1-deoxy-D-xylulose 5-phosphate Synthase Research Articles

An activity-based probe for antimicrobial target DXP synthase, a thiamin diphosphate-dependent enzyme

This work reports an alkyl acetylphosphonate (alkylAP) activity-based probe (ABP) for 1-deoxy-d-xylulose 5-phosphate synthase DXPS, a promising antimicrobial target. This essential thiamin diphosphate (ThDP)-dependent enzyme operates at a branchpoint in bacterial central metabolism and is believed to play key roles in pathogen adaptation during infection. How different bacterial pathogens harness DXPS activity to adapt and survive within host environments remains incompletely understood, and tools for probing DXPS function in different contexts of infection are lacking. Here, we have developed alkylAP-based ABP 1, designed to react with the ThDP cofactor on active DXPS to form a stable C2α-phosphonolactylThDP adduct which subsequently crosslinks to the DXPS active site upon photoactivation. ABP 1 displays low micromolar potency against DXPS and dose-dependent labeling of DXPS that is blocked by alkylAP-based inhibitors. The probe displays selectivity for DXPS over ThDP-dependent enzymes and is capable of detecting active DXPS in a complex proteome. These studies represent an important advance toward development of tools to probe DXPS function in different contexts of bacterial infection, and for drug discovery efforts on this target.

Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog.

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure-activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

Copper-catalyzed Cyclization Reactions in Pursuit of Molecules That Inhibit DXP synthase

Copper-catalyzed Cyclization Reactions in Pursuit of Molecules That Inhibit DXP synthase

Ruthenium-Catalyzed Cyclization Reactions in Pursuit of Molecules That Inhibit DXP Synthase

Ruthenium-Catalyzed Cyclization Reactions in Pursuit of Molecules That Inhibit DXP Synthase

DXP Synthase Function in a Bacterial Metabolic Adaptation and Implications for Antibacterial Strategies.

Pathogenic bacteria possess a remarkable ability to adapt to fluctuating host environments and cause infection. Disturbing bacterial central metabolism through inhibition of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) has the potential to hinder bacterial adaptation, representing a new antibacterial strategy. DXPS functions at a critical metabolic branchpoint to produce the metabolite DXP, a precursor to pyridoxal-5-phosphate (PLP), thiamin diphosphate (ThDP) and isoprenoids presumed essential for metabolic adaptation in nutrient-limited host environments. However, specific roles of DXPS in bacterial adaptations that rely on vitamins or isoprenoids have not been studied. Here we investigate DXPS function in an adaptation of uropathogenic E. coli (UPEC) to d-serine (d-Ser), a bacteriostatic host metabolite that is present at high concentrations in the urinary tract. UPEC adapt to d-Ser by producing a PLP-dependent deaminase, DsdA, that converts d-Ser to pyruvate, pointing to a role for DXPS-dependent PLP synthesis in this adaptation. Using a DXPS-selective probe, butyl acetylphosphonate (BAP), and leveraging the toxic effects of d-Ser, we reveal a link between DXPS activity and d-Ser catabolism. We find that UPEC are sensitized to d-Ser and produce sustained higher levels of DsdA to catabolize d-Ser in the presence of BAP. In addition, BAP activity in the presence of d-Ser is suppressed by β-alanine, the product of aspartate decarboxylase PanD targeted by d-Ser. This BAP-dependent sensitivity to d-Ser marks a metabolic vulnerability that can be exploited to design combination therapies. As a starting point, we show that combining inhibitors of DXPS and CoA biosynthesis displays synergy against UPEC grown in urine where there is increased dependence on the TCA cycle and gluconeogenesis from amino acids. Thus, this study provides the first evidence for a DXPS-dependent metabolic adaptation in a bacterial pathogen and demonstrates how this might be leveraged for development of antibacterial strategies against clinically relevant pathogens.

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction.

The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.

Computational and Synthetic Biology Approaches for the Biosynthesis of Antiviral and Anticancer Terpenoids from Bacillus subtilis.

