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Phyllostachys edulis is a spectacularly fast-growing species that completes its height growth within 2 months after the shoot emerges without producing leaves (fast-growing period, FGP). This phase was considered heterotrophic, with the carbon necessary for the growth being transferred from the mature culms via the rhizomes, although previous studies observed key enzymes and anatomical features related to C4-carbon fixation in developing culms. We tested whether C4-photosynthesis or dark-CO2 fixation through anaplerotic reactions significantly contributes to the FGP, resulting in differences in the natural abundance of δ 13C in bulk organic matter and organic compounds. Further, pulse-13CO2-labelling was performed on developing culms, either from the surface or from the internal hollow, to ascertain whether significant CO2 fixation occurs in developing culms. δ 13C of young shoots and developing culms were higher (−26.3 to −26.9 ‰) compared to all organs of mature bamboos (−28.4 to −30.1 ‰). Developing culms contained chlorophylls, most observed in the skin tissues. After pulse-13CO2-labelling, the polar fraction extracted from the skin tissues was slightly enriched in 13C, and only a weak 13C enrichment was observed in inner tissues. Main carbon source sustaining the FGP was not assimilated by the developing culm, while a limited anaplerotic fixation of respired CO2 cannot be excluded and is more likely than C4-photosynthetic carbon fixation. [ABSTRACT FROM AUTHOR]

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yield by approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

Pathways replenishing tricarboxylic acid cycle were divided into four major groups based on metabolite serving as source for oxaloacetic acid or other tricarboxylic acid cycle component synthesis. Using this metabolic map, the analysis of genetic potential for functioning of tricarboxylic acid cycle replenishment pathways was carried out for seven strains of purple non-sulfur bacterium Rhodopseudomonas palustris. The results varied from strain to strain. Published microarray data for phototrophic acetate cultures of Rps. palustris CGA009 were analyzed to validate activity of the putative pathways. All the results were compared with the results for another purple non-sulfur bacterium, Rhodobacter capsulatus SB1003 and species-specific differences were clarified. [ABSTRACT FROM AUTHOR]

Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19-fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum . [ABSTRACT FROM AUTHOR]

Increasing evidence has shown that the brain is a site of diabetic end-organ damage. This study investigates cerebral metabolism and the interactions between astrocytes and neurons at different stages of diabetes to identify the potential pathogenesis of diabetic encephalopathy. [1-C]glucose or [2-C]acetate is infused into 1- and 15-week diabetic rats, the brain extracts of which are analyzed by using H and C magnetic resonance spectroscopy. The C-labeling pattern and enrichment of cerebral metabolites are also investigated. The increased C incorporation in the glutamine, glutamate, and γ-aminobutyric acid carbons from [2-C]acetate suggests that the astrocytic mitochondrial metabolism is enhanced in 1-week diabetic rats. By contrast, the decreased labeling from [1-C]glucose reflected that the neuronal mitochondrial metabolism is impaired. As diabetes developed to 15 weeks, glutamine and glutamate concentrations significantly decreased. The increased labeling of glutamine C4 but unchanged labeling of glutamate C4 from [2-C]acetate suggests decreased astrocyte supply to the neurons. In addition, the enhanced pyruvate recycling pathway manifested by the increased lactate C2 enrichment in 1-week diabetic rats is weakened in 15-week diabetic rats. Our study demonstrates the overall metabolism disturbances, changes in specific metabolic pathways, and interaction between astrocytes and neurons during the onset and development of diabetes. These results contribute to the mechanistic understanding of diabetes pathogenesis and evolution. [ABSTRACT FROM AUTHOR]

Corynebacterium glutamicum ssp. lactofermentum strain JHI3-156 produces l-isoleucine (Ile). Overexpression of the Saccharomyces cerevisiae-derived NADH kinase gene ( POS5) and the endogenous glucose-6-phosphate dehydrogenase and NAD kinase genes ( zwf- ppnK) in JHI3-156 increased Ile production by 26 and 31 %, respectively. To decipher the global effect of POS5 and zwf- ppnK overexpression on Ile biosynthesis, proteomic analysis was conducted. Twenty-four differentially expressed proteins were identified in the POS5-overexpressing strain, most of which are related to inositol catabolism, central carbon metabolism, anaplerotic pathway, protein biosynthesis and the stress response. In the zwf- ppnK-overexpressing strain, seven differentially-expressed proteins, including PpnK and anaplerotic enzymes, were identified. This result indicates the involvement of a novel inositol catabolism step and the importance of the anaplerotic pathway in Ile biosynthesis. This finding will be helpful in the systematic metabolic engineering of C. glutamicum for Ile biosynthesis. [ABSTRACT FROM AUTHOR]

751I.751II.752III.753IV.754V.757VI.762VII.763VIII.765IX.765X.766766References766 Summary: In general, leaves are 13C‐depleted compared with all other organs (e.g. roots, stem/trunk and fruits). Different hypotheses are formulated in the literature to explain this difference. One of these states that CO2 respired by leaves in the dark is 13C‐enriched compared with leaf organic matter, while it is 13C‐depleted in the case of root respiration. The opposite respiratory fractionation between leaves and roots was invoked as an explanation for the widespread between‐organ isotopic differences. After summarizing the basics of photosynthetic and post‐photosynthetic discrimination, we mainly review the recent findings on the isotopic composition of CO2 respired by leaves (autotrophic organs) and roots (heterotrophic organs) compared with respective plant material (i.e. apparent respiratory fractionation) as well as its metabolic origin. The potential impact of such fractionation on the isotopic signal of organic matter (OM) is discussed. Some perspectives for future studies are also proposed. [ABSTRACT FROM AUTHOR]

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase ( MDH2), and pyruvate carboxylase ( PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38 ± 0.01 g g xylose consumed) was improved from that of the control strain (0.31 ± 0.01 g g xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1-MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1-MDH2-PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells. [ABSTRACT FROM AUTHOR]

External factors affecting the activity of isocitrate lyase (ICL) in Rhodobacter capsulatus B10 grown under controlled photoheterotrophic anaerobic conditions were investigated. The activity of this enzyme was found to depend on the history of the inoculum and on the growth phase on acetate medium. Intracellular degradation of ICL under unfavorable conditions was shown. However, transition of the growing culture from acetate to lactate did not result in active degradation of the enzyme. When transferred to acetate, Rba. capsulatus could grow without the lag phase and did not exhibit ICL activity, suggesting another anaplerotic pathway in Rba. capsulatus cells. Since emergence of the ICL activity in the cells grown on acetate results in an increase in its growth rate, the glyoxylate bypass plays an important role in acetate metabolism of Rba. Capsulatus. [ABSTRACT FROM AUTHOR]

Summary The excessive application of nitrogen fertilizer to maximize crop yields causes negative environmental effects such as pollution and ecological imbalance. To overcome this problem, researchers have attempted to improve the nitrogen assimilation capacity of crops. Maize Dof1 (ZmDof1) is a plant-specific transcription factor shown to promote nitrogen assimilation in Arabidopsis thaliana (Arabidopsis) even under nitrogen-deficient conditions. The present study examines the effect of the introduction of the ZmDof1 gene on carbon and nitrogen assimilation in rice. ZmDof1 induced the expression of phospho enolpyruvate carboxylase (PEPC) genes in transgenic rice plants and transactivated the PEPC promoters in protoplast transient assays, showing similar effects in rice as in Arabidopsis. Transgenic rice expressing ZmDof1 and grown in the presence of 360 μ m (nitrogen-sufficient) or 90 μ m (nitrogen-deficient) of nitrogen concentrations showed modulation of metabolite content and gene expression associated with the anaplerotic pathway for the TCA cycle, suggesting an increased carbon flow towards nitrogen assimilation. Furthermore, increases in carbon and nitrogen amounts per seedling were found in Dof1 rice grown under nitrogen-deficient conditions. Nitrogen deficiency also resulted in the predominant distribution of nitrogen to roots, accompanied by significant increases in root biomass and modification of the shoot-to-root ratio. Measurement of the CO2 gas exchange rate showed a significant increase in the net photosynthesis rate in Dof1 rice under nitrogen-deficient conditions. Taken these together, the present study displayed that ZmDof1 expression in rice could induce gene expressions such as PEPC genes, modulate carbon and nitrogen metabolites, increase nitrogen assimilation and enhance growth under low-nitrogen conditions. [ABSTRACT FROM AUTHOR]

Abstract: To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum. [Copyright &y& Elsevier]

