Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. 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.     

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis

Ribulose 5-phosphate is a precursor for riboflavin biosynthesis. Alteration of carbon flow into the pentose phosphate pathway will affect the availability of ribulose 5-phosphate and the riboflavin yield. We have modulated carbon flow in Bacillus subtilis through the gluconate bypass by over-expression of glucose dehydrogenase under the control of the constitutively expressed P43 promoter. Over-expression of glucose dehydrogenase resulted in low acid production (acetate and pyruvate). The substantial reduction in acid production is accompanied by increased riboflavin production and an increased rate of growth while glucose consumption remained unchanged. Metabolic analysis indicated that over-expression of glucose dehydrogenase increased intracellular pool of ribulose 5-phosphate. The high concentrations of ribulose 5-phosphate could explain the increased riboflavin production.     

Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis

Carbon flow in Bacillus subtilis through the pentose phosphate (PP) pathway was modulated by overexpression of glucose-6-phosphate dehydrogenase (G6PDH) under the control of the inducible Pxyl promoter in B. subtilis PY. Alteration of carbon flow into the PP pathway will affect the availability of ribulose-5-phosphate (Ru5P) and the riboflavin yield. Overexpression of G6PDH resulted in the glucose consumption rate increasing slightly, while the specific growth rate was unchanged. An improvement by 25% ± 2 of the riboflavin production was obtained. Compared to by-products formation in flask culture, low acid production (acetate and pyruvate) and more acetoin were observed. Metabolic analysis, together with carbon flux redistribution, indicated that the PP pathway fluxes are increased in response to overexpression of G6PDH. Moreover, increased flux of the PP pathway is associated with an increased intracellular pool of Ru5P, which is a precursor for riboflavin biosynthesis. The high concentrations of Ru5P could explain the increased riboflavin production.     

Enhancement of l-ornithine production by disruption of three genes encoding putative oxidoreductases in Corynebacterium glutamicum

Recently, Corynebacterium glutamicum has been shown to exhibit gluconate bypass activity, with two key enzymes, glucose dehydrogenase (GDH) and gluconate kinase, that provides an alternate route to 6-phosphogluconate formation. In this study, gene disruption analysis was used to examine possible metabolic functions of three proteins encoded by open reading frames having significant sequence similarity to GDH of Bacillus subtilis. Chromosomal in-frame deletion of three genes (NCgl0281, NCgl2582, and NCgl2053) encoding putative NADP⁺-dependent oxidoreductases led to the absence of GDH activity and correlated with increased specific glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. This finding suggested that enhanced carbon flux from glucose was directed toward the oxidative pentose phosphate (PP) pathway, when the mutant was cultivated with 6 % glucose. Consequently, the mutant showed 72.4 % increased intracellular NADPH and 66.3 % increased extracellular l-ornithine production. The enhanced activities of the oxidative PP pathway in the mutant explain both the increased intracellular NADPH and the high extracellular concentration of l-ornithine. Thus, the observed metabolic changes in this work corroborate the importance of NADPH in l-ornithine production from C. glutamicum.     

Fermentation and metabolic characteristics of Gluconacetobacter oboediens for different carbon sources

The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h⁻¹. The ¹³C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO₂ production rate and the biomass concentration decreased, where the ¹³C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as the feed gluconate concentration was increased.     

Pyruvate production by Enterococcus casseliflavus A-12 from gluconate in an alkaline medium

A newly isolated lactic acid bacterium, Enterococcus casseliflavus A-12, produced pyruvic acid (16 g/l) during aerobic culture in an alkaline medium containing sodium gluconate (50 g/l) as the carbon source. The production was dependent on the pH of the culture, the optimum initial pH being 10.0. With static culture, the organism produced lactic acid (2.7 g/l) from both gluconate and glucose. Pyruvate did not accumulate in growing cultures on glucose, but resting cells obtained from a culture on gluconate produced pyruvate from glucose as well as gluconate. The enzyme profiles of the organism, which grew on gluconate and glucose, suggested that gluconate was metabolized via the Entner-Doudoroff and Embdem-Meyerhof-Parnas pathways in aerobic culture, and that glucose was oxidized mainly via the latter pathway under both aerobic and anaerobic conditions. Gluconokinase, a key enzyme in the aerobic metabolism of gluconate, was partially purified from this strain and characterized.     

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.     

