Deoxyxylulose 5-phosphate Synthase Research Articles

Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase (DXS), a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway. Here we provide experimental insights into the underlying mechanism. Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro, causing a size shift. In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer. Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells. Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply, whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.

Effects of herbivory on carotenoid biosynthesis and breakdown.

The carotenoid content of plants may be impacted by stress with major consequences for photosynthesis and photoprotection. Most carotenoid stress research, however, has concentrated on abiotic stresses, and we know little about how biological stresses, such as herbivory, alter profiles of plant carotenoids and their degradation products. For example, carotenoid derivatives such as β-cyclocitral and β-ionone have been recently shown to act as signals in plant growth and protection against oxidative stress and herbivory. To understand how carotenoid composition is influenced by herbivory, changes in biosynthesis and degradation should be investigated. This chapter describes methods to simulate herbivory in a simple reproducible fashion and to assess carotenoid biosynthesis and degradation. Carotenoid biosynthesis depends on precursors provided by the methylerythritol 4-phosphate (MEP) pathway, which converts pyruvate and glyceraldehyde-3-phosphate to the five-carbon units used for construction of larger isoprenoids. We present protocols to quantify the activity of the first enzyme of the MEP pathway, deoxy-xylulose 5-phosphate synthase (DXS), usually assumed to be rate-controlling, and to estimate the concentration of the first intermediate of the pathway, deoxy-xylulose 5-phosphate (DXP). We also discuss procedures to measure the formation of volatile carotenoid breakdown products after herbivory. To monitor the activity of carotenoid-specific biosynthetic enzymes, such as phytoene synthase, protocols are available elsewhere in this volume (Wurtzel, 2022b).

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis.

ABSTRACTZymomonas mobilis is an ethanologenic bacterium currently being developed for production of advanced biofuels. Recent studies have shown that Z. mobilis can fix dinitrogen gas (N2) as a sole nitrogen source. During N2 fixation, Z. mobilis exhibits increased biomass-specific rates of ethanol production. In order to better understand the physiology of Z. mobilis during N2 fixation and during changes in ammonium (NH4+) availability, we performed liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomics and shotgun proteomics under three regimes of nitrogen availability: continuous N2 fixation, gradual NH4+ depletion, and acute NH4+ addition to N2-fixing cells. We report dynamic changes in abundance of proteins and metabolites related to nitrogen fixation, motility, ammonium assimilation, amino acid biosynthesis, nucleotide biosynthesis, isoprenoid biosynthesis, and Entner-Doudoroff (ED) glycolysis, providing insight into the regulatory mechanisms that control these processes in Z. mobilis. Our analysis identified potential physiological mechanisms that may contribute to increased specific ethanol production during N2 fixation, including decreased activity of biosynthetic pathways, increased protein abundance of alcohol dehydrogenase (ADHI), and increased thermodynamic favorability of the ED pathway. Of particular relevance to advanced biofuel production, we found that intermediates in the methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis were depleted during N2 fixation, coinciding with decreased protein abundance of deoxyxylulose 5-phosphate synthase (DXS), the first enzyme in the pathway. This implies that DXS protein abundance serves as a native control point in regulating MEP pathway activity in Z. mobilis. The results of this study will inform metabolic engineering to further develop Z. mobilis as a platform organism for biofuel production.IMPORTANCE Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol. Developing microbial systems to generate advanced biofuels requires metabolic engineering to reroute carbon away from ethanol and other native products and toward desired pathways, such as the MEP pathway for isoprenoid biosynthesis. However, rational engineering of microbial metabolism relies on understanding metabolic control points, in terms of both enzyme activity and thermodynamic favorability. In Z. mobilis, the factors that control glycolytic rates, ethanol production, and isoprenoid production are still not fully understood. In this study, we performed metabolomic, proteomic, and thermodynamic analysis of Z. mobilis during N2 fixation. This analysis identified key changes in metabolite levels, enzyme abundance, and glycolytic thermodynamic favorability that occurred during changes in NH4+ availability, helping to inform future efforts in metabolic engineering.

