The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

Conferring thermotolerant phenotype to wild-type Yarrowia lipolytica improves cell growth and erythritol production.

In microbial engineering, heat stress is an important environmental factor modulating cell growth, metabolic flux distribution and the synthesis of target products. Yarrowia lipolytica, as a GARS (generally recognized as safe) nonconventional yeast, has been widely used in the food industry, especially as the host of erythritol production. Biomanufacturing economics is limited by the high operational cost of cooling energy in large-scale fermentation. It is of great significance to select thermotolerant Y. lipolytica to reduce the cooling cost and elucidate the heat-resistant mechanism at molecular level. For this purpose, we performed adaptive evolution and obtained a thermotolerant strain named Y. lipolytica BBE-18. Transcriptome analysis allows us to identify four genes in thiamine metabolism pathway that are responsible for the complicated thermotolerant phenotype. The heat-resistant phenotype was validated with the model strain Y. lipolytica Po1f by overexpression of single and combined genes. Then, conferring the thermotolerant phenotype to the wild-type Y. lipolytica BBE-17 enable the strain to produce three-times more erythritol of the control strain with 3°C higher than optimal cultivation temperature. To our knowledge, this is the first report on engineering heat-resistant phenotype to improve the erythritol production in Y. lipolytica. However, due to the increase of culture temperature, a large amount of adenosine triphosphate is consumed to ensure the life activities of Y. lipolytica which limits the potential of cell synthetic products to a certain extent. Even so, this study provides a reference for Y. lipolytica to produce other products under high temperature.

Combinatorial Methylerythritol Phosphate Pathway Engineering and Process Optimization for Increased Menaquinone-7 Synthesis in Bacillus subtilis.

Vitamin K2 (menaquinone) is an essential vitamin existing in the daily diet, and menaquinone-7 (MK-7) is an important form of it. In a recent work, we engineered the synthesis modules of MK-7 in Bacillus subtilis, and the strain BS20 could produce 360 mg/l MK-7 in shake flasks, while the methylerythritol phosphate (MEP) pathway, which provides the precursor isopentenyl diphosphate for MK-7 synthesis, was not engineered. In this study, we overexpressed five genes of the MEP pathway in BS20 and finally obtained a strain (BS20DFHG) with MK-7 titer of 415 mg/l in shake flasks. Next, we optimized the fermentation process parameters (initial pH, temperature and aeration) in an 8-unit parallel bioreactor system consisting of 300-ml glass vessels. Based on this, we scaled up the MK-7 production by the strain BS20DFHG in a 50-l bioreactor, and the highest MK-7 titer reached 242 mg/l. Here, we show that the engineered strain BS20DFHG may be used for the industrial production of MK-7 in the future.

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds.

BACKGROUND: Deoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated. RESULTS: The accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn't in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a 'Three Faucets and Cisterns Model' about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a "Faucet", the biosynthetic capacity of intermediate metabolites as a "Cistern", and the carotenoid accumulations as the content of "Cistern". CONCLUSION: Our study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the "Three Faucets and Cisterns model" presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

The plastidial metabolite 2-C-methyl-D-erythritol-2,4-cyclodiphosphate modulates defence responses against aphids.

Feeding by insect herbivores such as caterpillars and aphids induces plant resistance mechanisms that are mediated by the phytohormones jasmonic acid (JA) and salicylic acid (SA). These phytohormonal pathways often crosstalk. Besides phytohormones, methyl-D-erythriol-2,4-cyclodiphosphate (MEcPP), the penultimate metabolite in the methyl-D-erythritol-4-phosphate pathway, has been speculated to regulate transcription of nuclear genes in response to biotic stressors such as aphids. Here, we show that MEcPP uniquely enhances the SA pathway without attenuating the JA pathway. Arabidopsis mutant plants that accumulate high levels of MEcPP (hds3) are highly resistant to the cabbage aphid (Brevicoryne brassicae), whereas resistance to the large cabbage white caterpillar (Pieris brassicae) remains unaltered. Thus, MEcPP is a distinct signalling molecule that acts beyond phytohormonal crosstalk to induce resistance against the cabbage aphid in Arabidopsis. We dissect the molecular mechanisms of MEcPP mediating plant resistance against the aphid B. brassicae. This shows that MEcPP induces the expression of genes encoding enzymes involved in the biosynthesis of several primary and secondary metabolic pathways contributing to enhanced resistance against this aphid species. A unique ability to regulate multifaceted molecular mechanisms makes MEcPP an attractive target for metabolic engineering in Brassica crop plants to increase resistance to cabbage aphids.

Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production.

Erythritol production is a unique response to hyperosmotic stress that is observed in a small group of yeasts, including Yarrowia lipolytica. This study investigated whether this unusual mechanism is regulated by the HOG pathway, well described in Saccharomyces cerevisiae. The gene YALI0E25135g was identified as the Y. lipolytica homologue of HOG1 and was found to be phosphorylated in response to hyperosmotic shock. Deletion of the gene caused a significant decrease in resistance to hyperosmotic stress and negatively affected erythritol production. Interestingly, the deletion strain yl-hog1Δ displayed significant morphological defects, with the cells growing in a filamentous form. Moreover, yl-hog1Δ cells were also resistant to the cell wall damaging agents Congo red and calcofluor white. Collectively, these results indicate that yl-Hog1 is crucial for the cellular response to hyperosmotic stress, plays a role in the induction of erythritol production, and potentially prevents cross-talk with different MAPK signalling pathways in the cell.

Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors.

Plant abietane diterpenoids (e.g. aethiopinone, 1- oxoaethiopinone, salvipisone and ferruginol), synthesized in the roots of several Salvia spp, have antibacterial, antifungal, sedative and anti-proliferative properties. Recently we have reported that content of these compounds in S. sclarea hairy roots is strongly depending on transcriptional regulation of genes belonging to the plastidial MEP-dependent terpenoid pathway, from which they mostly derive. To boost the synthesis of this interesting class of compounds, heterologous AtWRKY18, AtWRKY40, and AtMYC2 TFs were overexpressed in S. sclarea hairy roots and proved to regulate in a coordinated manner the expression of several genes encoding enzymes of the MEP-dependent pathway, especially DXS, DXR, GGPPS and CPPS. The content of total abietane diterpenes was enhanced in all overexpressing lines, although in a variable manner due to a negative pleiotropic effect on HR growth. Interestingly, in the best performing HR lines overexpressing the AtWRKY40 TF induced a significant 4-fold increase in the final yield of aethiopinone, for which we have reported an interesting anti-proliferative activity against resistant melanoma cells. The present results are also informative and instrumental to enhance the synthesis of abietane diterpenes derived from the plastidial MEP-derived terpenoid pathway in other Salvia species.

Erythritol production by yeasts: a snapshot of current knowledge.

Erythritol is a four-carbon sugar alcohol produced by microorganisms as an osmoprotectant. It could be used as a natural sweetener in the pharmaceutical and food industries. Here, a snapshot of current knowledge on erythritol metabolism and synthesis, optimization of its production and more precise process and producer strain improvement is presented.

Genetic diversity and structural analysis of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) from Plasmodium falciparum.

BACKGROUND & OBJECTIVES: Plasmodium parasite harbours unique methylerythritol phosphate (MEP) pathway which is obligatory for the biosynthesis of isoprenoids. In malaria parasites, the isoprenoids are indispensable during hepatic, erythrocytic and gametocytic stages. Owing to the criticality of MEP pathway and the potential of its enzymes to act as antimalarial drug target, this study comprehensively investigated the genetic diversity and structural composition of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), fourth enzyme of MEP pathway in Indian Plasmodium falciparum (PfIspE). METHODS: The study employed sequencing, modeling and bioinformatics approaches to examine the genetic diversity and associated structural polymorphism in the PfIspE gene amplified from the clinical blood samples collected from seven malaria endemic geographical regions of India. RESULTS: The sequence analysis showed that PfIspE gene is highly conserved with 100% sequence identity among all the P. falciparum Indian isolates as well as with the PfIspE gene of reference strain 3D7. Phylogenetic analysis suggested that PfIspE is highly evolved and differ sufficiently from human orthologue mevalonate kinase gene. Structural modeling studies revealed that PfIspE has conserved ATP and CDPME-binding domains. The active site was observed to be relatively rigid in architecture with >60% ß-pleated sheets. INTERPRETATION & CONCLUSION: The results of genetic, phylogeny and modeling studies strengthen the potential of PfIspE enzyme as a promising antimalarial drug target.

Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering.

Erythritol (1,2,3,4-butanetetrol) is a four-carbon sugar alcohol with sweetening properties that is used by the agrofood industry as a food additive. In this study, we demonstrated that metabolic engineering can be used to improve the production of erythritol from glycerol in the yeast Yarrowia lipolytica. The best results were obtained using a mutant that overexpressed GUT1 and TKL1, which encode a glycerol kinase and a transketolase, respectively, and in which EYK1, which encodes erythrulose kinase, was disrupted; the latter enzyme is involved in an early step of erythritol catabolism. In this strain, erythritol productivity was 75% higher than in the wild type; furthermore, the culturing time needed to achieve maximum concentration was reduced by 40%. An additional advantage is that the strain was unable to consume the erythritol it had created, further increasing the process's efficiency. The erythritol productivity values we obtained here are among the highest reported thus far.

