Isopentenyl Diphosphate Biosynthesis Research Articles

Comparison of the antibiotic resistance mechanisms in a gram-positive and a gram-negative bacterium by gene networks analysis.

Nowadays, the emergence of some microbial species resistant to antibiotics, both gram-positive and gram-negative bacteria, is due to changes in molecular activities, biological processes and their cellular structure in order to survive. The aim of the gene network analysis for the drug-resistant Enterococcus faecium as gram-positive and Salmonella Typhimurium as gram-negative bacteria was to gain insights into the important interactions between hub genes involved in key molecular pathways associated with cellular adaptations and the comparison of survival mechanisms of these two bacteria exposed to ciprofloxacin. To identify the gene clusters and hub genes, the gene networks in drug-resistant E. faecium and S. Typhimurium were analyzed using Cytoscape. Subsequently, the putative regulatory elements were found by examining the promoter regions of the hub genes and their gene ontology (GO) was determined. In addition, the interaction between milRNAs and up-regulated genes was predicted. RcsC and D920_01853 have been identified as the most important of the hub genes in S. Typhimurium and E. faecium, respectively. The enrichment analysis of hub genes revealed the importance of efflux pumps, and different enzymatic and binding activities in both bacteria. However, E. faecium specifically increases phospholipid biosynthesis and isopentenyl diphosphate biosynthesis, whereas S. Typhimurium focuses on phosphorelay signal transduction, transcriptional regulation, and protein autophosphorylation. The similarities in the GO findings of the promoters suggest common pathways for survival and basic physiological functions of both bacteria, including peptidoglycan production, glucose transport and cellular homeostasis. The genes with the most interactions with milRNAs include dpiB, rcsC and kdpD in S. Typhimurium and EFAU004_01228, EFAU004_02016 and EFAU004_00870 in E. faecium, respectively. The results showed that gram-positive and gram-negative bacteria have different mechanisms to survive under antibiotic stress. By deciphering their intricate adaptations, we can develop more effective therapeutic approaches and combat the challenges posed by multidrug-resistant bacteria.

Multienzyme Assembly on Caveolar Membranes In Cellulo

Assembling enzymes into complexes facilitates the transfer of intermediates, insulates intermediate leakage, streamlines the metabolic flux, and increases the production of the desired products during biosynthesis. Here, we report the construction of membrane-bound multienzyme complexes, multienzyme caveolar membranes (MCMs), based on sequential protein assemblies on a membrane scaffold. β-Cav1, an engineered caveolin-1 isoform beta, self-assembles to form caveolar membranes. Enzymes that catalyze the biosynthesis of isopentenyl diphosphate and dimethylallyl pyrophosphate to α-farnesene were assembled on caveolar membranes through noncovalent interactions or covalent protein reactions. Bacterial strains harboring MCMs gave α-farnesene production titers up to 10 times higher than the control strains without enzyme assembly. Isolated MCMs can produce farnesyl diphosphate (FPP) and α-farnesene ex vivo, indicating the structural and functional independence of MCMs in vitro, in cellulo, and ex vivo. This work shows an under-reported direction of multienzyme assembly: creating a hydrophobic microenvironment for the biosynthetic enzymes at nanoscales significantly increases the titer of the hydrophobic product, α-farnesene, for example.

Aryl Hydrocarbon Receptor (AhR) Activation by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) Dose-Dependently Shifts the Gut Microbiome Consistent with the Progression of Non-Alcoholic Fatty Liver Disease.

Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with non-alcoholic fatty liver disease (NAFLD) and recapitulated in a NAFLD-phenotype elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. TCDD induces hepatic fat accumulation and increases levels of secondary bile acids, including taurolithocholic acid and deoxycholic acid (microbial modified bile acids involved in host bile acid regulation signaling pathways). To investigate the effects of TCDD on the gut microbiota, the cecum contents of male C57BL/6 mice orally gavaged with sesame oil vehicle or 0.3, 3, or 30 µg/kg TCDD were examined using shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species (i.e., Lactobacillus reuteri). Increased species were also associated with dose-dependent increases in bile salt hydrolase sequences, responsible for deconjugation reactions in secondary bile acid metabolism. Increased L. reuteri levels were further associated with mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis and o-succinylbenzoate synthase, a menaquinone biosynthesis associated gene. Analysis of the gut microbiomes from cirrhosis patients identified an increased abundance of genes from the mevalonate-dependent IPP biosynthesis as well as several other menaquinone biosynthesis genes, including o-succinylbenzoate synthase. These results extend the association of lactobacilli with the AhR/intestinal axis in NAFLD progression and highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD.

Signs of the plastid: Enzymes involved in plastid-localized metabolic pathways in a eugregarine species

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study.

Comparative transcriptome analysis reveals key genes in the regulation of squalene and β-sitosterol biosynthesis in Torreya grandis

Torreya grandis (T. grandis, Taxaceae) is both economically and medicinally valuable species being rich in various bioactive compounds (e.g., squalene and β-sitosterol). However, the contents of these compounds are various and cultivar specific, and the complicated regulatory mechanisms of their biosynthesis in T. grandis are still unknown. To uncover the underlying molecular mechanisms that control the differences in the accumulation of squalene and β-sitosterol, a comprehensive transcriptome was constructed from nine different T. grandis cultivars. A total of more than 60,372 unigenes were obtained, of which over 60% were successfully annotated. Identification and expression analysis of the differentially-expressed genes (DEGs) showed that 39 candidate genes were involved in squalene and β-sitosterol biosynthesis in T. grandis seeds. In particular, the expression patterns of genes related to the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways indicates that both pathways promote the upstream biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in different T. grandis cultivars. Moreover, several key regulatory steps controlling the differential accumulation of squalene and β-sitosterol between T. grandis cultivars were also discussed.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

Plant Senescence: The Role of Volatile Terpene Compounds (VTCs)

Senescence is a natural, energy-dependent, physiological, developmental and an ecological process that is controlled by the plant’s own genetic program, allowing maximum recovery of nutrients from older organs for the survival of the plant, as such; it is classified as essential component of the growth and development of plants. In some cases, under one or many environmental stresses, senescence is triggered in plants. Despite many studies in the area, less consideration has been given to plant secondary metabolites, especially the role of VTCs on plant senescence. This review seeks to capture the biosynthesis and signal transduction of VTCs, the physiology of VTCs in plant development and how that is linked to some phytohormones to induce senescence. Much progress has been made in the elucidation of metabolic pathways leading to the biosynthesis of VTCs. In addition to the classical cytosolic mevalonic acid (MVA) pathway from acetyl-CoA, the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, originating from glyceraldehyde-3-phosphate (GAP) and pyruvate, leads to the biosynthesis of isoprenoid precursors, isopentenyl diphosphate and dimethyl allyl diphosphate. VTCs synthesis and emission are believed to be tightly regulated by photosynthetic carbon supply into MEP pathway. Thus, under abiotic stresses such as drought, high salinity, high and low temperature, and low CO2 that directly affect stomatal conductance and ultimately biochemical limitation to photosynthesis, there has been observed induction of VTC synthesis and emissions, reflecting the elicitation of MEP pathway. This reveals the possibility of important function(s) of VTCs in plant defense against stress by mobilizing resources from components of plants and therefore, senescence. Our current understanding of the relationship between environmental responses and senescence mostly comes from the study of senescence response to phytohormones such as abscisic acid, jasmonic acid, ethylene and salicylic acid, which are extensively involved in response to various abiotic and biotic stresses. These stresses affect synthesis and/or signaling pathways of phytohormones to eventually trigger expression of stress-responsive genes, which in turn appears to affect leaf senescence. Comparison of plant response to stresses in relation to patterns of VTCs and phytohormones biosynthesis indicates a considerable crosstalk between these metabolic processes and their signal to plant senescence.

Transcriptome analysis of Chlorella zofingiensis to identify genes and their expressions involved in astaxanthin and triacylglycerol biosynthesis

Glucose induces the green alga Chlorella zofingiensis to accumulate high amounts of astaxanthin and triacylglycerol (TAG). Here we used comparative transcriptome analysis to identify the genes and their expressions for the biosynthesis of the important metabolites. Transcriptome data revealed a great number of gene coding sequences involved in most primary and secondary metabolisms, including full coding sequences of all the genes for astaxanthin and TAG biosynthesis. The biosynthesis of IPP (isopentenyl diphosphate) by the MEP (methylerythritol 4-phosphate) pathway was found to be correlated to astaxanthin biosynthesis. The enhanced astaxanthin synthesis induced by glucose was closely related to the up-regulation of astaxanthin pathway genes coupled with the repression of side pathway genes. Most of the genes involved in fatty acid and TAG biosynthesis consist of more than one copy. Their global up-regulation triggered off the accumulation of fatty acids and TAG in C. zofingiensis. In addition, glucose up-regulated DGAT2 (diacylglycerol acyltransferase 2) but down-regulated PDAT (phospholipid: diacylglycerol acyltransferase), suggesting that the acyl-CoA-dependent pathway attributed to the accumulation of TAG. Astaxanthin and TAG accumulations were found to be coordinated. Our transcriptome datasets can serve as a valuable and informative platform for functional studies on carotenoids, lipids and other important metabolites.

Molecular mechanism of ethylene stimulation of latex yield in rubber tree (Hevea brasiliensis) revealed by de novo sequencing and transcriptome analysis.

BackgroundRubber tree (Hevea brasiliensis) is an important industrial crop cultivated in tropical areas for natural rubber production. Treatment of the bark of rubber trees with ehephon (an ethylene releaser) has been a routine measure to increase latex yield, but the molecular mechanism behind the stimulation of rubber production by ethylene still remains a puzzle. Deciphering the enigma is of great importance for improvement of rubber tree for high yield.ResultsDe novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 h (E8) and 24 h (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified.ConclusionsThe rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2587-4) contains supplementary material, which is available to authorized users.

Transcriptomic analysis reveals numerous diverse protein kinases and transcription factors involved in desiccation tolerance in the resurrection plant Myrothamnus flabellifolia.

The woody resurrection plant Myrothamnus flabellifolia has remarkable tolerance to desiccation. Pyro-sequencing technology permitted us to analyze the transcriptome of M. flabellifolia during both dehydration and rehydration. We identified a total of 8287 and 8542 differentially transcribed genes during dehydration and rehydration treatments respectively. Approximately 295 transcription factors (TFs) and 484 protein kinases (PKs) were up- or down-regulated in response to desiccation stress. Among these, the transcript levels of 53 TFs and 91 PKs increased rapidly and peaked early during dehydration. These regulators transduce signal cascades of molecular pathways, including the up-regulation of ABA-dependent and independent drought stress pathways and the activation of protective mechanisms for coping with oxidative damage. Antioxidant systems are up-regulated, and the photosynthetic system is modified to reduce ROS generation. Secondary metabolism may participate in the desiccation tolerance of M. flabellifolia as indicated by increases in transcript abundance of genes involved in isopentenyl diphosphate biosynthesis. Up-regulation of genes encoding late embryogenesis abundant proteins and sucrose phosphate synthase is also associated with increased tolerance to desiccation. During rehydration, the transcriptome is also enriched in transcripts of genes encoding TFs and PKs, as well as genes involved in photosynthesis, and protein synthesis. The data reported here contribute comprehensive insights into the molecular mechanisms of desiccation tolerance in M. flabellifolia.

Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents.

Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

Feedback inhibition of 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXS) regulates the 2‐C‐methyl‐D‐erythritol 4‐ phosphate (MEP) pathway

The 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway constitutes an important metabolic pathway for the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and other higher isoprenoids. The goal of this study was to understand the regulation of the MEP pathway. It was suggested from previous studies that the first enzyme of the MEP pathway, 1‐deoxy‐d‐xylulose 5‐phosphate synthase (DXS), could have significant regulatory role. In order to test that the DXS enzyme was cloned from Populus trichocarpa and the recombinant protein (PtDXS) was purified from E. coli. An LC‐MS/MS based assay was developed for studying the regulation of PtDXS activity. PtDXS was found to be inhibited by IDP and DMADP. Further studies show that both of these metabolites compete with thiamine diphosphate for binding with the enzyme. Computational analysis shows that IDP and DMADP bind with the enzyme in a manner very similar to the binding of thiamine diphosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway. This work was supported by the US National Science Foundation (Grant IOS‐0950574). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Identification and validation of a novel lead compound targeting 4-diphosphocytidyl-2-C-methylerythritol synthetase (IspD) of mycobacteria

Tuberculosis is a serious threat to world-wide public health usually caused in humans by Mycobacterium tuberculosis (M. tuberculosis). It exclusively utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the precursors of all isoprenoid compounds. The 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD; EC 2.7.7.60) is the key enzyme of the MEP pathway. It is also of interest as a new chemotherapeutic target, as the enzyme is absent in mammals and ispD is an essential gene for growth. A high-throughput screening method was therefore developed to identify compounds that inhibit IspD. This process was applied to identify a lead compound, domiphen bromide (DMB), that may effectively inhibit IspD. The inhibitory action of DMB was confirmed by over-expressing or down-regulating IspD in Mycobacterium smegmatis (M. smegmatis), demonstrating that DMB inhibit M. smegmatis growth additionally through an IspD-independent pathway. This also led to higher levels of growth inhibition when combined with IspD knockdown. This novel IspD inhibitor was also reported to exhibit antimycobacterial activity in vitro, an effect that likely occurs as a result of perturbation of cell wall biosynthesis.

A Whole-Cell Phenotypic Screening Platform for Identifying Methylerythritol Phosphate Pathway-Selective Inhibitors as Novel Antibacterial Agents

Isoprenoid biosynthesis is essential for survival of all living organisms. More than 50,000 unique isoprenoids occur naturally, with each constructed from two simple five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Two pathways for the biosynthesis of IPP and DMAPP are found in nature. Humans exclusively use the mevalonate (MVA) pathway, while most bacteria, including all Gram-negative and many Gram-positive species, use the unrelated methylerythritol phosphate (MEP) pathway. Here we report the development of a novel, whole-cell phenotypic screening platform to identify compounds that selectively inhibit the MEP pathway. Strains of Salmonella enterica serovar Typhimurium were engineered to have separately inducible MEP (native) and MVA (nonnative) pathways. These strains, RMC26 and CT31-7d, were then used to differentiate MVA pathway- and MEP pathway-specific perturbation. Compounds that inhibit MEP pathway-dependent bacterial growth but leave MVA-dependent growth unaffected represent MEP pathway-selective antibacterials. This screening platform offers three significant results. First, the compound is antibacterial and is therefore cell permeant, enabling access to the intracellular target. Second, the compound inhibits one or more MEP pathway enzymes. Third, the MVA pathway is unaffected, suggesting selectivity for targeting the bacterial versus host pathway. The cell lines also display increased sensitivity to two reported MEP pathway-specific inhibitors, further biasing the platform toward inhibitors selective for the MEP pathway. We demonstrate development of a robust, high-throughput screening platform that combines phenotypic and target-based screening that can identify MEP pathway-selective antibacterials simply by monitoring optical density as the readout for cell growth/inhibition.

Construction and analysis of EST libraries of the trans-polyisoprene producing plant, Eucommia ulmoides Oliver

