Uwe Sauer

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..Thus, E. coli actively limits its galactose catabolism at the expense of otherwise possible faster growth...

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..These principles form the forces by which evolution shapes metabolic fluxes in microorganisms' environmental context...

..coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis...

..Hence, alteration in one network constituent can be efficiently buffered by converse alterations in the other constituent, implying a passive mechanism to maintain metabolic homeostasis upon perturbations in enzyme capacity...

..In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes...

..Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs...

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..These principles are also implemented in the publicly available software FiatFlux, which directly calculates flux ratios from the mass spectra of amino acids...

..Co-utilization of glucose and malate resulted in a synergistic decrease of the respiratory tricarboxylic acid cycle flux...

..Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes...

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..Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis...

..In the future, this catalogue will enable comprehensive in silico calculability analyses to identify the most sensitive measurements and direct experimental design...

..In most yeasts, the PP pathway activity appears to be driven exclusively by the demand for NADPH...

..As a consequence, higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands, suggesting a transhydrogenase-like mechanism to close the balance of reducing equivalents...

..By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype...

..The presented protocols take 5-10 d, have been used extensively in the past decade and are exemplified here for the central metabolism of Escherichia coli...

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..This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture...

..A conceptual simplification of flux analysis is the analytical determination of metabolic flux ratios exclusively from MS data, which can then be used in a second step to estimate absolute in vivo fluxes...

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..metabolism with an extraordinary flexibility to cope with varying catabolic and anabolic demands, which raises two general questions: why do only a few bacteria contain both isoforms, and how do other organisms manage NADPH metabolism?..

..coli and B. subtilis, was the lack of cofactor specificity in the oxidative pentose phosphate pathway, which contrasts with the textbook view of the pentose phosphate pathway dehydrogenases as being NADP+ dependent...

..Analysis of available genome sequences demonstrated that orthologs of both yneI and gabD are broadly distributed across phylogenetic space...

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..While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration...

..Here we identify key experiments in a systems biology approach with a genome-scale model of Saccharomyces cerevisiae metabolism, thereby reducing the workload for experimental network analyses and functional genomics...

..Therefore, suitable internal standards, such as (13)C-labeled biomass extracts, are mandatory for quantitative metabolomics with any methods...

..Consequently, it is impossible to conclude that the "best" solution was identified or that no good solution exists, because parameter spaces typically cannot be explored in a reasonable amount of time...

..The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production...

..The observation of a trade-off within, but not between, populations suggests that the populations evolved different genetic solutions for growth in the selective environment, which in turn led to different physiological constraints...

..Examples are discussed that illustrate how microbial metabolism can be tailored for large-scale industrial processes with imperfect mixing and how the liver can be protected from metabolic insults or stimulated for better regeneration...

..This review highlights recent developments towards high-throughput flux analysis...

..Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase...

..Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis...

..The Cra/F1P duo seems to not only control the expression of the PTS(Fru) but also checks the activity of the PTS(Ntr) in vivo...

..These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes...

..Thus, at least for metabolism, persistence of the paralog fraction in the genome can be better explained with an array of different, often overlapping functional roles...

..While sensing of extracellular glucose concentrations has a general influence on the in vivo TCA cycle activity, the growth-rate-dependent increase in respiratory TCA cycle activity was independent of glucose sensing...

..The approach is flexible and paves the way to large-scale fluxome profiling of any biological system and condition...

..The improved resistance of our evolved strains was based on their capacity to remain viable in a toxic environment during the prolonged, furfural induced lag phase...

..The reporter method quantitatively resolved the expected mutant-specific metabolic phenotypes down to subpopulation fractions of about 1%...

..Beyond revising our picture of TOR signaling, we expect ensemble modeling to help elucidate other insufficiently characterized cellular circuits...

..Integration of metabolomics, (13)C-flux analysis and other data enable the condition-dependent characterization of regulatory circuits that ultimately govern the metabolic phenotype...

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..Taken together, the data suggest that IHF plays a role in the physiological shift that sets P. putida for entering stationary phase...

..cerevisiae and reviews their underlying mechanistic principles - both at the individual enzyme level and in the context of the entire metabolic network operation...

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..5-50 mg dry cell weight. We recommend this method for routine and yet comprehensive quantification of primary metabolites in a wide variety of biological matrices...

..The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B. subtilis biomass in fed-batch cultures...

..coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion...

..However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose...

..Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption...

..cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae...

..The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture...

..We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory...

..Since the product-corrected biomass yields were identical in mutants and parent, the results show that efficient ATP generation is not overly important for exponential growth of B. subtilis in batch culture...

..coli to compensate for genetic perturbations and the utility of combining multiple high throughput data sets to differentiate between causal and noncausal mechanistic changes...

..The metabolically most active clones were identified by screening for high specific glucose uptake rates during ammonium starvation-induced stationary phase in deep-well microtiter plates...

..Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms...

..With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline...

..It can therefore be concluded that the glucokinase pathway is a sine qua non condition for P. putida to grow with glucose...

..Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism...

..This study establishes an unequivocal relationship between gene and pathway enzymes in the metabolism of L-lysine, which is of crucial importance for the successful colonization of the rhizosphere of plants by this microorganism...

..This effect is presumably caused by the physiological function of arginine kinase as a short term energy buffer in the form of phosphoarginine, but a pH-buffering effect cannot be excluded...

..We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production...

..Intracellular acetate concentrations were significantly lower in evolved A. aceti, when compared to wild-type A. aceti and E. coli, indicating that cytoplasmatic anion accumulation is an important component of acetate toxicity...

..Network rigidity and robustness are probably universal functional design principles, whereas the standby mode may be more specific...

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..coli shake flasks and 1-ml deep-well microtiter plates. Due to its simplicity, speed, and robustness, 13C-constrained metabolic flux balancing is promising for routine and high-throughput analysis on a miniaturized scale...

..Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively...

..By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered...

..Presumably as a consequence of the unfavorable kinetics of an ATP-synthesizing anaplerotic PEP carboxykinase reaction, such pyruvate carboxylase mutants grow slowly or, as in the case of wild-type 168, not at all...

..General selection guidelines for improving multiple-stress resistance in S. cerevisiae are presented and discussed...

..Robustness may be a key to understanding cellular complexity, elucidating design principles, and fostering closer interactions between experimentation and theory...