Recent advancements in medicinal research have identified several antiviral and anticancer terpenoids that are usually deployed as a source of flavor, fragrances and pharmaceuticals. Under the current COVID-19 pandemic conditions, natural therapeutics with the least side effects are the need of the hour to save the patients, especially, which are pre-affected with other medical complications. Although plants are the major sources of terpenoids; however, for the environmental concerns, the global interest has shifted to the biocatalytic production of molecules from microbial sources. The gram-positive bacterium Bacillus subtilis is a suitable host in this regard due to its GRAS (generally regarded as safe) status, ease in genetic manipulations and wide industrial acceptability. The B. subtilis synthesizes its terpenoid molecules from 1-deoxy-d-xylulose-5-phosphate (DXP) pathway, a common route in almost all microbial strains. Here, we summarize the computational and synthetic biology approaches to improve the production of terpenoid-based therapeutics from B. subtilis by utilizing DXP pathway. We focus on the in-silico approaches for screening the functionally improved enzyme-variants of the two crucial enzymes namely, the DXP synthase (DXS) and Farnesyl Pyrophosphate Synthase (FPPS). The approaches for engineering the active sites are subsequently explained. It will be helpful to construct the functionally improved enzymes for the high-yield production of terpenoid-based anticancer and antiviral metabolites, which would help to reduce the cost and improve the availability of such therapeutics for the humankind.

Negative regulation of plastidial isoprenoid pathway by herbivore-induced β-cyclocitral in Arabidopsis thaliana

Insect damage to plants is known to up-regulate defense and down-regulate growth processes. While there are frequent reports about up-regulation of defense signaling and production of defense metabolites in response to herbivory, much less is understood about the mechanisms by which growth and carbon assimilation are down-regulated. Here we demonstrate that insect herbivory down-regulates the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in Arabidopsis (Arabidopsis thaliana), a pathway making primarily metabolites for use in photosynthesis. Simulated feeding by the generalist herbivore Spodoptera littoralis suppressed flux through the MEP pathway and decreased steady-state levels of the intermediate 1-deoxy-D-xylulose 5-phosphate (DXP). Simulated herbivory also increased reactive oxygen species content which caused the conversion of β-carotene to β-cyclocitral (βCC). This volatile oxidation product affected the MEP pathway by directly inhibiting DXP synthase (DXS), the rate-controlling enzyme of the MEP pathway in Arabidopsis and inducing plant resistance against S. littoralis βCC inhibited both DXS transcript accumulation and DXS activity. Molecular models suggested that βCC binds to DXS at the binding site for the thymine pyrophosphate cofactor and blocks catalysis, which was confirmed by direct assays of βCC with the purified DXS protein in vitro. Another intermediate of the MEP pathway, 2-C-methyl-D-erythritol-2, 4-cyclodiphosphate, which is known to stimulate salicylate defense signaling, showed greater accumulation and enhanced export out of the plastid in response to simulated herbivory. Together, our work implicates βCC as a signal of herbivore damage in Arabidopsis that increases defense and decreases flux through the MEP pathway, a pathway involved in growth and carbon assimilation.

Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase.

The product of 1-deoxy-d-xyluose 5-phosphate (DXP) synthase, DXP, feeds into the bacterial biosynthesis of isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate. DXP is essential for human pathogens but not utilized by humans; thus, DXP synthase is an attractive anti-infective target. The unique ThDP-dependent mechanism and structure of DXP synthase offer ideal opportunities for selective targeting. Upon reaction with pyruvate, DXP synthase uniquely stabilizes the predecarboxylation intermediate, C2α-lactylThDP (LThDP), in a closed conformation. Subsequent binding of d-glyceraldehyde 3-phosphate induces an open conformation that is proposed to destabilize LThDP, triggering decarboxylation. Evidence for the closed and open conformations has been revealed by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography, which indicate that H49 and H299 are involved in conformational dynamics and movement of the fork and spoon motifs away from the active site is important for the closed-to-open transition. Interestingly, H49 and H299 are critical for DXP formation and interact with the predecarboxylation intermediate in the closed conformation. H299 is removed from the active site in the open conformation of the postdecarboxylation state. In this study, we show that substitution at H49 and H299 negatively impacts LThDP formation by shifting the conformational equilibrium of DXP synthase toward an open conformation. We also present a method for monitoring the dynamics of the spoon motif that uncovered a previously undetected role for H49 in coordinating the closed conformation. Overall, our results suggest that H49 and H299 are critical for the closed, predecarboxylation state providing the first direct link between catalysis and conformational dynamics.

