Arginine increases genotoxicity induced by methyl methanesulfonate in human lymphocytes

Nitric oxide (NO) is a free radical that is produced in cells from l-arginine. NO is involved in the physiological control of different tissues, but it can act as a toxic mediator in the cells. In this study we investigated the effect of l-arginine on the genotoxicity induced by methyl methanesulfonate (MMS) in human lymphocytes. Blood was treated with N^G-nitro-l-arginine methyl ester (l-NAME) as an inhibitor of nitric oxide synthase for finding out the role of NO in this effect. Human whole blood was treated with l-arginine (50, 100 and 250 μM) and/or l-NAME, then it was treated in vitro with MMS after 24 h of culture. The lymphocytes were stimulated by phytohemagglutinin to find out the micronuclei in cytokinesis-blocked binucleated cells. DNA fragmentation of lymphocytes was detected by using a fluorescence microscope after propidium iodide staining. These data showed that arginine increased the frequency of MMS-induced micronuclei in lymphocytes. However, the genotoxicity was decreased by using l-NAME. Arginine and l-NAME have not shown any DNA damage in cultured human lymphocytes. In conclusion, addition of l-arginine to MMS as an alkylating agent caused an increase of DNA damage in human lymphocytes. This enhancement of genotoxicity was reduced by NAME as NO inhibitor. It is thus cleared that an increase of DNA damage by arginine and MMS is related to NO production.     

Gene Expression of Lupeol Synthase and Biosynthesis of Nitric Oxide in Cell Suspension Cultures of Betula platyphylla in Response to a Phomopsis Elicitor

The current work aimed to characterize the generation of nitric oxide (NO) and gene expression of lupeol synthase (LUS) in Betula platyphylla cells exposed to a Phomopsis elicitor. The effects of nitrate reductase (NR) and NO synthase (NOS), the two key enzymes responsible for endogenous NO biosynthesis in plants, were also investigated. NO production in B. platyphylla cell cultures exhibited a biphasic pattern, reaching the Wrst plateau within 1.0–10 h of exposure to the Phomopsis elicitor. LUS gene expression was found to increase abruptly 10 h after Phomopsis induction, reaching its highest level (18.08) at 24 h. The maximum levels of NOS and NR activities in elicitor-treated cells were found to be 1.7-fold and 6.9-fold those of untreated cells, respectively. Pharmacological experiments showed that Phomopsis elicitor-induced NO production and LUS gene expression level were significantly suppressed by the NOS inhibitor N^G-nitro-l-Arg methyl ester (l-NAME), the NR inhibitor sodium azide (NaN₃), and the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). NaNO₂ and l-arginine (the substrates that produce NO via NR and NOS) and NO donor sodium nitroprusside (SNP) were found to increase both NO production and LUS gene expression. These results suggest that the increase in LUS gene expression due to fungal elicitor-induced NO may involve the NR and NOS biosynthetic pathways.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

The effects of cyclooxygenase and nitric oxide synthase inhibition on oxidative stress in isolated rat heart

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas still exist about potential impact of these drugs on cardiovascular system. The present study was aimed to estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA], and SC-560) on oxidative stress in isolated rat heart, with special focus on l-arginine/NO system. The hearts of male Wistar albino rats (total number n = 96, each group 12 rats, 8 weeks old, body mass 180–200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40–120 cmH₂O). After control experiments the hearts were perfused with the following drugs: 100 μmol/l ASA (Aspirin), alone or in combination with 30 μmol/l l-NAME, 0.3 μmol/l meloxicam (movalis) with or without 30 μmol/l l-NAME, 3 μmol/l meloxicam (alone or in combination with 30 μmol/l l-NAME), 30 μmol/l l-NAME, and administration of 0.25 μmol/l SC-560. In samples of coronary venous effluent the following oxidative stress markers were measured spectrophotometrically: index of lipid peroxidation (measured as thiobarbituric acid reactive substances [TBARS]), superoxide anion radical release (O₂ ^?), and hydrogen peroxide (H₂O₂). While ASA was found to have an adverse influence on redox balance in coronary circulation, and coronary perfusion, meloxicam and SC-560 do not negatively affect the intact model of the heart. Furthermore, all effects were modulated by NOS inhibition. It seems that interaction between COX and l-arginine/NO system truly exists in coronary circulation, and can be one of the possible causes for achieved effects. That means: those effects induced by different inhibitors of COX are modulated by subsequent inhibition of NOS.     

