Estimation of P-to-O ratio inBacillus subtilis and its influence on maximum riboflavin yield

Sauer, Uwe; Bailey, James E.

doi:10.1002/(sici)1097-0290(19990920)64:6<750::aid-bit15>3.0.co;2-s

Cited by 40 publications

(28 citation statements)

References 23 publications

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“…However, when the flux through caa 3 oxidase reaction was restricted to zero under the same conditions to reflect the experimentally observed repression of caa 3 oxidase in the presence of glucose (see below), the in silico model could correctly compute the experimental growth rates of B. subtilis. In B. subtilis, four different terminal oxidases are known to exist, which catalyze the transfer of electrons to O 2 with different energy-coupling efficiencies (16,24) (Fig. 2D).…”

Section: Resultsmentioning

confidence: 99%

“…The model has been used to obtain information on the intracellular flux distribution based on fractional carbon isotope labeling (15) and for the estimation of the P/O ratio (16) and the prediction of maximum theoretical yields of commercial biochemical products (14). Here we describe the reconstruction of a highly detailed and validated genome-scale metabolic network for B. subtilis, which reconciles established genomic and physiological data with high-throughput phenotyping data generated herein.…”

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confidence: 99%

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Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data

Oh¹,

Palsson²,

Park

et al. 2007

Journal of Biological Chemistry

402

356

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In this report, a genome-scale reconstruction of Bacillus subtilis metabolism and its iterative development based on the combination of genomic, biochemical, and physiological information and high-throughput phenotyping experiments is presented. The initial reconstruction was converted into an in silico model and expanded in a four-step iterative fashion. First, network gap analysis was used to identify 48 missing reactions that are needed for growth but were not found in the genome annotation. Second, the computed growth rates under aerobic conditions were compared with high-throughput phenotypic screen data, and the initial in silico model could predict the outcomes qualitatively in 140 of 271 cases considered. Detailed analysis of the incorrect predictions resulted in the addition of 75 reactions to the initial reconstruction, and 200 of 271 cases were correctly computed. Third, in silico computations of the growth phenotypes of knock-out strains were found to be consistent with experimental observations in 720 of 766 cases evaluated. Fourth, the integrated analysis of the large-scale substrate utilization and gene essentiality data with the genomescale metabolic model revealed the requirement of 80 specific enzymes (transport, 53; intracellular reactions, 27) that were not in the genome annotation. Subsequent sequence analysis resulted in the identification of genes that could be putatively assigned to 13 intracellular enzymes. The final reconstruction accounted for 844 open reading frames and consisted of 1020 metabolic reactions and 988 metabolites. Hence, the in silico model can be used to obtain experimentally verifiable hypothesis on the metabolic functions of various genes.Bacillus subtilis has been the organism of choice for the production of several important industrial products, including antibiotics, enzymes, nucleosides, and vitamins. Several aspects of the biochemistry, genetics, and physiology of B. subtilis have been studied extensively making B. subtilis the best characterized prokaryote second only to Escherichia coli (1, 2). In addition various "-omics" data sets such as transcriptomic (3), proteomic (4), and metabolomic (5) are available for B. subtilis. This wealth of experimental data enables the development of a genome-scale metabolic in silico model that can be used not only for quantitative interpretation and structured integration of such extensive data sets but also as a tool for hypothesis generation and engineering of B. subtilis metabolism.To date, constraint-based reconstruction and analysis (COBRA) 4 of cellular metabolism has been employed successfully to develop organism-specific genome-scale in silico models that have enabled numerous applications (6). Unlike other modeling strategies such as kinetic (7), stochastic (8), and cybernetic (9) methods, the COBRA approach does not attempt to compute precisely what a biochemical network does; rather, it seeks to distinguish between the network states that are achievable from those that are not, based on a detailed reconstruction of...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data

Oh¹,

Palsson²,

Park

et al. 2007

Journal of Biological Chemistry

402

356

View full text Add to dashboard Cite

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“…Transport of one electron from FADH, however, translocates only one proton (49). Consequently, all cultures were assumed to exhibit a maximal P/O of unity (the exact value depends on the amount of FADH that is produced in the tricarboxylic acid [TCA] cycle), as was previously suggested for B. subtilis (10,44). This P-to-O ratio corresponds to the generation of 1 ATP per NADH and 0.5 ATP per FADH, so that complete oxidation of glucose to carbon dioxide yields 15 ATP.…”