Enamide Prodrugs of Acetyl Phosphonate Deoxy-d-xylulose-5-phosphate Synthase Inhibitors as Potent Antibacterial Agents.

To fight the growing threat of antibiotic resistance, new antibiotics are required that target essential bacterial processes other than protein, DNA/RNA, and cell wall synthesis, which constitute the majority of currently used antibiotics. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase is a vital enzyme in bacterial central metabolism, feeding into the de novo synthesis of thiamine diphosphate, pyridoxal phosphate, and essential isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. While potent and selective inhibitors of DXP synthase in vitro activity have been discovered, their antibacterial activity is modest. To improve the antibacterial activity of selective alkyl acetylphosphonate (alkylAP) inhibitors of DXP synthase, we synthesized peptidic enamide prodrugs of alkylAPs inspired by the natural product dehydrophos, a prodrug of methyl acetylphosphonate. This prodrug strategy achieves dramatic increases in activity against Gram-negative pathogens for two alkylAPs, butyl acetylphosphonate and homopropargyl acetylphosphonate, decreasing minimum inhibitory concentrations against Escherichia coli by 33- and nearly 2000-fold, respectively. Antimicrobial studies and LC-MS/MS analysis of alkylAP-treated E. coli establish that the increased potency of prodrugs is due to increased accumulation of alkylAP inhibitors of DXP synthase via transport of the prodrug through the OppA peptide permease and subsequent amide hydrolysis. This work demonstrates the promise of targeting DXP synthase for the development of novel antibacterial agents.

Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate synthase, a central metabolic enzyme in bacteria

The underexploited antibacterial target 1-deoxy-d-xyluose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP). DXP is an essential intermediate in the biosynthesis of ThDP, pyridoxal phosphate, and isoprenoids in many pathogenic bacteria. DXP synthase catalyzes a distinct mechanism in ThDP decarboxylative enzymology in which the first enzyme-bound pre-decarboxylation intermediate, C2α-lactyl-ThDP (LThDP), is stabilized by DXP synthase in the absence of d-GAP, and d-GAP then induces efficient LThDP decarboxylation. Despite the observed LThDP accumulation and lack of evidence for C2α-carbanion formation in the absence of d-GAP, CO2 is released at appreciable levels under these conditions. Here, seeking to resolve these conflicting observations, we show that DXP synthase catalyzes the oxidative decarboxylation of pyruvate under conditions in which LThDP accumulates. O2-dependent LThDP decarboxylation led to one-electron transfer from the C2α-carbanion/enamine to O2, with intermediate ThDP-enamine radical formation, followed by peracetic acid formation en route to acetate. Thus, LThDP formation and decarboxylation and DXP formation were studied under anaerobic conditions. Our results support a model in which O2-dependent LThDP decarboxylation and peracetic acid formation occur in the absence of d-GAP, decreasing the levels of pyruvate and O2 in solution. The relative pyruvate and O2 concentrations then dictate the extent of LThDP accumulation, and its buildup can be observed when [pyruvate] > [O2]. The finding that O2 acts as a structurally distinct trigger of LThDP decarboxylation supports the hypothesis that a mechanism involving small molecule-dependent LThDP decarboxylation equips DXP synthase for diverse, yet uncharacterized cellular functions.

Targeting the Unique Mechanism of Bacterial 1-Deoxy-d-xylulose-5-phosphate Synthase.