The role of l-arginine-nitric oxide pathway in bacterial translocation

This study investigated the nitric oxide (NO) role as a mediator of arginine on bacterial translocation (BT) and gut damage in mice after intestinal obstruction (IO). The effects of pretreatment with arginine with or without NO inhibition on the systemic and local immunological response were also assessed. Mice were categorized into four groups. Group ARG received chow containing 2 % arginine, while group ARG + l-NAME received the same diet plus l-NAME (N-nitro-l-arginine methyl ester) by gavage. The IO and Sham groups were fed standard chow. After 7 days, animals were gavaged with radiolabeled Escherichia coli, anesthetized and subjected to IO, except the Sham group. Animals were euthanized after 18 h, and BT was evaluated in the mesenteric lymph nodes, blood, liver, spleen and lungs. In another experiment, the intestinal injury was assessed regarding intestinal permeability and ileum histological analyses. Intestinal secretory immunoglobulin A (sIgA) levels, serum IFN-γ and IL-10 cytokines were assessed. Arginine reduced BT, but NO inhibition enhanced BT compared with the ARG group (p < 0.05). Intestinal permeability in the ARG and ARG + l-NAME groups was similar but decreased when compared with the IO group (p < 0.05). Histological preservation was observed. Arginine treatment increased IL-10 and sIgA levels when compared with the Sham and IO groups (p < 0.05). The cytokines and sIgA concentrations were similar in the ARG + l-NAME and Sham groups. Arginine appeared to reduce BT and its effects on the modulation of cytokines and secretory IgA in mice after IO are mediated by NO production.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48 h) PASMC were stimulated with IL-1β alone or with NO donor, l-arginine and/or NO synthase inhibitor l-NAME for 4 and 24 h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24 h. VEGF release was inhibited by l-NAME (531.8 ± 52 pg/ml), but restored and further elevated by l-arginine (1,529 ± 287 pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by l-NAME (1.1-fold) and augmented by l-arginine (3.8-fold) at 4 h. Restoration of a NO pathway by l-arginine in l-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [³H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.     

Different vasoactive effects of chronic endothelial and neuronal NO-synthase inhibition in young Wistar rats

While the unequivocal pattern of endothelial nitric oxide synthase (eNOS) inhibition in cardiovascular control is recognized, the role of NO produced by neuronal NOS (nNOS) remains unclear. The aim of this study was to compare the effects of chronic treatment with 7-nitroindazole (7-NI, nNOS inhibitor) and N^G-nitro-l-arginine methylester (l-NAME, general and predominantly eNOS inhibitor) on cardiovascular system of young normotensive rats. Wistar rats (4 weeks old) were used: controls and rats administered either 7-NI (10 mg/kg bw/day) or l-NAME (50 mg/kg bw/day) in drinking water for 6 weeks. The systolic blood pressure (sBP) was measured by plethysmographic method, and the vasoactivity of isolated arteries was recorded. 7-NI-treatment did not affect sBP; however, the sBP was increased after l-NAME-treatment. l-NAME inhibited acetylcholine-induced relaxation of thoracic aorta (TA), whereas it remained unchanged after 7-NI-treatment. The response of TA to sodium nitroprusside was increased in both experimental groups. The expression of eNOS and nNOS in TA was unchanged in both experimental groups, whereas the activity of NOS was decreased in l-NAME-treated group. Noradrenaline- and angiotensin II-induced contractions of TA were reduced in l-NAME-treated group; however, the contractions remained unchanged in 7-NI-treated group. In all groups, the endogenous angiotensin II participated in adrenergic contraction of TA; this contribution was significantly increased in l-NAME-treated group. Neurogenic contractions in mesenteric artery (MA) remained unchanged after 7-NI-treatment, but increased after l-NAME-treatment. Results show that NO deficiency induced by administration of 7-NI and l-NAME had different cardiovascular effects: eNOS and nNOS triggered distinct signaling pathways in young normotensive rats.     

l-Proline uptake in Saccharomyces cerevisiae mitochondria can contribute to bioenergetics during nutrient stress as alternative mitochondrial fuel

l-Proline (pyrrolidine-2-carboxylic acid) is a distinctive metabolite both biochemically and biotechnologically and is currently recognized to have a cardinal role in gene expression and cellular signaling pathways in stress response. Proline-fueled mitochondrial metabolism involves the oxidative conversion of l-Proline to l-Glutamate in two enzymatic steps by means of Put1p and Put2p that help Saccharomyces cerevisiae to respond to changes in the nutritional environment by initiating the breakdown of l-Proline as a source for nitrogen, carbon, and energy. Compartmentalization of l-Proline catabolic pathway implies that extensive l-Proline transport must take place between the cytosol where its biogenesis via Pro1p, Pro2p, Pro3p occurs and mitochondria. l-Proline uptake in S. cerevisiae purified and active mitochondria was investigated by swelling experiments, oxygen uptake and fluorimetric measurement of a membrane potential generation (ΔΨ). Our results strongly suggest that l-Proline uptake occurs via a carried-mediated process as demonstrated by saturation kinetics and experiments with N-ethylmaleimide, a pharmacological compound that is a cysteine-modifying reagent in hydrophobic protein domains and that inhibited mitochondrial transport. Plasticity of S. cerevisiae cell biochemistry according to background fluctuations is an important factor of adaptation to stress. Thus l-Proline → Glutamate route feeds Krebs cycle providing energy and anaplerotic carbon for yeast survival.     

