Response of fluxome and metabolome to temperature-induced recombinant protein synthesis in Escherichia coli

The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:plambdaFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.     

Metabolic flux redistribution in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> in response to osmotic stress

Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes--trehalose first and proline at a later stage of the gradient--increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-- glycolysis and the tricarboxylic acid cycle--while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and alpha-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutamicum.     

RNA synthesis during morphogenesis of the fungusMucor racemosus

Bacteroides succinogenes produces acetate and succinate as major products of carbohydrate fermentation. An investigation of the enzymes involved indicated that pyruvate is oxidized by a flavin-dependent pyruvate cleavage enzyme to acetyl-CoA and CO₂. Active CO₂ exchange is associated with the pyruvate oxidation system. Reduction of flavin nucleotides is CoASH-dependent and does not require ferredoxin. Acetyl-CoA is further metabolized via acetyl phosphate to acetate and ATP. Reduced flavin nucleotide is used to reduce fumarate to succinate by a particulate flavin-specific fumarate reductase reaction which may involve cytochrome b. Phosphoenolpyruvate (PEP) is carboxylated to oxalacetate by a GDP-specific PEP carboxykinase. Oxalacetate, in turn, is converted to malate by a pyridine nucleotide-dependent malate dehydrogenase. The organism has a NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The data suggest that reduced pyridine nucleotides generated during glycolysis are oxidized in malate formation and that the electrons generated during pyruvate oxidation are used to reduce fumarate to succinate.     

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.     

Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3-propanediol

Metabolic fluxes estimated from stable-isotope studies provide a key to understanding cell physiology and regulation of metabolism. A limitation of the classical method for metabolic flux analysis (MFA) is the requirement for isotopic steady state. To extend the scope of flux determination from stationary to nonstationary systems, we present a novel modeling strategy that combines key ideas from isotopomer spectral analysis (ISA) and stationary MFA. Isotopic transients of the precursor pool and the sampled products are described by two parameters, D and G parameters, respectively, which are incorporated into the flux model. The G value is the fraction of labeled product in the sample, and the D value is the fractional contribution of the feed for the production of labeled products. We illustrate the novel modeling strategy with a nonstationary system that closely resembles industrial production conditions, i.e. fed-batch fermentation of Escherichia coli that produces 1,3-propanediol (PDO). Metabolic fluxes and the D and G parameters were estimated by fitting labeling distributions of biomass amino acids measured by GC/MS to a model of E. coli metabolism. We obtained highly consistent fits from the data with 82 redundant measurements. Metabolic fluxes were estimated for 20 time points during course of the fermentation. As such we established, for the first time, detailed time profiles of in vivo fluxes. We found that intracellular fluxes changed significantly during the fed-batch. The intracellular flux associated with PDO pathway increased by 10%. Concurrently, we observed a decrease in the split ratio between glycolysis and pentose phosphate pathway from 70/30 to 50/50 as a function of time. The TCA cycle flux, on the other hand, remained constant throughout the fermentation. Furthermore, our flux results provided additional insight in support of the assumed genotype of the organism.     

A method for accounting for maintenance costs in flux balance analysis improves the prediction of plant cell metabolic phenotypes under stress conditions

Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade‐off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper‐osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper‐osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance.     

敲除frdB基因对大肠杆菌厌氧混合酸发酵的影响

以SPUEC101(产琥珀酸)为出发菌,利用RED同源重组技术敲除延胡索酸还原酶基因frdB,得到重组菌株SPUEC103(△frdB),通过减少延胡索酸生成琥珀酸的通量,实现延胡索酸的积累。实验结果表明:敲除frdB基因后,缺陷菌株生长速率降低,利用葡萄糖的能力也有所降低,同时敲除frdB基因较大程度地改变琥珀酸、延胡索酸等的分布,在两阶段发酵中,当发酵培养基中添加30 g/L的葡萄糖时,琥珀酸和延胡索酸得率最高,对比SPUEC101,SPUEC103的琥珀酸产量产率由24.6%下降为15.4%,并有延胡索酸和少量的苹果酸生成,分别为0.182±0.002 g/L和0.023±0.002 g/L,同时丙酮酸和乙酸含量也略有升高,分别由1.87±0.02 g/L、0.012±0.002 g/L上升到2.36±0.03 g/L、0.862±0.012 g/L。     

