Biomedical subjects

Uwe Sauer

Publications and source records attributed to Uwe Sauer.

At least 37 records · Page 2Linked to original sources

PubMed2005

Multiple and interconnected pathways for L-lysine catabolism in Pseudomonas putida KT2440.

L-lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to delta-aminovaleramide and the hydrolysis of the latter to delta-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Delta1-piperidine-2-carboxylate into aminoadipate. Based on L-[U-13C, U-15N]lysine experiments, we quantified the relative use of pathways in the wild type and its isogenic mutants. The fate of 13C label of L-lysine indicates that in addition to the existing connection between the D- and L-lysine pathways at the early steps of the catabolism of L-lysine mediated by a lysine racemase, there is yet another interconnection at the lower end of the pathways in which aminoadipate is channeled to yield glutarate. 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.

2-Aminoadipic Acid↗

PubMed2005

Experimental identification and quantification of glucose metabolism in seven bacterial species.

The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, gamma-proteobacteria, and alpha-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. 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.

Aerobiosis↗

PubMed2005

Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli.

Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.

Anaerobiosis↗

PubMed2004

The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria.

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. 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.

Bacteria↗

PubMed2004

Model-independent fluxome profiling from 2H and 13C experiments for metabolic variant discrimination.

We introduce a conceptually novel method for intracellular fluxome profiling from unsupervised statistical analysis of stable isotope labeling. Without a priori knowledge on the metabolic system, we identified characteristic flux fingerprints in 10 Bacillus subtilis mutants from 132 2H and 13C tracer experiments. Beyond variant discrimination, independent component analysis automatically mapped several fingerprints to their metabolic determinants. The approach is flexible and paves the way to large-scale fluxome profiling of any biological system and condition.

Amino Acids↗

PubMed2004

Robustness of cellular functions.

Robustness, the ability to maintain performance in the face of perturbations and uncertainty, is a long-recognized key property of living systems. Owing to intimate links to cellular complexity, however, its molecular and cellular basis has only recently begun to be understood. Theoretical approaches to complex engineered systems can provide guidelines for investigating cellular robustness because biology and engineering employ a common set of basic mechanisms in different combinations. Robustness may be a key to understanding cellular complexity, elucidating design principles, and fostering closer interactions between experimentation and theory.

Animals↗

PubMed2004

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains.

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.

Cellulose↗

PubMed2004

High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints.

13C-constrained flux balancing analysis based on gas chromatography-mass spectrometry data is presented here as a simple and robust method for the estimation of intracellular carbon fluxes. In this approach, the underdetermined system of metabolite balances deduced from stoichiometric relations and measured extracellular rates is complemented with 13C constraints from metabolic flux ratio analysis. Fluxes in central carbon metabolism of exponentially growing Escherichia coli were estimated by 13C-constrained flux balancing from three different 13C-labeled glucose experiments. The best resolution of the network was achieved using 13C constraints derived from [U-13C]glucose and [1-13C]glucose experiments. The corresponding flux estimate was in excellent agreement with a solution that was independently obtained with a comprehensive isotopomer model. This new methodology was also demonstrated to faithfully capture the intracellular flux distribution in E. 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.

Carbon Isotopes↗

PubMed2004

High-throughput phenomics: experimental methods for mapping fluxomes.

Many technologies have been developed to help explain the phenotypic consequences of genetic and/or environmental modifications in areas like functional genomics, pharmaceutical research and metabolic engineering. The missing link in contemporary functional analyses that focus on the analysis of cellular components is the capacity to directly observe functional units. By linking genes and proteins to higher level biological functions, the molecular fluxes through metabolic networks (the fluxome) determine the cellular phenotype. Quantitative monitoring of such whole network operations by methods of metabolic flux analysis, thus bridges the gap by providing a global perspective of the integrated regulation at the transcriptional, translational and metabolic level. This review highlights recent developments towards high-throughput flux analysis.

Animals↗

PubMed2004

Inverse metabolic engineering with phosphagen kinase systems improves the cellular energy state.

Inverse metabolic engineering attempts to identify or construct desired phenotypes of applied interest to endow them on appropriate host organisms. A particular desirable phenotype is the ATP homeostasis exhibited by animal cells with high and variable ATP turnover through temporal and spatial energy buffering. This buffering is achieved by phosphagen kinase systems that consist of a specific kinase and its cognate phosphagen, which functions as a large pool of 'high-energy phosphates' that are used to replenish ATP during periods of high energetic demand. This review discusses recent advances and potentials of inverse metabolic engineering of cell types that do not normally contain such systems--bacteria, yeast, plants, and liver--with creatine or arginine kinase systems. 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.

Adenosine Triphosphate↗

PubMed2004

The phosphoenolpyruvate carboxykinase also catalyzes C3 carboxylation at the interface of glycolysis and the TCA cycle of Bacillus subtilis.

Quantitative physiological characterization and isotopic tracer experiments revealed that pyruvate kinase mutants of Bacillus subtilis produced significantly more CO(2) from glucose in the tricarboxylic acid cycle than is explained by the remaining conversion of phosphoenolpyruvate (PEP) to pyruvate catalyzed by the phosphotransferase system. We show here that this additional catabolic flux into the tricarboxylic acid cycle was catalyzed by the PEP carboxykinase. In contrast to its normal role in gluconeogenesis, PEP carboxykinase can operate in the reverse direction from PEP to oxaloacetate upon knockout of pyruvate kinase in a riboflavin-producing B. subtilis strain and in wild-type 168. At least in the industrial strain, we demonstrate the additional capacity of PEP carboxykinase to function as a substitute anaplerotic reaction when the normal pyruvate carboxylase is inactivated. 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.