Interference with Clp protease impairs carotenoid accumulation during tomato fruit ripening.

Profound metabolic and structural changes are required for fleshy green fruits to ripen and become colorful and tasty. In tomato (Solanum lycopersicum), fruit ripening involves the differentiation of chromoplasts, specialized plastids that accumulate carotenoid pigments such as β-carotene (pro-vitamin A) and lycopene. Here, we explored the role of the plastidial Clp protease in chromoplast development and carotenoid accumulation. Ripening-specific silencing of one of the subunits of the Clp proteolytic complex resulted in β-carotene-enriched fruits that appeared orange instead of red when ripe. Clp-defective fruit displayed aberrant chromoplasts and up-regulated expression of nuclear genes encoding the tomato homologs of Orange (OR) and ClpB3 chaperones, most probably to deal with misfolded and aggregated proteins that could not be degraded by the Clp protease. ClpB3 and OR chaperones protect the carotenoid biosynthetic enzymes deoxyxylulose 5-phosphate synthase and phytoene synthase, respectively, from degradation, whereas OR chaperones additionally promote chromoplast differentiation by preventing the degradation of carotenoids such as β-carotene. We conclude that the Clp protease contributes to the differentiation of chloroplasts into chromoplasts during tomato fruit ripening, acting in co-ordination with specific chaperones that alleviate protein folding stress, promote enzyme stability and accumulation, and prevent carotenoid degradation.

Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota).

Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products.

Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity.

Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.

Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.

Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid.

Isopentenyl Diphosphate Inhibition of Thiamin Diphosphate Enzymes, Especially Deoxyxylulose 5‐Phosphate Synthase

Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate compete with thiamin diphosphate (ThDP) thereby reducing the activity of deoxyxylulose 5-phosphate synthase (DXS). Studies of this phenom...

Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis.

The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.

Analysis of metabolic pathway of terpenoids in Blumea balsamifera

In order to provide a theoretical basis for the regulation of active ingredient, the terpenoids metabolic pathway and specific enzymes in Blumea balsamifera are investigated. Basing on transcriptome information, B. balsamifera terpenoids metabolic pathway was analyzed in KEGG data base. Four metabolic pathway of terpenoids were found in KEGG data base. They were terpenoid backbone biosynthesis, monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, contained 103, 10, 29,59 genes, respectively. Through the analysis of the enzyme and product in the pathway, the result showed that there were 8 kinds of monoterpenes, 3 kinds of diterpenes, 3 kinds of triterpenes and sesquiterpenes. The mainly key enzymes were deoxyxylulose 5-phosphate synthase, HMG-CoA reductase and allyl transferase system. In B. balsamifera, there were relatively few monoterpenes synthetic enzymes, while the type of products was much more than other terpenes. This may be relate to the non-specific catalytic characteristic of monoterpene synthase. It is expected to improve the yield of terpenoids in B. balsamifera by analysis the pathways and regulation the key enzymes.

Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control

Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress.

Differential Oxidative Metabolism and 5-Ketoclomazone Accumulation Are Involved in Echinochloa phyllopogon Resistance to Clomazone

Echinochloa phyllopogon (late watergrass) is a major weed of California rice (Oryza sativa) that has evolved cytochrome P450-mediated metabolic resistance to different herbicides with multiple modes of action. E. phyllopogon populations from Sacramento Valley rice fields have also recently shown resistance to the herbicide clomazone. Clomazone is a proherbicide that must be metabolized to 5-ketoclomazone, which is the active compound that inhibits deoxyxylulose 5-phosphate synthase, a key enzyme of the nonmevalonate isoprenoid pathway. This study evaluated the differential clomazone metabolism within strains of the same species to investigate whether enhanced oxidative metabolism also confers clomazone resistance in E. phyllopogon. Using reverse-phase liquid chromatography-tandem mass spectrometry techniques in the multireaction monitoring mode, we elucidated that oxidative biotransformations are involved as a mechanism of clomazone resistance in this species. E. phyllopogon plants hydroxylated mostly the isoxazolidinone ring of clomazone, and clomazone hydroxylation activity was greater in resistant than in susceptible plants. The major clomazone metabolites resulted from monohydroxylation and dihydroxylation of the isoxazolidinone ring. Resistant plants accumulated 6- to 12-fold more of the monohydroxylated metabolite than susceptible plants, while susceptible plants accumulated 2.5-fold more of the phytotoxic metabolite of clomazone, 5-ketoclomazone. Our results demonstrate that oxidative metabolism endows multiple-herbicide-resistant E. phyllopogon with cross-resistance to clomazone through enhanced herbicide degradation and lower accumulation of the toxic metabolite in resistant versus susceptible plants.

Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark‐grown Arabidopsis seedlings

Carotenoids are plastidial isoprenoids essential for plant life. In Arabidopsis thaliana carotenoid biosynthesis is strongly upregulated when seedlings that germinate in the dark (etiolated) emerge from the soil and light derepresses photomorphogenesis, causing etioplasts to become chloroplasts. We found that carotenoid biosynthesis is also induced when deetiolation is derepressed in the absence of actual light, eventually resulting in improved greening (chlorophyll accumulation) upon illumination. The increased production of carotenoids in the dark correlates with an upregulated activity of phytoene synthase (PSY; the first committed enzyme of carotenogenesis) and the induction of PSY gene expression in cotyledons (where carotenoids accumulate in dark-grown seedlings). The metabolic precursors for carotenoid synthesis under these conditions are mostly supplied by the plastidial methylerythritol 4-phosphate (MEP) pathway. Accumulation of flux-controlling MEP pathway enzymes, such as deoxyxylulose 5-phosphate synthase (DXS), is post-transcriptionally increased when deetiolation is derepressed in the dark. Unlike the situation observed in light-grown plants, however, the sole overexpression of DXS in dark-grown seedlings does not increase carotenoid accumulation. By contrast, induced expression of a PSY-encoding transgene results in increased carotenoid levels and a concomitant post-transcriptional accumulation of DXS. These data provide evidence for a feedback mechanism by which PSY controls metabolic flux to the carotenoid pathway in plants.

Regulation of isoprene emission from poplar leaves throughout a day

Isoprene is a biogenic hydrocarbon that significantly affects tropospheric chemistry. Numerous plant species, including many trees, emit isoprene. Isoprene is synthesized by isoprene synthase (IspS), from dimethylallyl diphosphate (DMADP) made by the methylerythritol 4-phosphate (MEP) pathway. It has been demonstrated that in developing leaves, isoprene emission is regulated by transcriptional control of IspS, while in mature leaves subjected to changing growth temperature, regulation of emission is shared between IspS and DMADP supply from the MEP pathway. Isoprene emission also varies throughout a day, with circadian regulation implicated. This study investigated changes in isoprene emission capacity, and expression of IspS and the enzymes of the MEP pathway throughout several days, with Populus trichocarpa grown at different temperatures to induce different levels of isoprene emission. Isoprene emission capacity exhibited ultradian regulation, with a period of about 12 h; peak capacity was observed at 0300 and 1500 h daily. Several of the enzymes of the MEP pathway had previously been suggested to have regulatory roles in the production of other plastidic terpenoids, and transcript accumulation for these enzymes, combined with in silico promoter analyses, supported a regulatory role for deoxyxylulose 5-phosphate synthase (DXS) in particular.

Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways

Carotenoids are isoprenoid pigments of industrial and nutritional interest. Although they are produced in non-carotenogenic Escherichia coli engineered with the appropriate biosynthetic genes, only a limited pool of their metabolic precursors is available in these bacteria. We have compared the production of carotenoids (lycopene) in strains in which the supply of precursors was enhanced either by upregulating the endogenous pathway via overexpression of deoxyxylulose 5-phosphate synthase (DXS) or by incorporating an exogenous MVA+ operon. In strains expressing DXS under the control of a leaky IPTG-inducible promoter, lycopene accumulation was increased up to 8-fold in the absence of inducer. Addition of IPTG, however, negatively affected lycopene production. Although induction of too high levels of the MVA+ operon enzymes also appeared to cause interference with cell metabolism, supplementation with mevalonate (to be metabolized into carotenoid precursors) resulted in a 10-fold increase in lycopene levels in cells with a near wild-type background. An additional 2-fold increase (up to 228 mg/l) was obtained using an engineered BL21 strain. These results confirm that the MVA+ pathway is most convenient to upregulate the production of carotenoids (lycopene) production in E. coli but that factors other than precursor supply should be considered for high pigment accumulation levels.