Possible Involvement of PPAR-γ in the Anticonvulsant Effect of Aegle marmelos (L.) Correa

Aegle marmelos is well documented for antihyperglycemic effect and PPAR-γ activation has been suggested to be the molecular mechanism of its action. Also, the plant has been used in Ayurveda as a brain tonic and has been postulated to have antidepressant activities. The present study was designed to investigate the anticonvulsant effects of A. marmelos leaf extract (AME) in pentylenetetrazole and maximal electroshock induced convulsions; involvement of PPAR-γ, nitric oxide pathway and effect of chronic AME treatment on post-ictal depression. AME was administered at doses of 50, 100 and 200 mg kg^?1 in PTZ and MES model. Severity of convulsions was noted in both the models. Pretreatment with bisphenol A diglycidyl ether (BADGE) was used to study the involvement of PPAR-γ and l-arginine and N-nitro-l-arginine methyl ester hydrochloride (l-NAME) to study the involvement of nitric oxide (NO). Chronic treatment with AME interspersed with sub maximal doses of PTZ (50 mg kg^?1) on every fifth day up to 15 days was given to study post-ictal depression using forced swimming and actophotometer. AME showed significant increase in the onset time and decrease in the duration of convulsions in PTZ and MES models dose dependently. In MES a dose of 100 mg kg^?1 had effect comparable to phenytoin. Pretreatment with BADGE and l-arginine reversed the protective effect while l-NAME did not alter the protective effect, thereby indicating possible involvement of PPAR-γ and inhibition of NO. Chronic AME treatment ameliorated the post-seizure depression significantly as evidenced by increase in the locomotor activity and decrease in the immobility time.     

Enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different Corynebacterium glutamicum

During l-glutamate production, phosphoenolpyruvate carboxylase and pyruvate carboxylase (PCx) play important roles in supplying oxaloacetate to the tricarboxylic acid cycle. To explore the significance of PCx for l-glutamate overproduction, the pyc gene encoding PCx was amplified in Corynebacterium glutamicum GDK-9 triggered by biotin limitation and CN1021 triggered by a temperature shock, respectively. In the fed-batch cultures, GDK-9pXMJ19pyc exhibited 7.4 % lower l-alanine excretion and no improved l-glutamate production. In contrast, CN1021pXMJ19pyc finally exhibited 13 % lower l-alanine excretion and identical l-glutamate production, however, 8.5 % higher l-glutamate production was detected during a short period of the fermentation. It was indicated that pyc overexpression in l-glutamate producer strains, especially CN1021, increased the supply of oxaloacetate for l-glutamate synthesis and decreased byproduct excretion at the pyruvate node.     

Bacillus Anthracis Endospores Regulate Ornithine Decarboxylase and Inducible Nitric Oxide Synthase Through ERK1/2 and p38 Mitogen-Activated Protein Kinases

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (^?NO) from inducible nitric oxide synthase (NOS2) by competing for l-arginine, producing l-ornithine at the expense of ^?NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of l-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores’ germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.     

Nitric oxide regulates tissue transglutaminase localization and function in the vasculature

The multifunctional enzyme tissue transglutaminase (TG2) contributes to the development and progression of several cardiovascular diseases. Extracellular rather than intracellular TG2 is enzymatically active, however, the mechanism by which it is exported out of the cell remains unknown. Nitric oxide (NO) is shown to constrain TG2 externalization in endothelial and fibroblast cells. Here, we examined the role of both exogenous and endogenous (endothelial cell-derived) NO in regulating TG2 localization in vascular cells and tissue. NO synthase inhibition in endothelial cells (ECs) using N-nitro l-arginine methyl ester (l-NAME) led to a time-dependent decrease in S-nitrosation and increase in externalization of TG2. Laminar shear stress led to decreased extracellular TG2 in ECs. S-nitrosoglutathione treatment led to decreased activity and externalization of TG2 in human aortic smooth muscle and fibroblast (IMR90) cells. Co-culture of these cells with ECs resulted in increased S-nitrosation and decreased externalization and activity of TG2, which was reversed by l-NAME. Aged Fischer 344 rats had higher tissue scaffold-associated TG2 compared to young. NO regulates intracellular versus extracellular TG2 localization in vascular cells and tissue, likely via S-nitrosation. This in part, explains increased TG2 externalization and activity in aging aorta.     