Corynebacterium glutamicum is a biotin auxotrophic bacterium in which glutamate production is induced under biotin-limited conditions. During glutamate production, anaplerotic reactions catalyzed by phosphoenolpyruvate carboxylase (PEPC) and a biotin-containing enzyme pyruvate carboxylase (PC) are believed to play an important role in supplying oxaloacetate in the tricarboxylic acid cycle. To understand the distinct roles of PEPC and PC on glutamate production by C. glutamicum, we observed glutamate production induced under biotin-limited conditions in the disruptants of the genes encoding PEPC (ppc) and PC (pyc), respectively. The pyc disruptant retained the ability to produce high amounts of glutamate, and lactate was simultaneously produced probably due to the increased intracellular pyruvate levels. On the other hand, the ppc knockout mutant could not produce glutamate. Additionally, glutamate production in the pyc disruptant was enhanced by overexpression of ppc rather than disruption of the lactate dehydrogenase gene (ldh), which is involved in lactate production. Metabolic flux analysis based on the 13C-labeling experiment and measurement of 13C-enrichment in glutamate using nuclear magnetic resonance spectroscopy revealed that the flux for anaplerotic reactions in the pyc disruptant was lower than that in the wild type, concomitantly increasing the flux for lactate formation. Moreover, overexpression of ppc increased this flux in both the pyc disruptant and the wild type. Our results suggest that the PEPC-catalyzed anaplerotic reaction is necessary for glutamate production induced under biotin-limited conditions, because PC is not active during glutamate production, and overexpression of ppc effectively enhances glutamate production under biotin-limited conditions. [Copyright &y& Elsevier]

Abstract: Arabidopsis thaliana was transformed with a Sorghum C4 PEPC cDNA under the control of the 35S promoter. Primary transformants were isolated and classified on a seed PEPC activity basis: A lines (two-fold increase) and B lines (four-fold increase). Primary transformants also expressed the C4 PEPC in leaves (up to a 10-fold increase in activity). In seeds, ectopically expressed PEPC impacted on dry weight and total protein content that were increased by up to 30%. In the stable progeny of A and B lines, the increase in PEPC activity was amplified in seeds; however, a decrease in PEPC content and activity (inversely proportional to seed levels) was observed in maternal tissues (leaves and siliques). This was due to the absence of the exogenous C4 PEPC and a partial decrease in the endogenous C3 PEPC forms (mRNA, protein and activity). There was no apparent compensation by a PEPC hyperphosphorylation. In these plants, the anaplerotic pathway flux was altered (notably, a decrease in organic acids and glutamine levels) as judged by 1H NMR metabolite profiling. Whereas in the corresponding seeds (four- to eight-fold increase in PEPC activity) there was no increase in protein and seed mass, but a moderate decrease in lipid content was observed (compared to control plants). Under normal culture conditions, the transformants (primary and progeny) did not show any apparent growth phenotype or modification in seed production per plant. However, the A and B lines exhibited severe growth defects when salt stressed by LiCl. [Copyright &y& Elsevier]

An ambitious aim in plant breeding and biotechnology is to increase the protein content of crop seeds used for food and feed. Using an approach to manipulate assimilate partitioning, we succeeded in elevating the protein content in legume seeds up to 50%. Transgenic bean plants were generated which express a Corynebacterium glutamicum phosphoenolpyruvate carboxylase (PEPC)in a seed-specific manner. The bacterial enzyme was not feedback inhibited by malate. Transgenic seeds showed a higher [14C]-CO2 uptake and about a threefold increased incorporation of labelled carbon into proteins. Changed metabolite profiles of maturing cotyledons indicated a shift of metabolic fluxes from sugars/ starch into organic acids and free amino acids. These changes were consistent with an increased carbon flow through the anaplerotic pathway catalysed by PEPC. Consequently, transgenic seeds accumulated up to 20% more protein per gram seed dry weight. Additionally, seed dry weight was higher by 20%-30%. We conclude that PEPC in seeds is a promising target for molecular plant breeding. [ABSTRACT FROM AUTHOR]

The article explores the fate of the phosphoenolpyruvate (PEP) in oxidative stress conditions in plants. It mentions that the ozone can affect the pathways in which PEP is connected. It notes the importance to determine how PEP partitioning is regulated during ozone exposure in illuminated leaves. It also emphasizes that a reduction in the different pathways downstream of PEP can lead to a decline in the scavenging power against reactive oxygen species (ROS).