Examining Ancient Inter-domain Horizontal Gene Transfer

Details of the genomic changes that occurred in the ancestors of Eukarya, Archaea and Bacteria are elusive. Ancient interdomain horizontal gene transfer (IDHGT) amongst the ancestors of these three domains has been difficult to detect and analyze because of the extreme degree of divergence of genes in these three domains and because most evidence for such events are poorly supported. In addition, many researchers have suggested that the prevalence of IDHGT events early in the evolution of life would most likely obscure the patterns of divergence of major groups of organisms let alone allow the tracking of horizontal transfer at this level. In order to approach this problem, we mined the E. coli genome for genes with distinct paralogs. Using the 1,268 E. coli K-12 genes with 40% or higher similarity level to a paralog elsewhere in the E. coli genome we detected 95 genes found exclusively in Bacteria and Archaea and 86 genes found in Bacteria and Eukarya. These genes form the basis for our analysis of IDHGT. We also applied a newly developed statistical test (the node height test), to examine the robustness of these inferences and to corroborate the phylogenetically identified cases of ancient IDHGT. Our results suggest that ancient inter domain HGT is restricted to special cases, mostly involving symbiosis in eukaryotes and specific adaptations in prokaryotes. Only three genes in the Bacteria + Eukarya class (Deoxyxylulose-5-phosphate synthase (DXPS), fructose 1,6-phosphate aldolase class II protein and glucosamine-6-phosphate deaminase) and three genes–in the Bacteria + Archaea class (ABC-type FE3+ -siderophore transport system, ferrous iron transport protein B, and dipeptide transport protein) showed evidence of ancient IDHGT. However, we conclude that robust estimates of IDHGT will be very difficult to obtain due to the methodological limitations and the extreme sequence saturation of the genes suspected of being involved in IDHGT.

Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.

Genetic Engineering of a Zeaxanthin-rich Potato by Antisense Inactivation and Co-suppression of Carotenoid Epoxidation

Zeaxanthin is an important dietary carotenoid but its abundance in our food is low. In order to provide a better supply of zeaxanthin in a staple crop, two different potato ( Solanum tuberosum L.) varieties were genetically modified. By transformation with sense and antisense constructs encoding zeaxanthin epoxidase, zeaxanthin conversion to violaxanthin was inhibited. Both approaches (antisense and co-suppression) yielded potato tubers with higher levels of zeaxanthin. Depending on the transgenic lines and tuber development, zeaxanthin content was elevated 4 to 130-fold reaching values up to 40 μg/g dry weight. As a consequence of the genetic manipulation, the amount of violaxanthin was diminished dramatically and in some cases the monoepoxy intermediate antheraxanthin accumulated. Between one and eight copies of the sense or antisense epoxidase gene fragments were integrated into the genome. In addition, most of the transformants with higher zeaxanthin levels showed also increased total carotenoid contents (up to 5.7-fold) and some of them exhibited reduced amounts of lutein. The increase in total carotenoids suggests that the genetic modification affects the regulation of the whole carotenoid biosynthetic pathway in potato tubers. Northern blot analysis demonstrated that upregulation of carotenogenesis in the transgenics is accompanied by substantial higher phytoene synthase transcript levels in 6-week-old tubers and a very slight increase of the β-carotene hydroxylase transcript. The amount of the deoxyxylulose 5-phosphate synthase mRNA was very similar in wild type and transformed tubers. Abscisic acid content of tubers remained unchanged whereas α-tocopherol was 2 to 3 fold elevated in the transformants.