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Regulatory effects on central carbon metabolism from poly-3-hydroxybutryate synthesis

Poly-3-hydroxybutyrate (PHB) synthesis in Escherichia coli elicits regulatory responses that affect product yield and productivity. We used controlled, steady-state cultures (chemostats) of a genetically stable strain to determine growth-independent metabolic flux regulation. We measured flux and steady-state intracellular metabolite concentrations across different dilution rates (0.05, 0.15, 0.3 h⁻¹), limitations (glucose, gluconate and nitrogen), and operon copy counts of the PHB pathway (0, 6, 17, and 29). As PHB flux increases, specific substrate consumption and lactate secretion increase while formate and acetate secretion decreases in N-limited, glucose-fed conditions.To understand the regulatory mechanisms that resulted in these macroscopic changes, we used a flux balance analysis model to analyze intracellular redox conditions. Our model shows that under N-limited conditions, synthesis of PHB creates excess reducing equivalents. Cells, under these conditions, secrete more reduced metabolites in order to recycle reducing equivalents. By switching to a more oxidized substrate (gluconate) that decreased excess reducing equivalents, PHB flux yield increased 1.6 fold compared to glucose-fed fermentations. High flux of PHB (~1.2 mmol/g DCW h) was maintained under these steady-state, oxidized conditions. These results imply redox imbalance is a driving force in industrial production of PHB, and substrates that are more oxidized than glucose can increase productivity.     

Utilization of gluconate by Escherichia coli. Induction of gluconate kinase and 6-phosphogluconate dehydratase activities

1. A mutant of Escherichia coli, devoid of phosphopyruvate synthetase, glucosephosphate isomerase and 6-phosphogluconate dehydrogenase activities, grew readily on gluconate and inducibly formed an uptake system for gluconate, gluconate kinase and 6-phosphogluconate dehydratase while doing so. 2. This mutant also grew on glucose 6-phosphate and inducibly formed 6-phosphogluconate dehydratase; however, the formation of the gluconate uptake system and gluconate kinase was not induced under these conditions. 3. The use of the Entner–Doudoroff pathway for the dissimilation of 6-phosphogluconate, derived from either gluconate or glucose 6-phosphate, by this mutant was also demonstrated by the accumulation of 2-keto-3-deoxy-6-phosphogluconate (3-deoxy-6-phospho-l-glycero-2-hexulosonate) from both these substrates in a similar mutant that also lacked phospho-2-keto-3-deoxygluconate aldolase activity. 4. Glucose 6-phosphate inhibits the continued utilization of fructose by cultures of the mutants growing on fructose, as it does in wild-type E. coli. 5. The mutants do not use glucose for growth. This is shown to be due to insufficiency of phosphopyruvate, which is required for glucose uptake.     

Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) gluconate 6-phosphatase (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.Abbreviations GOD glucose oxidase - GD glucose dehydrogenase - PP pentose phosphate - EM Embden-Meyerhof - TCA tricarboxylic acid     

The kinetics and physiology of stipitatic acid and gluconate production by carbon sufficient cultures of Penicillium stipitatum growing in continuous culture

The yield from glucose of ammonia-grown carbon-limited continuous cultures of Penicillium stipitatum was ca. 20% higher than that of nitrate-grown cultures at all growth rates examined. However, the yield from oxygen was similar during growth on both nitrogen sources. Under phosphate limitation the specific rate of gluconic acid and stipitatic acid production increased with growth rate, but the former product accounted for virtually 100% of the excreted carbon. Stipitatic acid was not produced under nitrogen limitation, and glucose supplied to the culture in excess of that required for growth was virtually quantatively converted into gluconic acid. Productivities of 11.4 g gluconic acid/L/h were stably maintained in continuous culture. Under conditions of glucose excess the enzyme glucose oxidase was excreted into the culture. The specific activity of this extracellular enzyme increased when the input glucose concentration to the culture was progressively increased. The excretion of a protein under nitrogen limitation suggests that this enzyme plays an important role under these conditions. Indeed, it was demonstrated that nitrogen-limited cultures did not overmetabolize gluconate at either pH 6.5 or 3.5, although up to 29 g/L gluconate was present in the culture. The Y(gluconate) and YO(2) of C- and N-limited gluconate-grown cultures were similar indicating that the rapid conversion of glucose to gluconate probably affords a means of regulating carbon flow in this organism. Nitrogen-limited cultures of P. stipitatum overmetabolized glucose to a much greater extent than acetate, fructose, or gluconate.     