Section: Vol 183 2001mentioning

confidence: 99%

Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

2001

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The energetic efficiency of microbial growth is significantly reduced in cultures growing under glucose excess compared to cultures growing under glucose limitation, but the magnitude to which different energy-dissipating processes contribute to the reduced efficiency is currently not well understood. We introduce here a new concept for balancing the total cellular energy flux that is based on the conversion of energy and carbon fluxes into energy equivalents, and we apply this concept to glucose-, ammonia-, and phosphate-limited chemostat cultures of riboflavin-producing Bacillus subtilis. Based on [U-13 C 6 ]glucose-labeling experiments and metabolic flux analysis, the total energy flux in slow-growing, glucose-limited B. subtilis is almost exclusively partitioned in maintenance metabolism and biomass formation. In excess-glucose cultures, in contrast, uncoupling of anabolism and catabolism is primarily achieved by overflow metabolism, while two quantified futile enzyme cycles and metabolic shifts to energetically less efficient pathways are negligible. In most cultures, about 20% of the total energy flux could not be assigned to a particular energy-consuming process and thus are probably dissipated by processes such as ion leakage that are not being considered at present. In contrast to glucoseor ammonia-limited cultures, metabolic flux analysis revealed low tricarboxylic acid (TCA) cycle fluxes in phosphate-limited B. subtilis, which is consistent with CcpA-dependent catabolite repression of the cycle and/or transcriptional activation of genes involved in overflow metabolism in the presence of excess glucose. ATPdependent control of in vivo enzyme activity appears to be irrelevant for the observed differences in TCA cycle fluxes.The very basis of microbial growth resides in balanced fluxes through anabolic and catabolic reactions. These metabolic fluxes are highly variable and change with the environmental conditions and the rate of growth, since faster-growing cells demand a higher rate of metabolism. To delineate these influences, metabolic flux responses are typically studied in chemostat cultures that are maintained under different nutrient limitations. When microorganisms are limited for their energy source (usually the carbon source), catabolism is tightly coupled to anabolism and high biomass yields on the carbon source are achieved (40). Compared to those with carbon (C) limitation, excess-C cultures exhibit generally high rates of carbon consumption and low yields of biomass and thus have a low energetic growth efficiency (11,31,32,57). Most frequently excess-C cultures in chemostats are limited by other cellular macroelements such as nitrogen (N) and phosphorus (P) but also potassium, the predominant intracellular cation (11). Potassium or P limitation invokes usually a strong uncoupling of catabolic and anabolic processes that lead consequently to low biomass yields on the energy source, while N limitation has more moderate effects on cellular physiology (31, 68). Although mostly studied in the gr...

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“…Unlike NADPH production, metabolic production of ATP is not directly accessible from the flux distribution because the stoichiometry of ATP generation via respiration and ATPase is not known exactly and may vary with environmental conditions (49). It is not unreasonable, however, to assume identical efficiencies of ATP generation in all the carbon-limited samples investigated here, thus allowing for a direct comparison of ATP production in the five experiments.…”

Section: Vol 68 2002 Carbon Fluxes In Riboflavin-producing B Subtimentioning

confidence: 99%

“…It is not unreasonable, however, to assume identical efficiencies of ATP generation in all the carbon-limited samples investigated here, thus allowing for a direct comparison of ATP production in the five experiments. Specifically, we used a P-to-O ratio of about 1, which corresponds to the generation of 1 ATP molecule per NADH (13,49). Depending on the estimated flux through the succinate dehydrogenase that produces the energetically less valuable FADH, however, the Pto-O ratio may be lower than 1.…”

Section: Vol 68 2002 Carbon Fluxes In Riboflavin-producing B Subtimentioning

confidence: 99%

Intracellular Carbon Fluxes in Riboflavin-Producing Bacillus subtilis during Growth on Two-Carbon Substrate Mixtures

Dauner

Sonderegger

Hochuli

et al. 2002

Appl Environ Microbiol

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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.During batch growth on mixtures of carbon substrates, bacteria frequently first consume almost exclusively their preferred substrate. Consumption of any other substrate(s) occurs only after depletion of the preferred one, leading to a diauxic pattern of growth. The primary molecular mechanisms that inhibit the simultaneous utilization of substrates are catabolite repression (9) and inducer exclusion (47). Under carbon-limited conditions in chemostat cultures, in contrast, mixtures of carbon sources are often utilized simultaneously at low and intermediate dilution rates (D) (18). Thus, for the most frequently used paradigm catabolite repression sugar, glucose, such coutilization occurs at concentrations below a critical repression level (29). Consequently, many catabolic enzymes are relieved from repression so that alternative substrates can be catabolized (32,34).While many biotechnological processes operate on a single carbon source, substrate mixtures may help to diagnose potential bottlenecks in the biosynthetic pathways to a desired product (10). For the production of riboflavin (vitamin B 2 ), which is a commercially important additive in the feed and food industries, such cofeeding experiments were used to identify potential limitations in building block supply (55) (Fig. 1). Generally, cofeeding experiments are evaluated on the basis of physiological analysis, with a primary focus on production. Much information on the underlying metabolic network response, however, is not accessible from extracellular physiological data but requires knowledge of intracellular carbon fluxes.The classical approach to analyzing intracellular car...

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Estimation of P-to-O ratio inBacillus subtilis and its influence on maximum riboflavin yield

Cited by 40 publications

References 23 publications

Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data

Genome-scale Reconstruction of Metabolic Network in Bacillus subtilis Based on High-throughput Phenotyping and Gene Essentiality Data

Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

Intracellular Carbon Fluxes in Riboflavin-Producing Bacillus subtilis during Growth on Two-Carbon Substrate Mixtures

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