NH4+对L-色氨酸发酵的影响

目的:探究NH₄⁺浓度对大肠杆菌E.coli TRTH发酵生产L-色氨酸的影响。方法:通过外源添加试验,利用30 L发酵罐进行分批补料发酵试验,考察E. coli TRTH发酵生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、耗糖速率、发酵液中NH₄⁺浓度及质粒稳定性变化。建立了大肠杆菌合成L-色氨酸的代谢流平衡模型,应用 MATLAB 软件计算出E. coli TRTH发酵中后期代谢网络的代谢流分布。结果:发酵结果显示,利用NaOH和氨水混合补料,控制NH₄⁺浓度在120 mmol/L以下,菌体能够以较长时间和较高比生长速率保持对数生长,最终菌体生物量和L-色氨酸产量分别提高了12.16%和19.80%。随着NH₄⁺浓度的增加,发酵液中丙酮酸、乳酸及乙酸浓度均略有增加,细胞质粒稳定性下降。控制NH₄⁺浓度在120 mmol/L以下,E. coli TRTH发酵生产L-色氨酸的代谢流量分析结果表明,EMP途径的代谢流量降低7.31%,PP途径的代谢流量增加7.14%,TCA循环的代谢流量降低22.04%。结论:高浓度的NH₄⁺导致菌体生长提前结束,耗糖速率降低,产酸受阻,控制NH₄⁺浓度在120 mmol/L以下,解除了NH₄⁺对菌体生长和产物生成的抑制,使得菌体生物量和L-色氨酸产量大幅提高,实现了高密度发酵培养的目的。     

Comparative growth analysis of the facultative anaerobes Bacillus subtilis,Bacillus licheniformis,and Escherichia coli

Bacillus subtilis anaerobic respiration and fermentative growth capabilities were compared to two other facultative anaerobes, Bacillus licheniformis and Escherichia coli. In glycerol defined medium, B. subtilis grew with nitrate, but not nitrite or fumarate, while B. licheniformis grew with nitrate or fumarate, but not nitrite. Growth of E. coli occurred in glycerol defined medium with either nitrate, nitrite, or fumarate. In order to grow by fermentation, B. subtilis required both glucose and pyruvate, while B. licheniformis and E. coli were capable of using either glucose or pyruvate.     

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.     

A kinetic model of Escherichia coli core metabolism satisfying multiple sets of mutant flux data

In contrast to stoichiometric-based models, the development of large-scale kinetic models of metabolism has been hindered by the challenge of identifying kinetic parameter values and kinetic rate laws applicable to a wide range of environmental and/or genetic perturbations. The recently introduced ensemble modeling (EM) procedure provides a promising remedy to address these challenges by decomposing metabolic reactions into elementary reaction steps and incorporating all phenotypic observations, upon perturbation, in its model parameterization scheme. Here, we present a kinetic model of Escherichia coli core metabolism that satisfies the fluxomic data for wild-type and seven mutant strains by making use of the EM concepts. This model encompasses 138 reactions, 93 metabolites and 60 substrate-level regulatory interactions accounting for glycolysis/gluconeogenesis, pentose phosphate pathway, TCA cycle, major pyruvate metabolism, anaplerotic reactions and a number of reactions in other parts of the metabolism. Parameterization is performed using a formal optimization approach that minimizes the discrepancies between model predictions and flux measurements. The predicted fluxes by the model are within the uncertainty range of experimental flux data for 78% of the reactions (with measured fluxes) for both the wild-type and seven mutant strains. The remaining flux predictions are mostly within three standard deviations of reported ranges. Converting the EM-based parameters into a Michaelis–Menten equivalent formalism revealed that 35% of K_m and 77% of k_cat parameters are within uncertainty range of the literature-reported values. The predicted metabolite concentrations by the model are also within uncertainty ranges of metabolomic data for 68% of the metabolites. A leave-one-out cross-validation test to evaluate the flux prediction performance of the model showed that metabolic fluxes for the mutants located in the proximity of mutations used for training the model can be predicted more accurately. The constructed model and the parameterization procedure presented in this study pave the way for the construction of larger-scale kinetic models with more narrowly distributed parameter values as new metabolomic/fluxomic data sets are becoming available for E. coli and other organisms.     

Phenotypic characterization of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under osmotic stress conditions using elementary mode analysis

Corynebacterium glutamicum, a soil bacterium, is used to produce amino acids such as lysine and glutamate. C. glutamicum is often exposed to osmolality changes in its medium, and the bacterium has therefore evolved several adaptive response mechanisms to overcome them. In this study we quantify the metabolic response of C. glutamicum under osmotic stress using elementary mode analysis (EMA). Further, we obtain the optimal phenotypic space for the synthesis of lysine and formation of biomass. The analysis demonstrated that with increasing osmotic stress, the flux towards trehalose formation and energy-generating pathways increased, while the flux of anabolic reactions diminished. Nodal analysis indicated that glucose-6-phosphate, phosphoenol pyruvate, and pyruvate nodes were capable of adapting to osmotic stress, whereas the oxaloacetic acid node was relatively unresponsive. Fewer elementary modes were active under stress indicating the rigid behavior of the metabolism in response to high osmolality. Optimal phenotypic space analysis revealed that under normal conditions the organism optimized growth during the initial log phase and lysine and trehalose formation during the stationary phase. However, under osmotic stress, the analysis demonstrated that the organism operates under suboptimal conditions for growth, and lysine and trehalose formation.     