Improvement of pyruvate production based on regulation of intracellular redox state in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

The commercial demand for pyruvate has been expanding. However, some challenges need to be overcome in the microbial production of pyruvate, such as low glucose consumption caused by excessive accumulation of NADH. In this study, weakening or block of the TCA cycle, overexpression of foreign NADH oxidase, and carbon sources with different oxidation state was attempted to decrease NADH accumulation in engineered strain YP211. Results showed that blocking or weakening TCA cycle could not lower the intracellular redox state in strain YP211.Overexpressing NADH oxidase from Lactococcus lactis significantly decreased the intracellular NADH content and increased the consumption rate of glucose. However, the yield of pyruvate did not increase significantly. Compared with glucose as carbon source, sodium gluconate with a higher oxidation state resulted in a significant decrease of NADH/NAD⁺, and the concentration and yield of pyruvate increased by 62 and 6%, respectively. In the fed-batch fermentation, the yield of pyruvate increased to 0.78 g/g gluconate, and the concentration of pyruvate reached 78.8 g/L. It was suggested that sodium gluconate was a more ideal carbon source for strain YP211, which could effectively decrease NADH content and improve the pyruvate production.     

Enhancement of riboflavin production by deregulating gluconeogenesis in Bacillus subtilis

The regulation of metabolic flux through glycolytic versus the gluconeogenic pathway plays an important role in central carbon metabolism. In this study, we made an attempt to enhance riboflavin production by deregulating gluconeogenesis in Bacillus subtilis. To this end, gapB (code for NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase), fbp (code for fructose-1,6-bisphosphatase) and pckA (code for phosphoenolpyruvate carboxykinase) were overexpressed in parental strain B. subtilis RH33. Compared with RH33, overexpression of fbp and gapB resulted in approximately 18.0 and 14.2 % increased riboflavin production, respectively, while overexpression of pckA obtained the opposite result. Significant enhancement of riboflavin titers up to 4.89 g/l was obtained in shake flask cultures when gapB and fbp were co-overexpressed, nevertheless the specific growth rate decreased slightly and the specific glucose uptake rate remained almost unchanged. An improvement by 21.9 and 27.8 % of the riboflavin production was achieved by co-overexpression of gapB and fbp in shake flask and fed-batch fermentation, respectively. These results imply that deregulation of gluconeogenesis is an effective strategy for production of metabolites directly stemming from the pentose phosphate pathway as well as other NADPH-demanding compounds with glucose as carbon source in B. subtilis.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium

Escherichia coli overexpressing a NAD(+)-dependent formate dehydrogenase (FDH) from Candida boidinii was grown in chemostat culture on various carbon sources at 0.05 h(-1) dilution rate, under anaerobic conditions using defined medium and compared to a control without the heterologous FDH pathway. Metabolic fluxes, NADH/NAD(+) ratios and NAD(H/(+)) levels were determined under a range of intracellular NADH availability. The effect of NADH manipulation on the distribution of metabolic fluxes in E. coli was assessed under steady-state conditions. The heterologous FDH pathway converts 1 mol of formate into 1 mol of NADH and carbon dioxide, in contrast with the native FDH where no cofactor involvement is present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed and reached 4.6 mol NADH/mol of substrate when sorbitol was used as a carbon source in a complex medium. In the current study, it was found that higher NADH yields and NADH/NAD(+) ratios were achieved with our in vivo NADH regeneration system compared to a control lacking the new FDH pathway in the three carbon sources (glucose, gluconate and sorbitol) examined suggesting a more reduced intracellular environment. The total NAD(H/(+)) amounts were very similar for all the combinations studied. It was also found that the ethanol to acetate ratio increased with increased NADH availability. This ratio increased from 1.05 for the control strain in glucose to 9.45 for the strain expressing the heterologous NAD(+)-dependent FDH in sorbitol.     

Comparative study of riboflavin production from two microorganisms: Eremothecium ashbyii and Ashbya gossypii

The conversion of the various substrates to riboflavin via fermentation by Eremothecium ashbyii and Ashbya gossypii was investigated. Optimum operation temperature and initial pH of the medium were determined as 30°C and 6.5 respectively for both microorganisms. In order to examine the effects of different substrates and their initial concentrations, glucose, glycerol, sunflower oil, whey and various combinations of these were utilized in the experiments. Maximum specific growth rates and riboflavin yields were obtained in the media which contained glucose and sunflower oil together as the substrate.     

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.     

Fermentation of Xylose Causes Inefficient Metabolic State Due to Carbon/Energy Starvation and Reduced Glycolytic Flux in Recombinant Industrial Saccharomyces cerevisiae

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose.