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.     

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.     

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.     

Production of fungal alpha-amylase by Saccharomyces kluyveri in glucose-limited cultivations

Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.     

Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts

In a quantitative comparative study, we elucidated the glucose metabolism in fourteen hemiascomycetous yeasts from the Genolevures project. The metabolic networks of these different species were first established by (13)C-labeling data and the inventory of the genomes. This information was subsequently used for metabolic-flux ratio analysis to quantify the intracellular carbon flux distributions in these yeast species. Firstly, we found that compartmentation of amino acid biosynthesis in most species was identical to that in Saccharomyces cerevisiae. Exceptions were the mitochondrial origin of aspartate biosynthesis in Yarrowia lipolytica and the cytosolic origin of alanine biosynthesis in S. kluyveri. Secondly, the control of flux through the TCA cycle was inversely correlated with the ethanol production rate, with S. cerevisiae being the yeast with the highest ethanol production capacity. The classification between respiratory and respiro-fermentative metabolism, however, was not qualitatively exclusive but quantitatively gradual. Thirdly, the flux through the pentose phosphate (PP) pathway was correlated to the yield of biomass, suggesting a balanced production and consumption of NADPH. Generally, this implies the lack of active transhydrogenase-like activities in hemiascomycetous yeasts under the tested growth condition, with Pichia angusta as the sole exception. In the latter case, about 40% of the NADPH was produced in the PP pathway in excess of the requirements for biomass production, which strongly suggests the operation of a yet unidentified mechanism for NADPH reoxidation in this species. In most yeasts, the PP pathway activity appears to be driven exclusively by the demand for NADPH.     

Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture

The central carbon metabolism of the nystatin-producing strain Streptomyces noursei ATCC 11455 was evaluated by 13C-labelling experiments. A batch fermentation was examined during the idiophase by GC-MS measurements of the labelling patterns of amino acids in the biomass. The labelling patterns of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation.     

Production of a heterologous proteinase A by Saccharomyces kluyveri

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.     

Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels

The dynamics of galactose metabolism in Saccharomyces cerevisiae was studied by analyzing the metabolic response of the CEN.PK 113-7D wild-type strain when exposed to a galactose pulse during aerobic growth in a galactose-limited steady-state cultivation at a dilution rate of 0.097 h(-1). A fast sampling technique and subsequent methanol-chloroform/solid phase extractions were applied for in vivo measurements of the dynamic changes of the AMP, ADP, ATP levels and the sugar phosphates of the Leloir pathway. The ATP level was found to be significantly lower for yeast growing under galactose limitation (0.37 +/- 0.05 micromol/g CDW) than what has been reported for growth under glucose limitation. The galactose pulse of 5.58 mM was consumed within 40 min (t = 40) and 7 min after the pulse was added cell growth stopped. Subsequently, the cells started to grow and at t = 30 the specific growth rate had recovered to half the steady-state growth rate (0.047 h(-1)). To evaluate the change in flux distribution at steady state and during the galactose transient, a stoichiometric model describing the aerobic metabolism of S. cerevisiae was set up for quantification of the metabolic fluxes. At t = 7 the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium. During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux. During the pulse an enhanced level of Gal-1P was measured, which may be responsible for a toxic metabolic response in S. cerevisiae. The increase in the Gal-1P concentration is intensified by the low affinity of Gal7 towards Gal-1P and, hence, under the physiological conditions examined Gal7 seems to exert control over flux through the Leloir pathway.     

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.     

13C Metabolic Flux Analysis of acetate conversion to lipids by Yarrowia lipolytica

Volatile fatty acids (VFAs) are an inexpensive and renewable carbon source that can be generated from gas fermentation and anaerobic digestion of fermentable wastes. The oleaginous yeast Yarrowia lipolytica is a promising biocatalyst that can utilize VFAs and convert them into triacylglycerides (TAGs). However, currently there is limited knowledge on the metabolism of Y. lipolytica when cultured on VFAs. To develop a better understanding, we used acetate as the sole carbon source to culture two strains, a control strain and a previously engineered strain for lipid overaccumulation. For both strains, metabolism during the growth phase and lipid production phase were investigated by metabolic flux analysis using two parallel sodium acetate tracers. The resolved flux distributions demonstrate that the glyoxylate shunt pathway is constantly active and the flux through gluconeogenesis varies depending on strain and phase. In particular, by regulating the activities of malate transport and pyruvate kinase, the cells divert only a portion of the glyoxylate shunt flux required to satisfy the needs for anaplerotic reactions and NADPH production through gluconeogenesis and the oxidative pentose phosphate pathway (PPP). Excess flux flows back to the tricarboxylic acid (TCA) cycle for energy production. As with the case of glucose as the substrate, the primary source for lipogenic NADPH is derived from the oxidative PPP.     

Robustness and Plasticity of Metabolic Pathway Flux among Uropathogenic Isolates of Pseudomonas aeruginosa

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.     

Kinetic analysis of a trehalase-overexpressing strain grown on trehalose: a new tool for respiro-fermentative transition studies in Saccharomyces cerevisiae

AIMS: The aim was to demonstrate the use of a trehalase-overexpressing Saccharomyces cerevisiae strain grown on trehalose as a valuable tool in the studies of respiro-fermentative transition at a reduced scale. METHODS AND RESULTS: A trehalase-overexpressing strain was cultivated in synthetic medium on trehalose under aerobic conditions. This strain grew at a maximum specific growth rate of 0.16 h(-1) and showed a pure oxidative metabolism. Glucose pulse experiments were carried out in this system in order to quantify the short-term Crabtree effect. These data were then compared with glucose pulse experiments carried out in the conventional way with the wild-type strain in glucose-limited chemostats. Glucose-pulse experiments in aerobic batch cultures grown on trehalose led to a metabolic respiro-fermentative transition similar to the one observed in glucose-limited chemostats. CONCLUSIONS: This cultivation system allowed us to quantitatively mimic at the flask scale the Crabtree effect observed in conventional chemostat studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is of primary interest in S. cerevisiae studies in which: (i) the implementation of oxidative growth is required (as with studies of the Crabtree effect and heterologous protein production); (ii) small-scale culture systems are required (e.g. high-throughput mutant screening and isotopic labelling experiments).     

Physiology of Sporeforming Bacteria Associated with Insects: Metabolism of Bacillus popilliae Grown in Third-Instar Popillia japonica Newman Larvae

The timing and relative participation of concurrent pathways of carbohydrate metabolism as well as the extent of terminal respiratory activity were determined by radiorespirometry with 14-C substrates and by enzyme assays for vegetative and sporulating cells of the bacterium Bacillus popilliae cultured in whole, intact Popillia japonica (Japanese beetle) larvae. During vegetative proliferation, the pentose phosphate pathway predominates in the bacterial cells with minor involvement of the Embden-Meyerhof-Parnas pathway. As the cells proceed through sporulation, pentose phosphate and Embden-Meyerhof-Parnas activity remains constant. No tricarboxylic cycle activity is evident during growth and sporulation of B. popilliae. The results demonstrate (i) predominantly aerobic metabolism for carbohydrate assimilation within in vivo sporulating cells, (ii) a major contrast to the metabolism of other aerobic sporeforming bacteria that exhibit derepression of tricarboxylic acid cycle enzymatic activity at the onset of sporulation, and (iii) no causal necessity of the cycle to B. popilliae sporogeny.