Enhanced performance of the methylerythritol phosphate pathway by manipulation of redox reactions relevant to IspC, IspG, and IspH.

The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway.

Description of 'Candidatus Marispirochaeta associata' and reclassification of Spirochaeta bajacaliforniensis, Spirochaeta smaragdinae and Spirochaeta sinaica to a new genus Sediminispirochaeta gen. nov. as Sediminispirochaeta bajacaliforniensis comb. nov., Sediminispirochaeta smaragdinae comb. nov. and Sediminispirochaeta sinaica comb. nov.

Strain JC231 was isolated from a coastal saline habitat of Gujarat and was identified based on 16S rRNA gene sequence analysis as a member belonging to the genus Spirochaeta and showed highest sequence similarity (<91 %) with Spirochaeta bajacaliforniensis DSM 16054T and other members of the family Spirochaetaceae. Intensive attempts to culture strain JC231 in pure culture have failed and were associated with only one species of a Desulfovibrio. However, presence of fosmidomycin inhibited the growth of Desulfovibrio sp. and strain JC231 was characterized in its presence. Strain JC231 was an obligate anaerobe, helical shaped and Gram-stain-negative with catalase and oxidase negative. Draft genome sequence analysis of strain JC231 indicated the full complement of genes for both 2-C-methyl-d-erythritol 4-phosphate and 3-hydroxy-3-methylglutaryl-CoA pathways of terpenogenesis. C14 : 0, iso-C15 : 0, C16 : 0, iso-C15 : 1H/C13 : 0 3OH and iso-C14 : 0 are the major (>5 %) fatty acids. Strain JC231 contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and six unidentified lipids (L1-L6). G+C content of strain JC231 was 55.7 mol%. Distinct morphological, physiological and genotypic differences from the previously described taxa support the classification of strain JC231 as a representative of a new genus and species in the family Spirochaetaceae, for which the name 'CandidatusMarispirochaeta associata' is proposed. Strain JC231 is deposited as a defined co-culture with Desulfovibrio sp. JC271 to DSMZ (DSM 29857) and KCTC (KCTC 15472). Based on phenotypic, genotypic and phylogenetic analyses, we also propose the reclassification of Spirochaeta bajacaliforniensis as Sediminispirochaeta bajacaliforniensis gen. nov., comb. nov., Spirochaeta smaragdinae as Sediminispirochaeta smaragdinae comb. nov. and Spirochaeta sinaica as Sediminispirochaeta sinaica comb. nov.

Biosynthesis of ß-carotene in engineered E. coli using the MEP and MVA pathways.

BACKGROUND: ß-carotene is a carotenoid compound that has been widely used not only in the industrial production of pharmaceuticals but also as nutraceuticals, animal feed additives, functional cosmetics, and food colorants. Currently, more than 90% of commercial ß-carotene is produced by chemical synthesis. Due to the growing public concern over food safety, the use of chemically synthesized ß-carotene as food additives or functional cosmetic agents has been severely controlled in recent years. This has reignited the enthusiasm for seeking natural ß-carotene in large-scale fermentative production by microorganisms. RESULTS: To increase ß-carotene production by improving the isopentenyl pyrophosphate (IPP) and geranyl diphospate (GPP) concentration in the cell, the optimized MEP (methylerythritol 4-phosphate) pathway containing 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl pyrophosphate isomerase (FNI) from Bacillus subtilis, geranyl diphosphate synthase (GPPS2) from Abies grandis have been co-expressed in an engineered E. coli strain. To further enhance the production of ß-carotene, the hybrid MVA (mevalonate) pathway has been introduced into an engineered E. coli strain, co-expressed with the optimized MEP pathway and GPPS2. The final genetically modified strain, YJM49, can accumulate 122.4±6.2 mg/L ß-carotene in flask culture, approximately 113-fold and 1.7 times greater than strain YJM39, which carries the native MEP pathway, and YJM45, which harbors the MVA pathway and the native MEP pathway, respectively. Subsequently, the fermentation process was optimized to enhance ß-carotene production with a maximum titer of 256.8±10.4 mg/L. Finally, the fed-batch fermentation of ß-carotene was evaluated using the optimized culture conditions. After induction for 56 h, the final engineered strain YJM49 accumulated 3.2 g/L ß-carotene with a volumetric productivity of 0.37 mg/(L · h · OD600) in aerobic fed-batch fermentation, and the conversion efficiency of glycerol to ß-carotene (gram to gram) reached 2.76%. CONCLUSIONS: In this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of ß-carotene was successfully assembled in E. coli BL21(DE3) with the optimized MEP (methylerythritol 4-phosphate) pathway, the gene for GPPS2 from Abies grandis, the hybrid MVA (mevalonate) pathway and ß-carotene synthesis genes from Erwinia herbicola.