The bacterial metabolite 1-deoxy-d-xyulose 5-phosphate (DXP) is essential in bacterial central metabolism feeding into isoprenoid, thiamin diphosphate (ThDP), and pyridoxal phosphate de novo biosynthesis. Halting its production through the inhibition of DXP synthase is an attractive strategy for the development of novel antibiotics. Recent work has revealed that DXP synthase utilizes a unique random sequential mechanism that requires formation of a ternary complex among pyruvate-derived C2α-lactylthiamin diphosphate (LThDP), d-glyceraldehyde 3-phosphate (d-GAP), and enzyme, setting it apart from all other known ThDP-dependent enzymes. Herein, we describe the development of bisubstrate inhibitors bearing an acetylphosphonate (AP) pyruvate mimic and a distal negative charge mimicking the phosphoryl group of d-GAP, designed to target the unique form of DXP synthase that binds LThDP and d-GAP in a ternary complex. A d-phenylalanine-derived triazole acetylphosphonate (d-PheTrAP) emerged as the most potent inhibitor in this series, displaying slow, tight-binding inhibition with a Ki* of 90 ± 10 nM, forward ( k1) and reverse ( k2) isomerization rates of 1.1 and 0.14 min-1, respectively, and exquisite selectivity (>15000-fold) for DXP synthase over mammalian pyruvate dehydrogenase. d-PheTrAP is the most potent, selective DXP synthase inhibitor described to date and represents the first inhibitor class designed specifically to exploit the unique E-LThDP-GAP ternary complex in ThDP enzymology.

Targeting DXP Synthase using TPP Mimics

A global emergence of antibiotic‐resistant bacteria, largely due to the misuse and over use of antibiotics, has led to an increase in demand for the development of new antibacterial agents. In order to develop new antibiotics, researchers need to identify new targets that have high specificity for certain bacterial pathways. The enzyme D‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the MEP (methylerythritolphosphate) pathway in human pathogens, which produces the biological molecules necessary to regulate numerous cellular functions, including central metabolism. Inhibition of DXP synthase would significantly reduce the rate of DXP production and the MEP pathway and thus is an ideal candidate for antibiotic development. DXP synthase requires the cofactor, thiamin pyrophosphate (TPP) for its activity. Preventing TPP from complexing with DXP synthase will inhibit the activity of the enzyme and consequently inhibit the MEP pathway. To test the efficacy of DXP synthase inhibitors both DXP synthase and the sequential enzyme of the MEP pathway, DXP reductoisomerase (IspC), were purified from E.coli using nickel affinity chromatography. Conversion from DXP to 2‐C‐Methyl‐D‐erythritol 4‐phosphate (MEP) by IspC coincides with NADPH oxidation that can be measured using spectroscopic techniques. The establishment of this in vitro system will allow us to test the efficacy novel DXP synthase inhibitors.Support or Funding InformationThis project was funded by SJFC internal funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase.

1-Deoxy-d-xylulose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate. DXP is at a metabolic branch point in bacteria, feeding into the methylerythritol phosphate pathway to indispensable isoprenoids and acting as a precursor for biosynthesis of essential cofactors in central metabolism, pyridoxal phosphate and ThDP, the latter of which is also required for DXP synthase catalysis. DXP synthase follows a unique random sequential mechanism and possesses an unusually large active site. These features have guided the design of sterically demanding alkylacetylphosphonates (alkylAPs) toward the development of selective DXP synthase inhibitors. alkylAPs studied here display selective, low μM inhibitory activity against DXP synthase. They are weak inhibitors of bacterial growth in standard nutrient rich conditions. However, bacteria are significantly sensitized to most alkylAPs in defined minimal growth medium, with minimal inhibitory concentrations (MICs) ranging from low μM to low mM and influenced by alkyl-chain length. The longest analog (C8) displays the weakest antimicrobial activity and is a substrate for efflux via AcrAB-TolC. The dependence of inhibitor potency on growth environment emphasizes the need for antimicrobial screening conditions that are relevant to the in vivo microbial microenvironment during infection. DXP synthase expression and thiamin supplementation studies offer support for DXP synthase as an intracellular target for some alkylAPs and reveal both the challenges and intriguing aspects of these approaches to study target engagement.

