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1.
Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains 总被引:1,自引:0,他引:1
The recombinant xylose fermenting strain Saccharomyces cerevisiae TMB3001 can grow on xylose, but the xylose utilisation rate is low. One important reason for the inefficient fermentation of xylose to ethanol is believed to be the imbalance of redox co-factors. In the present study, a metabolic flux model was constructed for two recombinant S. cerevisiae strains: TMB3001 and CPB.CR4 which in addition to xylose metabolism have a modulated redox metabolism, i.e. ammonia assimilation was shifted from being NADPH to NADH dependent by deletion of gdh1 and over-expression of GDH2. The intracellular fluxes were estimated for both strains in anaerobic continuous cultivations when the growth limiting feed consisted of glucose (2.5 g L-1) and xylose (13 g L-1). The metabolic network analysis with 13C labelled glucose showed that there was a shift in the specific xylose reductase activity towards use of NADH as co-factor rather than NADPH. This shift is beneficial for solving the redox imbalance and it can therefore partly explain the 25% increase in the ethanol yield observed for CPB.CR4. Furthermore, the analysis indicated that the glyoxylate cycle was activated in CPB.CR4. 相似文献
2.
Robert B. Helling 《Journal of bacteriology》1998,180(17):4571-4575
Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog α-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis. 相似文献
3.
Fabien Coze Fran?oise Gilard Guillaume Tcherkez Marie-Jo?lle Virolle Armel Guyonvarch 《PloS one》2013,8(12)
Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and 13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest. 相似文献
4.
Kasper Mller Bjarke Christensen Jochen Frster Jure Pikur Jens Nielsen Lisbeth Olsson 《Biotechnology and bioengineering》2002,77(2):186-193
The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose. 相似文献
5.
Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides 下载免费PDF全文
As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions. 相似文献
6.
Jin Hou Goutham N. Vemuri Xiaoming Bao Lisbeth Olsson 《Applied microbiology and biotechnology》2009,82(5):909-919
During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A
deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation.
However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor
imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the
native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains
was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic
NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol
to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall,
although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of
NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated. 相似文献
7.
8.
Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)-glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of 15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicum strains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4+ was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type. 相似文献
9.
Cline GW Lepine RL Papas KK Kibbey RG Shulman GI 《The Journal of biological chemistry》2004,279(43):44370-44375
Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process. 相似文献
10.
Maria Rudawska Barbara Kieliszewska-Rokicka Jean-Claude Debaud rzej Lewandowski Gilles Gay 《Physiologia plantarum》1994,92(2):279-285
Ammonium assimilation enzymes from several strains of ectendo- and ectomycorrhizal fungi were assayed after three weeks culture on a buffered synthetic medium containing ammonium as sole nitrogen source. Activity of NADP-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) of ectomycorrhizal strains was very low despite excellent mycelial growth. Only ectendomycorrhizal fungus MrgX isolated from roots of Pinus sylvestris showed high GDH activity. Similar results were obtained when the enzyme extracts were subjected to starch gel electrophoresis. Growth of the fungi, except ectendomycorrhizal MrgX, was arrested when inhibitors of glutamine synthetase (GS, EC 6.3.1.2) or glutamate synthase (GOGAT. EC 1.4.7.1) (methionine sulphoximine or albizine, respectively) were included in the culture medium. Glutamine synthetase activity was found in all fungi tested. The results suggest that the GS pathway for ammonium assimilation is potentially operative in ectomycorrhizal fungi and imply only a minor role for GDH in ammonium assimilation by the studied ectomycorrhizal symbionts of pine. Some physiological and ecological implications of these results are discussed. 相似文献
11.
Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point
Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT). In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15)N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT) strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks. 相似文献
12.
Glutamate dehydrogenase (GDH E.C. 1.4.1.2.4), glutamine synthetase (GS E.C. 6.3.1.2) and glutamate synthase (glutamine oxoglutarate amino transferase, GOGAT E.C. 2.6.1.53) activities, protein and organic nitrogen contents and growth of roots and shoots of maize seedlings raised in dark at 25±2°C in half strength Hoagland’s solution containing different ammonium salts as source of nitrogen, were determined to assess the contribution of alternate pathways in ammonium assimilation. Ammonium nitrate or in some cases ammonium chloride appeared to be the best source for both root and shoot growth and for increase in protein, total nitrogen and the enzymes of ammonium assimilation. In roots, NH4-nitrogen appeared to be assimilated by both GDH as well as GS-GOGAT pathways specially in the dark grown seedlings, while in shoots it was primarily by GS-GOGAT pathway. 相似文献
13.
14.