Eucommia ulmoides Oliver is one of a few woody plants capable of producing abundant quantities of trans-polyisoprene rubber in their leaves, barks, and seed coats. One cDNA library each was constructed from its outer stem tissue and inner stem tissue. They comprised a total of 27,752 expressed sequence tags (ESTs) representing 10,520 unigenes made up of 4,302 contigs and 6,218 singletons. Homologues of genes coding for rubber particle membrane proteins that participate in the synthesis of high-molecular poly-isoprene in latex were isolated, as well as those encoding known major latex proteins (MLPs). MLPs extensively shared ESTs, indicating their abundant expression during trans-polyisoprene rubber biosynthesis. The six mevalonate pathway genes which are implicated in the synthesis of isopentenyl diphosphate (IPP), a starting material of poly-isoprene biosynthesis, were isolated, and their role in IPP biosynthesis was confirmed by functional complementation of suitable yeast mutants. Genes encoding five full-length trans-isoprenyl diphosphate synthases were also isolated, and two among those synthesized farnesyl diphosphate from IPP and dimethylallyl diphosphate, an assumed intermediate of rubber biosynthesis. This study should provide a valuable resource for further studies of rubber synthesis in E. ulmoides.

Molecular characterization of the 1‐Deoxy‐D‐xylulose‐5‐ phosphate Synthase (DXS) from marine bacterium, Kocuria gwangalliensis

In addition to the ubiquitous mevalonate pathway, Kocuria gwangalliensis utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. 2‐C‐methyl‐D‐erythritol‐4‐phosphate synthase (MEP synthase) catalyzes the rearrangement of 1‐D‐deoxyxylulose‐ 5‐phosphate (DXP) to methylerythritol‐4‐ phosphate(MEP) as the first pathway‐specific reaction in the MEP biosynthetic pathway. In the present study, the gene coding for 1‐ Deoxy‐D‐xylulose‐5‐phosphate Synthase (DXS) was isolated from the marine bacterium, Kocuria gwangalliensis. The nucleotide sequence of DXS consisted of 2013 bp encoding 671 amino acids residues and turned out to be well conserved during evolution. DXS gene was subcloned into the pColdI expression vector which allows the expression of the recombinant DXS protein with C‐terminal fusion His‐tag and designated to pColdI‐DXS plasmid. The predicted size of recombinant DXS protein is approximately 73 kDa including a C‐terminal His‐Tag fusion. The resulting plasmid was transformed into the E. coli strain BL21(DE3) codon plus cell and the expression of the recombinant DXS protein was induced by the addition of IPTG at a final concentration of 0.1 mM. The expression patterns of recombinant DXS protein were analyzed by 10% SDS‐PAGE.

Genome-wide identification and characterization of novel genes involved in terpenoid biosynthesis in Salvia miltiorrhiza

Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.

Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP.

A plastidial sodium-dependent pyruvate transporter

Pyruvate serves as a metabolic precursor for many plastid-localized biosynthetic pathways, such as those for fatty acids, terpenoids and branched-chain amino acids. In spite of the importance of pyruvate uptake into plastids (organelles within cells of plants and algae), the molecular mechanisms of this uptake have not yet been explored. This is mainly because pyruvate is a relatively small compound that is able to passively permeate lipid bilayers, which precludes accurate measurement of pyruvate transport activity in reconstituted liposomes. Using differential transcriptome analyses of C(3) and C(4) plants of the genera Flaveria and Cleome, here we have identified a novel gene that is abundant in C(4) species, named BASS2 (BILE ACID:SODIUM SYMPORTER FAMILY PROTEIN 2). The BASS2 protein is localized at the chloroplast envelope membrane, and is highly abundant in C(4) plants that have the sodium-dependent pyruvate transporter. Recombinant BASS2 shows sodium-dependent pyruvate uptake activity. Sodium influx is balanced by a sodium:proton antiporter (NHD1), which was mimicked in recombinant Escherichia coli cells expressing both BASS2 and NHD1. Arabidopsis thaliana bass2 mutants lack pyruvate uptake into chloroplasts, which affects plastid-localized isopentenyl diphosphate synthesis, as evidenced by increased sensitivity of such mutants to mevastatin, an inhibitor of cytosolic isopentenyl diphosphate biosynthesis. We thus provide molecular evidence for a sodium-coupled metabolite transporter in plastid envelopes. Orthologues of BASS2 can be detected in all the genomes of land plants that have been characterized so far, thus indicating the widespread importance of sodium-coupled pyruvate import into plastids.