Implication of gluconate kinase activity in l-ornithine biosynthesis in Corynebacterium glutamicum

With the purpose of generating a microbial strain for l-ornithine production in Corynebacterium glutamicum, genes involved in the central carbon metabolism were inactivated so as to modulate the intracellular level of NADPH, and to evaluate their effects on l-ornithine production in C. glutamicum. Upon inactivation of the 6-phosphoglucoisomerase gene (pgi) in a C. glutamicum strain, the concomitant increase in intracellular NADPH concentrations from 2.55 to 5.75?mmol?g^?1 (dry cell weight) was accompanied by reduced growth rate and l-ornithine production, suggesting that l-ornithine production is not solely limited by NADPH availability. In contrast, inactivation of the gluconate kinase gene (gntK) led to a 51.8?% increase in intracellular NADPH concentration, which resulted in a 49.9?% increase in l-ornithine production. These results indicate that excess NADPH is not necessarily rate-limiting, but is required for increased l-ornithine production in C. glutamicum.     

Mitigation of Lead-Induced Neurotoxicity by the Naringin: Erythrocytes as Neurons Substitute Markers

This study aimed to investigate the effect of lead (Pb) on neuronal nitric oxide synthase (nNOS) activity using erythrocytes as neurons surrogate markers. Moreover, the protective effect of naringin (NAR) against lead acetate (PbAc)-induced neurotoxicity was investigated. Human erythrocytes were incubated with l-arginine (l-Arg), N ^ω-nitro-l-Arginine methyl ester ( l-NAME), NAR, PbAc, PbAc + l-Arg, PbAc + NAR, or PbAc + l-Arg +NAR. The present results revealed that incubation of erythrocytes with PbAc inhibited NOS activity and decreased nitrite levels as an index for nitric oxide (NO) production to values similar that of l-NAME as known NOS inhibitor. Likewise, PbAc induced a significant decrease in activities of ATPases and acetylcholinesterase compared to control cells. Furthermore, PbAc exposure significantly increased protein carbonyl content (PCC) and malondialdehyde (MDA) levels while significantly decrease the levels of reduced glutathione (GSH). On the contrary, incubation of erythrocytes with PbAc in the presence of l-Arg + NAR synergistically ameliorated the investigated parameters compared to erythrocytes incubated with PbAc alone. These data suggest that NAR can restore NO bioavailability in a situation of Pb-induced cellular damage. This attributed to antioxidant activity and restoration NOS activity.     

Construction of an l-serine producing Escherichia coli via metabolic engineering

l-Serine is a nonessential amino acid, but plays a crucial role as a building block for cell growth. Currently, l-serine production is mainly dependent on enzymatic or cellular conversion. In this study, we constructed a recombinant Escherichia coli that can fermentatively produce l-serine from glucose. To accumulate l-serine, sdaA encoding the l-serine dehydratase, iclR encoding the isocitrate lyase regulator, and arcA encoding the aerobic respiration control protein were deleted in turn. In batch fermentation, the engineered E. coli strain YF-5 exhibited obvious l-serine accumulation but poor cell growth. To restore cell growth, aceB encoding the malate synthase was knocked out, and the engineered strain was then transformed with plasmid that overexpressed serA ^FR , serB, and serC genes. The resulting strain YF-7 produced 4.5 g/L l-serine in batch cultivation and 8.34 g/L l-serine in fed-batch cultivation.     

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.     

Engineered Bacillus subtilis 168 produces l-malate by heterologous biosynthesis pathway construction and lactate dehydrogenase deletion

In the present work, Bacillus subtilis was engineered to produce l-malate. Initially, the study revealed that the slight fumarase activity under anaerobic conditions is extremely favourable for l-malate one-step fermentation accumulation. Subsequently, an efficient heterologous biosynthesis pathway formed by Escherichia coli phosphoenolpyruvate carboxylase and Saccharomyces cerevisiae malate dehydrogenase was introduced into B. subtilis, which led to 6.04?±?0.19?mM l-malate production. Finally, the l-malate production was increased 1.5-fold to 9.18?±?0.22?mM by the deletion of lactate dehydrogenase. Under two-stage fermentation conditions, the engineered B. subtilis produced up to 15.65?±?0.13?mM l-malate, which was 86.3?% higher than that under anaerobic fermentation conditions. Though the l-malate production by the recombinant was low, this is the first attempt to produce l-malate in engineered B. subtilis and paves the way for further improving l-malate production in B. subtilis.     

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF ^wt or pheA ^fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF ^wt and pheA ^fbr improved the titer of l-Phe to 4.46 ± 0.06 g l^?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA ^fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l^?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.     

Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability

The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the l-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved l-ornithine production by Corynebacterium glutamicum. Production of l-ornithine requires 2 moles of NADPH per mole of l-ornithine. Thus, the effect of NADPH availability on l-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of l-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l^?1 of l-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.