Evaluating the activity of bacterial enzyme DXP synthase a potential target for newer antibiotics

An emerging medical crisis involving the development of antibiotic resistant bacteria in the human body is compromising treatment of infections, leaving patients in critical condition. Infections that are becoming unresponsive to current treatment are creating a need for new antibiotics. In order to develop new antibiotics, researchers need to identify new targets that have high specificity for certain bacterial pathways. The enzyme D‐xylulose 5‐phosphate (DXP) synthase catalyzes the first step in the MEP (methylerythritol phosphate) pathway in human pathogens, which produces the biological molecules necessary to regulate numerous cellular functions, including central metabolism. Inhibition of DXP synthase would prevent or heavily reduce the rate of DXP production, which can then prevent or delay the continuation of the MEP pathway and thus is an ideal candidate for antibiotic development. In order to test the effectiveness of potential inhibitors of DXP synthase it is first necessary to establish a system to monitor the activity of DXP. We have purified Histidine tagged versions of DXP synthase and 1‐Deoxy‐D‐Xylulose 5‐Phosphate Reductoisomerase (IspC). The activity of DXP synthase cannot be directly measured using spectroscopic techniques, however it can be coupled to the activity of IspC, which in the process of converting DXP into MEP oxidizes NADPH. The establishment of this in vitro system will enable us to test the efficacy of novel inhibitors of DXP that are currently being developed.Support or Funding InformationSupported by internal grants from St. John Fisher College School of the Arts and Wegmans school of Pharmacy

Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.

Isolation and expression analyses of methyl-d-erythritol 4-phosphate (MEP) pathway genes from Haematococcus pluvialis

Haematococcus pluvialis is a green alga known to accumulate the keto-carotenoid astaxanthin under stress conditions. In H. pluvialis, carotenoids are derived from isopentenyl diphosphate (IPP), which is synthesized via the non-mevalonate methyl-d-erythritol 4-phosphate (MEP) pathway. The present study revealed that several treatments caused changes in pigment profiles and the expression levels of genes encoding MEP pathway enzymes. Additionally, photosynthesis fluorescence was monitored. Generally, under stress conditions, there was an increase in astaxanthin, along with a decrease in total chlorophyll and photo capacity. Six IPP biosynthetic genes were cloned from H. pluvialis. Expression analysis revealed that these transcripts were upregulated under stress culture conditions. However, the extent of MEP pathway gene expression varied with the stress conditions. 4-Diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) synthase (CMS) and CDP-ME kinase (CMK) exhibited significantly higher transcriptional expression under nitrogen starvation treatments. While 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS), CMS, CMK 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) synthase (HDS), and HMBPP reductoisomerase (HDR) showed significant upregulation on the second day under nitrogen starvation and high light (HL-N). The enhanced expression of these genes was also observed on the third day under high light. The high expression of MEP pathway genes was correlated with the accumulation of astaxanthin under HL-N stress. This is the first report of the isolation of IPP biosynthetic genes and their differential expression in H. pluvialis under stress conditions. The present study revealed the influence of stress conditions on the expression of MEP pathway genes and changes in pigment profiles.

Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine

Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5′-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5′-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28–34mg/l PN from 4-HO-Thr while the wild-type parent produced only 12mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.

A Specific Process to Purify 2-Methyl-D-Erythritol-4-Phosphate Enzymatically Converted from D-Glyceraldehyde-3-Phosphate and Pyruvate

A one-pot enzymatic cascade was established to synthesize MEP, one of the key intermediates in the MEP terpenoid biosynthetic pathway. D-GAP and sodium pyruvate were converted to MEP in a reaction catalyzed by DXP synthase and DXP reductoisomerase (DXR) in the presence of the coenzymes ThPP, NADPH, and Mg2+. The product was then isolated by using a specific two-step purification process and MEP was obtained in a yield of nearly 60% and high purity. Importantly, MEP prepared by this way was totally free from contamination by minor amounts of DXP that was not completely convertible by DXR.

Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender

Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.