Redistribution of metabolic fluxes in Escherichia coli with fermentative lactate dehydrogenase overexpression and deletion

Under anaerobic conditions, competition for pyruvate between the branch point enzymes pyruvate formate lyase (PFL, Km = 2 mM) and fermentative lactate dehydrogenase (LDH, Km = 7.2 mM) determines the partition of carbon flux. Two Escherichia coli mutant strains, one deficient in ackA, pta, and ldhA and the other overexpressing LDH, were constructed to systematically analyze the effects of these perturbations in the existing pathways on the redistribution of carbon fluxes. Deletion of the lactate and acetate synthesis pathways was detrimental to cell growth. Carbon flux is forced through ethanol and formate production pathways, resulting in a concomitant increase in those fluxes. In addition, overexpression of LDH simultaneously increases the common flux as well as the flux to the competing acetyl-CoA branch. Overexpression of lactate dehydrogenase (ldhA) in the parent strain increases the lactate synthesis rate from 0.19 to 0.40 mmol/g-biomass-h when the LDH activities increases from 1.3 to 15.3 units. Even an increase of more than 10 times in the LDH activity fails to divert a large fraction of the carbon flux to lactate; the majority of the flux still channels through the acetyl-CoA branch. Overexpression of LDH in the parent strain simultaneously increases the common flux as well as the flux through the acetyl-CoA branch. Subsequently, the flux amplification factors (or deviation indices which can be related to the flux control coefficients) are positive for all three fluxes occurring at the pyruvate node.     

废糟液全循环对自絮凝酵母糖酵解途径关键酶、胁迫相关代谢物及胞内组分的影响

旨在研究废糟液直接全循环对絮凝酵母乙醇发酵、糖酵解关键酶以及细胞组成的影响。在一有效容积1.5 L的搅拌式生物反应器中,使用葡萄糖为220 g/L,添加8 g/L酵母粉和6 g/L蛋白胨的培养基,以0.04 h?1的稀释率进行自絮凝颗粒酵母乙醇连续发酵。每隔3天将收集到的发酵液集中精馏处理,得到的废糟液用于配制发酵培养基。装置运行近20 d,实验结果表明,随着废液循环批次的增加,系统乙醇和生物量浓度明显降低,糖酵解途径3个关键限速酶:己糖激酶、6-磷酸果糖激酶和丙酮酸激酶不同程度受到抑制。为了应对废糟液中高沸点副产物积累导致的环境胁迫,维持细胞正常代谢,甘油和菌体胞内蛋白生物合成加强,碳水化合物积累减弱。这些研究结果对进一步研究高沸点副产物积累对酵母细胞乙醇发酵影响的机理和菌种的代谢工程改造,具有重要意义。     

The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli

In a previous study, an Escherichia coli strain lacking the key enzymes (acetate kinase and phosphotransacetylase, ACK-PTA) of the major acetate synthesis pathways reduced acetate accumulation. The ackA-pta mutant strain also exhibits an increased lactate synthesis rate. Metabolic flux analysis suggested that the majority of excessive carbon flux was redirected through the lactate formation pathway rather than the ethanol synthesis pathway. This result indicated that lactate dehydrogenase may be competitive at the pyruvate node. However, a 10-fold overexpression of the fermentative lactate dehydrogenase (ldhA) gene in the wild-type parent GJT001 was not able to divert carbon flux from acetate. The carbon flux through pyruvate and all its end products increases at the expense of flux through biosynthesis and succinate. Intracellular pyruvate measurements showed that strains overexpressing lactate dehydrogenase (LDH) depleted the pyruvate pool. This observation along with the observed excretion of pyruvate in the ackA-pta strain indicates the significance of intracellular pyruvate pools. In the current study, we focus on the role of the intracellular pyruvate pool in the redirection of metabolic fluxes at this important node. An increasing level of extracellular pyruvate leads to an increase in the intracellular pyruvate pool. This increase in intracellular pyruvate affects carbon flux distribution at the pyruvate node. Partitioning of the carbon flux to acetate at the expense of ethanol occurs at the acetyl-CoA node while partitioning at the pyruvate node favors lactate formation. The increased competitiveness of the lactate pathway may be due to the allosteric activation of LDH as a result of increased pyruvate levels. The interaction between the reactions catalyzed by the enzymes PFL (pyruvate formate lyase) and LDH was examined.     

Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival.

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.     

Continuous modeling of metabolic networks with gene regulation in yeast and in vivo determination of rate parameters

A continuous model of a metabolic network including gene regulation to simulate metabolic fluxes during batch cultivation of yeast Saccharomyces cerevisiae was developed. The metabolic network includes reactions of glycolysis, gluconeogenesis, glycerol and ethanol synthesis and consumption, the tricarboxylic acid cycle, and protein synthesis. Carbon sources considered were glucose and then ethanol synthesized during growth on glucose. The metabolic network has 39 fluxes, which represent the action of 50 enzymes and 64 genes and it is coupled with a gene regulation network which defines enzyme synthesis (activities) and incorporates regulation by glucose (enzyme induction and repression), modeled using ordinary differential equations. The model includes enzyme kinetics, equations that follow both mass-action law and transport as well as inducible, repressible, and constitutive enzymes of metabolism. The model was able to simulate a fermentation of S. cerevisiae during the exponential growth phase on glucose and the exponential growth phase on ethanol using only one set of kinetic parameters. All fluxes in the continuous model followed the behavior shown by the metabolic flux analysis (MFA) obtained from experimental results. The differences obtained between the fluxes given by the model and the fluxes determined by the MFA do not exceed 25% in 75% of the cases during exponential growth on glucose, and 20% in 90% of the cases during exponential growth on ethanol. Furthermore, the adjustment of the fermentation profiles of biomass, glucose, and ethanol were 95%, 95%, and 79%, respectively. With these results the simulation was considered successful. A comparison between the simulation of the continuous model and the experimental data of the diauxic yeast fermentation for glucose, biomass, and ethanol, shows an extremely good match using the parameters found. The small discrepancies between the fluxes obtained through MFA and those predicted by the differential equations, as well as the good match between the profiles of glucose, biomass, and ethanol, and our simulation, show that this simple model, that does not rely on complex kinetic expressions, is able to capture the global behavior of the experimental data. Also, the determination of parameters using a straightforward minimization technique using data at only two points in time was sufficient to produce a relatively accurate model. Thus, even with a small amount of experimental data (rates and not concentrations) it was possible to estimate the parameters minimizing a simple objective function. The method proposed allows the obtention of reasonable parameters and concentrations in a system with a much larger number of unknowns than equations. Hence a contribution of this study is to present a convenient way to find in vivo rate parameters to model metabolic and genetic networks under different conditions.     

13C-metabolic flux analysis of Actinobacillus succinogenes fermentative metabolism at different NaHCO3 and H2 concentrations

Actinobacillus succinogenes naturally produces high concentrations of succinate, a potential intermediary feedstock for bulk chemical productions. A. succinogenes responds to high CO(2) and H(2) concentrations by producing more succinate and by producing less formate, acetate, and ethanol. To determine how intermediary fluxes in A. succinogenes respond to CO(2) and H(2) perturbations, (13)C-metabolic flux analysis was performed in batch cultures at two different NaHCO(3) concentrations, with and without H(2), using a substrate mixture of [1-(13)C]glucose, [U-(13)C]glucose, and unlabeled NaHCO(3). The resulting amino acid, organic acid, and glycogen isotopomers were analyzed by gas chromatography-mass spectrometry and NMR. In all conditions, exchange flux was observed through malic enzyme and/or oxaloacetate decarboxylase. The presence of an exchange flux between oxaloacetate, malate, and pyruvate indicates that, in addition to phosphoenolpyruvate, oxaloacetate, and malate, pyruvate is a fourth node for flux distribution between succinate and alternative fermentation products. High NaHCO(3) concentrations decreased the amount of flux shunted by C(4)-decarboxylating activities from the succinate-producing C(4) pathway to the formate-, acetate-, and ethanol-producing C(3) pathway. In addition, pyruvate carboxylating flux increased in response to high NaHCO(3) concentrations. C(3)-pathway dehydrogenase fluxes increased or decreased appropriately in response to the different redox demands imposed by the different NaHCO(3) and H(2) concentrations. Overall, these metabolic flux changes allowed A. succinogenes to maintain a constant growth rate and biomass yield in all conditions. These results are discussed with respect to A. succinogenes' physiology and to metabolic engineering strategies to increase the flux to succinate.     

Altered fermentative metabolism in Chlamydomonas reinhardtii mutants lacking pyruvate formate lyase and both pyruvate formate lyase and alcohol dehydrogenase

Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H(2) production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H(2) production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.