Bacillus subtilis↗

PubMed2004

TCA cycle activity in Saccharomyces cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates.

Metabolic responses of Saccharomyces cerevisiae to different physical and chemical environmental conditions were investigated in glucose batch culture by GC-MS-detected mass isotopomer distributions in proteinogenic amino acids from (13)C-labelling experiments. For this purpose, GC-MS-based metabolic flux ratio analysis was extended from bacteria to the compartmentalized metabolism of S. cerevisiae. Generally, S. cerevisiae was shown to have low catabolic fluxes through the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. Notably, respiratory TCA cycle fluxes exhibited a strong correlation with the maximum specific growth rate that was attained under different environmental conditions, including a wide range of pH, osmolarity, decoupler and salt concentrations, but not temperature. At pH values of 4.0 to 6.0 with near-maximum growth rates, the TCA cycle operated as a bifurcated pathway to fulfil exclusively biosynthetic functions. Increasing or decreasing the pH beyond this physiologically optimal range, however, reduced growth and glucose uptake rates but increased the 'cyclic' respiratory mode of TCA cycle operation for catabolism. Thus, the results indicate that glucose repression of the TCA cycle is regulated by the rates of growth or glucose uptake, or signals derived from these. 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.

Biomass↗

PubMed2004

Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala.

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. 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.

Acetates↗

PubMed2004

Molecular basis for anaerobic growth of Saccharomyces cerevisiae on xylose, investigated by global gene expression and metabolic flux analysis.

Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. 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.

Adenosine Triphosphate↗

PubMed2004

Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae.

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. 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.

Aldehyde Oxidoreductases↗

PubMed2004

The Bacillus subtilis yqjI gene encodes the NADP+-dependent 6-P-gluconate dehydrogenase in the pentose phosphate pathway.

Despite the importance of the oxidative pentose phosphate (PP) pathway as a major source of reducing power and metabolic intermediates for biosynthetic processes, almost no direct genetic or biochemical evidence is available for Bacillus subtilis. Using a combination of knockout mutations in known and putative genes of the oxidative PP pathway and 13C-labeling experiments, we demonstrated that yqjI encodes the NADP+-dependent 6-P-gluconate dehydrogenase, as was hypothesized previously from sequence similarities. Moreover, YqjI was the predominant isoenzyme during glucose and gluconate catabolism, and its role in the oxidative PP pathway could not be played by either of two homologues, GntZ and YqeC. This conclusion is in contrast to the generally held view that GntZ is the relevant isoform; hence, we propose a new designation for yqjI, gndA, the monocistronic gene encoding the principal 6-P-gluconate dehydrogenase. Although we demonstrated the NAD+-dependent 6-P-gluconate dehydrogenase activity of GntZ, gntZ mutants exhibited no detectable phenotype on glucose, and GntZ did not contribute to PP pathway fluxes during growth on glucose. Since gntZ mutants grew normally on gluconate, the functional role of GntZ remains obscure, as does the role of the third homologue, YqeC. Knockout of the glucose-6-P dehydrogenase-encoding zwf gene was primarily compensated for by increased glycolytic fluxes, but about 5% of the catabolic flux was rerouted through the gluconate bypass with glucose dehydrogenase as the key enzyme.

Amino Acid Sequence↗

PubMed2003

The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli.

Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiological function that could potentially transfer electrons directly from NADH to NADP+ and vice versa. Using defined mutants and metabolic flux analysis, we identified the proton-translocating transhydrogenase PntAB as a major source of NADPH in E. coli. During standard aerobic batch growth on glucose, 35-45% of the NADPH that is required for biosynthesis was produced via PntAB, whereas pentose phosphate pathway and isocitrate dehydrogenase contributed 35-45% and 20-25%, respectively. The energy-independent transhydrogenase UdhA, in contrast, was essential for growth under metabolic conditions with excess NADPH formation, i.e. growth on acetate or in a phosphoglucose isomerase mutant that catabolized glucose through the pentose phosphate pathway. Thus, both isoforms have divergent physiological functions: energy-dependent reduction of NADP+ with NADH by PntAB and reoxidation of NADPH by UdhA. Expression appeared to be modulated by the redox state of cellular metabolism, because genetic and environmental manipulations that increased or decreased NADPH formation down-regulated pntA or udhA transcription, respectively. The two transhydrogenase isoforms provide E. coli primary 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?

Down-Regulation↗

PubMed2003

Long-term continuous evolution of acetate resistant Acetobacter aceti.

Elevated concentrations of cytotoxic acetate are found in many environmental niches, and few species are relatively resistant to acetate. In particular the high-level acetate resistance of so-called acetic acid bacteria that occurs in industrial settings must be constantly selected for. To investigate the nature of such high-level resistance, we grew the moderately acetate-resistant Acetobacter aceti wild-type and acetate-sensitive Escherichia coli in long-term continuous cultures with increasing acetate concentrations at near neutral pH. While E. coli did not acquire any significant resistance after 125 generations of selection, A. aceti evolved the capability to grow at acetate concentrations exceeding 50 g/L within 240 generations. This phenotype was found to be stable for several generations in the absence of selective pressure, hence must be genetically determined. 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.

Acetates↗