Engineering of cyanobacteria for the photosynthetic production of limonene from CO2.

Isoprenoids, major secondary metabolites in many organisms, are utilized in various applications. We constructed a model photosynthetic production system for limonene, a volatile isoprenoid, using a unicellular cyanobacterium that expresses the plant limonene synthase. This system produces limonene photosynthetically at a nearly constant rate and that can be efficiently recovered using a gas-stripping method. This production does not affect the growth of the cyanobacteria and is markedly enhanced by overexpression of three enzymes in the intrinsic pathway to provide the precursor of limonene, geranyl pyrophosphate. The photosynthetic production of limonene in our system is more or less sustained from the linear to stationary phase of cyanobacterial growth for up to 1 month.

Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.

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.

Combined phosphate and nitrogen limitation generates a nutrient stress transcriptome favorable for arbuscular mycorrhizal symbiosis in Medicago truncatula.

Arbuscular mycorrhizal (AM) symbiosis is stimulated by phosphorus (P) limitation and contributes to P and nitrogen (N) acquisition. However, the effects of combined P and N limitation on AM formation are largely unknown. Medicago truncatula plants were cultivated in the presence or absence of Rhizophagus irregularis (formerly Glomus intraradices) in P-limited (LP), N-limited (LN) or combined P- and N-limited (LPN) conditions, and compared with plants grown in sufficient P and N. The highest AM formation was observed in LPN, linked to systemic signaling by the plant nutrient status. Plant free phosphate concentrations were higher in LPN than in LP, as a result of cross-talk between P and N. Transcriptome analyses suggest that LPN induces the activation of NADPH oxidases in roots, concomitant with an altered profile of plant defense genes and a coordinate increase in the expression of genes involved in the methylerythritol phosphate and isoprenoid-derived pathways, including strigolactone synthesis genes. Taken together, these results suggest that low P and N fertilization systemically induces a physiological state of plants favorable for AM symbiosis despite their higher P status. Our findings highlight the importance of the plant nutrient status in controlling plant-fungus interaction.

Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera.

Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-D-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-D-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon-intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.

Cloning and functional characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the methylerythritol phosphate pathway for isoprenoid biosynthesis.

BACKGROUND: The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans. RESULTS: This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli. GENERAL SIGNIFICANCE: Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities. CONCLUSION: The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.

Analysis of the isoprenoid biosynthesis pathways in Listeria monocytogenes reveals a role for the alternative 2-C-methyl-D-erythritol 4-phosphate pathway in murine infection.

Most bacteria synthesize isoprenoids through one of two essential pathways which provide the basic building block, isopentyl diphosphate (IPP): either the classical mevalonate pathway or the alternative non-mevalonate 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. However, postgenomic analyses of the Listeria monocytogenes genome revealed that this pathogen possesses the genetic capacity to produce the complete set of enzymes involved in both pathways. The nonpathogenic species Listeria innocua naturally lacks the last two genes (gcpE and lytB) of the MEP pathway, and bioinformatic analyses strongly suggest that the genes have been lost through evolution. In the present study we show that heterologous expression of gcpE and lytB in L. innocua can functionally restore the MEP pathway in this organism and confer on it the ability to induce Vgamma9 Vdelta2 T cells. We have previously confirmed that both pathways are functional in L. monocytogenes and can provide sufficient IPP for normal growth in laboratory media (M. Begley, C. G. Gahan, A. K. Kollas, M. Hintz, C. Hill, H. Jomaa, and M. Eberl, FEBS Lett. 561:99-104, 2004). Here we describe a targeted mutagenesis strategy to create a double pathway mutant in L. monocytogenes which cannot grow in the absence of exogenously provided mevalonate, confirming the requirement for at least one intact pathway for growth. In addition, murine studies revealed that mutants lacking the MEP pathway were impaired in virulence relative to the parent strain during intraperitoneal infection, while mutants lacking the classical mevalonate pathway were not impaired in virulence potential. In vivo bioluminescence imaging also confirmed in vivo expression of the gcpE gene (MEP pathway) during murine infection.