sesquiterpenoids are synthesized by retrocerebral endocrine organs, the corpora allata, via the classical mevalonate (MVA) pathway. One of these compounds, juvenile hormone III (JH III), has also been identified in the sedge Cyperus iria. In higher plants, biosynthesis of the sesquiterpenoid backbone may proceed through two distinct pathways: the MVA pathway or the 2C-methyl erythritol 4-phosphate pathway or through a combination of both pathways. Cell suspension cultures of C. iria were used to elucidate the biosynthetic pathway of JH III in the plant. Enzyme inhibition and labeling studies conclusively demonstrated that the biosynthesis of the sesquiterpenoid backbone of JH III proceeds via the MVA pathway. Inhibitor and precursor feeding studies also suggest that later steps of JH III biosynthesis in C. iria are similar to the insect pathway and that the final enzymatic reaction in JH III biosynthesis is catalyzed by a cytochrome P(450) monooxygenase.     

Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection

A reversed-phase HPLC method for the simultaneous analysis of nine structurally similar withanolides, namely, 27-hydroxy withanone, 17-hydroxy withaferin A, 17-hydroxy-27-deoxy withaferin A, withaferin A, withanolide D, 27-hydroxy withanolide B, withanolide A, withanone and 27-deoxywithaferin A, has been developed using a linear binary gradient solvent system comprising methanol and water containing 0.1% acetic acid. Both photodiode array and evaporative light scattering detection were used to profile the extract compositions and to quantify the withanolides therein. Homogeneity and purity of each peak was ascertained by comparative evaluation of the on-line UV spectra of the eluted compounds with those of the reference compounds. The method has been validated with respect to various parameters of performance quality including computation regression analysis based on calibration curves, peak resolution factor, asymmetry factor, tailing factor, RSD (%) of retention time and peak area response, limit of quantivation, limit of detection, precision and recovery. The developed method has been applied to the analysis of leaf and root tissues of Withania somnifera for withanolide content.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.     

Biosynthesis of withanolides in Acnistus breviflorus

Administration of [2?¹⁴C]mevalonolactone to excised leaves of Acnistus breviflorus produced labelled withaferin A and jaborosalactone A. The former was degraded leading to the isolation of glyceric acid from C-25–C-27 of the withanolide. These carbons represented only 2 % of the total radioactivity of withaferin A. The relative radioactivity of these carbons indicated that C-26 is directly derived from C-2 of mevalonolactone suggesting that the 25-pro-R-methyl group of cholesterol or any other sterol intermediate had been oxidized to form the lactone ring of the withanolide. The total radioactivity value found for C-25–C-27 was much lower than the expected 20 % of the total value for the withanolide indicating that the side chain of the sterol precursor had been partially cleaved during the biosynthetic process.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis

Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.     

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.     

The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways

Lateral gene transfer (LGT) is a major force in microbial genome evolution. Here, we present an overview of lateral transfers affecting genes involved in isopentenyl diphosphate (IPP) synthesis. Two alternative metabolic pathways can synthesize this universal precursor of isoprenoids, the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway and the mevalonate (MVA) pathway. We have surveyed recent genomic data and the biochemical literature to determine the distribution of the genes composing these pathways within the bacterial domain. The scattered distribution observed is incompatible with a simple scheme of vertical transmission. LGT (among and between bacteria, archaea and eukaryotes) more parsimoniously explains many features of this pattern. This alternative scenario is supported by phylogenetic analyses, which unambiguously confirm several cases of lateral transfer. Available biochemical data allow the formulation of hypotheses about selective pressures favouring transfer. The phylogenetic diversity of the organisms involved and the range of possible causes and effects of these transfer events make the IPP biosynthetic pathways an ideal system for studying the evolutionary role of LGT.     

Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis

The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis. The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C. jejuni and Agrobacterium tumefaciens. The monofunctional enzymes (2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations. We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.     

Structures of withanosides I, II, III, IV, V, VI, and VII, new withanolide glycosides, from the roots of Indian Withania somnifera DUNAL. and inhibitory activity for tachyphylaxis to clonidine in isolated guinea-pig ileum.

Seven new withanolide glycosides called withanosides I, II, III, IV, V, VI, and VII were isolated from an Indian natural medicine, Ashwagandha, the roots of Indian Withania somnifera DUNAL. (Solanaceae), together with four known compounds, withaferin A, 5 alpha,20 alpha(F)(R)-dihydroxy-6 alpha,7 alpha-epoxy-1-oxowitha-2,24-dienolide, physagulin D, and coagulin Q. The structures of withanosides I, II, III, IV, V, VI, and VII were determined based on chemical and physicochemical evidence. Principal constituents, withanoside VI (10 and 30 microM) and withaferin A (10 microM), attenuated the tachyphylaxis to clonidine on electrically stimulated guinea-pig ileum in vitro.     

Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from ¹³C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-¹³C]- and [4-¹³C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer ¹³C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.     

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-¹³C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.     

Amino Acid Biosynthesis in the Halophilic Archaeon Haloarcula hispanica

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.