Roles of SpoT and FNR in NH4+ assimilation and osmoregulation in GOGAT (glutamate synthase)-deficient mutants of Escherichia coli. 总被引:1,自引:0,他引:1 下载免费PDF全文
An osmosensitive mutant of Escherichia coli was isolated and shown to harbor two mutations that were together necessary for osmosensitivity. One (ossB) was an insertion mutation in the gltBD operon, which encodes the enzyme glutamate synthase (GOGAT), involved in ammonia assimilation and L-glutamate biosynthesis. The other (ossA) was in the fnr gene, encoding the regulator protein FNR for anaerobic gene expression. Several missense or deletion mutations in fnr and gltBD behaved like ossA and ossB, respectively, in conferring osmosensitivity. A mutation affecting the DNA-binding domain of FNR was recessive to fnr+ with respect to the osmotolerance phenotype but was dominant-negative for its effect on expression of genes in anaerobic respiration. Our results may most simply be interpreted as suggesting the requirement for monomeric FNR during aerobic growth of E. coli in high-osmolarity media, presumably for L-glutamate accumulation via the GOGAT-independent pathway (catalyzed by glutamate dehydrogenase [GDH]), but the mechanism of FNR action is not known. We also found that the spoT gene (encoding guanosine 3',5'-bispyrophosphate [ppGpp] synthetase II/ppGpp-3' pyrophosphohydrolase), in multiple copies, overcomes the defect in NH4+ assimilation associated with GOGAT deficiency and thereby suppresses osmosensitivity in gltBD fnr strains. Enhancement of GDH activity in these derivatives appears to be responsible for the observed suppression. Its likely physiological relevance was established by the demonstration that growth of gltBD mutants (that are haploid for spoT+) on moderately low [NH4+] was restored with the use of C sources poorer than glucose in the medium. Our results raise the possibility that SpoT-mediated accumulation of ppGpp during C-limited growth leads to GDH activation and that the latter enzyme plays an important role in N assimilation in situ hitherto unrecognized from studies on laboratory-grown cultures. 相似文献
15.
Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS
glutamine synthetase
- GOGAT
glutamate synthase
- GDH
glutamate dehydrogenase 相似文献
16.
R Poulsen B Nøhr J Douthwaite S Hansen LV Iversen JJ Visser J Ruijter GJ 《The FEBS journal》2005,272(6):1313-1325
Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms. 相似文献
17.
Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose. 相似文献
18.
19.
Antje Berger Katrin Dohnt Petra Tielen Dieter Jahn Judith Becker Christoph Wittmann 《PloS one》2014,9(4)
Pseudomonas aeruginosa is a human pathogen that frequently causes urinary tract and catheter-associated urinary tract infections. Here, using 13C-metabolic flux analysis, we conducted quantitative analysis of metabolic fluxes in the model strain P. aeruginosa PAO1 and 17 clinical isolates. All P. aeruginosa strains catabolized glucose through the Entner-Doudoroff pathway with fully respiratory metabolism and no overflow. Together with other NADPH supplying reactions, this high-flux pathway provided by far more NADPH than needed for anabolism: a benefit for the pathogen to counteract oxidative stress imposed by the host. P. aeruginosa recruited the pentose phosphate pathway exclusively for biosynthesis. In contrast to glycolytic metabolism, which was conserved among all isolates, the flux through pyruvate metabolism, the tricarboxylic acid cycle, and the glyoxylate shunt was highly variable, likely caused by adaptive processes in individual strains during infection. This aspect of metabolism was niche-specific with respect to the corresponding flux because strains isolated from the urinary tract clustered separately from those originating from catheter-associated infections. Interestingly, most glucose-grown strains exhibited significant flux through the glyoxylate shunt. Projection into the theoretical flux space, which was computed using elementary flux-mode analysis, indicated that P. aeruginosa metabolism is optimized for efficient growth and exhibits significant potential for increasing NADPH supply to drive oxidative stress response. 相似文献
20.
Moreira dos Santos M Raghevendran V Kötter P Olsson L Nielsen J 《Metabolic engineering》2004,6(4):433-363
The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two strains were engineered by overexpression of malic enzyme. In one of them, malic enzyme was overexpressed as its wild-type mitochondrial form, and in the other strain a short form lacking the mitochondrial targeting sequence was overexpressed. The recombinant strains were analyzed in aerobic batch and continuous cultivations, and the basic growth characteristics were generally not affected to a great extent, even though pleiotropic effects of the manipulations could be seen by the altered in vitro activities of selected enzymes of the central metabolism. Moreover, the decreased pentose-phosphate pathway flux and the ratios of redox cofactors showed that a net transhydrogenase effect was obtained, which can be directed to the cytosol or the mitochondria. This may find application in redirecting fluxes for improving specific biotechnological applications. 相似文献