Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.     

Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum

L-Valine can be formed successfully using C. glutamicum strains missing an active pyruvate dehydrogenase enzyme complex (PDHC). Wild-type C. glutamicum and four PDHC-deficient strains were compared by (13)C metabolic flux analysis, especially focusing on the split ratio between glycolysis and the pentose phosphate pathway (PPP). Compared to the wild type, showing a carbon flux of 69% ± 14% through the PPP, a strong increase in the PPP flux was observed in PDHC-deficient strains with a maximum of 113% ± 22%. The shift in the split ratio can be explained by an increased demand of NADPH for l-valine formation. In accordance, the introduction of the Escherichia coli transhydrogenase PntAB, catalyzing the reversible conversion of NADH to NADPH, into an L-valine-producing C. glutamicum strain caused the PPP flux to decrease to 57% ± 6%, which is below the wild-type split ratio. Hence, transhydrogenase activity offers an alternative perspective for sufficient NADPH supply, which is relevant for most amino acid production systems. Moreover, as demonstrated for L-valine, this bypass leads to a significant increase of product yield due to a concurrent reduction in carbon dioxide formation via the PPP.     

Increasing available NADH supply during succinic acid production by Corynebacterium glutamicum

A critical factor in the biotechnological production of succinic acid with Corynebacterium glutamicum is the sufficient supply of NADH. It is conceivable that cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the succinic acid yield. PntAB genes from Escherichia coli can directly catalyze the reversible hydride transfer and adjust the dynamic balance between NADP(H) and NAD(H). Hence, we studied the physiological effect of coenzyme systems by expressing the membrane‐bound transhydrogenase pntAB genes. We have shown experimentally that the pntAB genes could function as an alternative source of NADH. In an anaerobic fermentation with C. glutamicum NC‐3‐pntAB, a 16% higher succinic acid yield and a 57% higher production from glucose were obtained by pntAB expression. Moreover, the formation of by‐products was significantly decreased. The concomitant increase in the consumption of intracellular NADPH from 0.6 to 1.2 mmol/g CDW and the increased NADH/NAD⁺ ratio resulted from introduction of pntAB, suggesting that the membrane‐bound transhydrogenase converted excess NADPH to NADH for succinic acid production. Finally, we explored whether the transhydrogenase had different effects on the succinic acid formation on different carbon sources. The succinic acid yield was increased in the presence of pntAB by 16% on glucose, 7% on sucrose, and without large influence on fructose and xylose. The results of this study demonstrated that the effectiveness of cofactor manipulation could be a promising strategy applied in metabolic engineering. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:12–19, 2015     

Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and alpha-ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.     

Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint-based modeling and experimental verification

Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.     

Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H(2)-producing conditions

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H(2)) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on (13)C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H(2)-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H(2) yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-β-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD(+). Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H(2) production.     

A theoretical analysis of the biosynthesis of actinorhodin in a hyper-producing Streptomyces lividansstrain cultivated on various carbon sources

A stoichiometric equation for the biosynthesis of actinorhodin (ACT) was derived taking into consideration both the requirements of the carbon precursors (acetyl-CoA) and reducing power (NADPH). The estimate for reducing power was derived from a detailed molecular analysis of each step in the ACT biosynthetic pathway. Even though ACT is slightly more oxidized than most carbon substrates, e.g. glucose, reducing power (NADPH and NADH) is necessary due to reducing steps and to monooxygenase steps. The equation was used to evaluate, in a metabolic network context, the experimental results from batch fermentations with eight different carbon sources using a Streptomyces lividans 1326 derived strain containing the pathway-specific activator gene ( actII-ORF4) on a multicopy plasmid (pIJ68). The yield of ACT on the various carbon sources ranged from 0.04 to 0.18 Cmol ACT/Cmol carbon source in the stationary phase. Glucose was the best carbon source and supported a yield of 25% of the maximum theoretical yield. There are no obvious constraints in the primary metabolic pathways that can explain why the various carbon sources allowed different levels of ACT production, because their potential for supplying acetyl-CoA and NADPH are far from fully utilized. For the observed ACT yields, there is an excess production of NADPH that has to be reoxidized either by a transhydrogenase or a NADPH oxidase. This study discusses the central metabolic pathways, focusing on providing precursors for ACT synthesis.     

Elementary mode analysis to study the preculturing effect on the metabolic state of Lactobacillus rhamnosus during growth on mixed substrates

Quantification of metabolism through elementary modes offers insights into the working of a metabolic network. We have determined the fluxes of elementary modes through linear optimization using the stoichiometry of the elementary modes as a constraint. We apply this methodology to obtain insights into the effect of preculturing on growth of Lactobacillus rhamnosus on medium containing mixed substrates. L. rhamnosus, a microaerophilic organism, produces flavor compounds such as diacetyl and acetoin during growth on glucose and citrate. The uptake of citrate has been shown to be sensitive to preculturing states of the cells. Elementary modes demonstrated that citrate was utilized by the organism as a sole carbon source. Further, both glucose and citrate was catabolized by this organism through aerobic and anaerobic routes. The flux analysis indicated that only 21 elementary modes were operational during growth of L. rhamnosus on glucose and citrate. Glucose specifically accounted for 6 elementary modes, while the remaining 15 involved citrate as substrate. The modes associated with glucose were mainly operational when cells were precultured on glucose. It was observed that all the 21 modes contributed to the fluxes when the cells were precultured on citrate. The NADH recycling through lactate formation and oxygen uptake were dependent on the preculturing state. The analysis also demonstrated that preculturing on citrate yielded better productivity of diacetyl and acetoin.     

Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids. By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified. This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source. The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system. Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand. The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization.     

Identification of metabolic model: Citrate production from glucose by Candida lipolytica

The concept of mass balance was used to analyze the metabolic pathways of citrate production by Candida lipolytica from glucose. Specific rates of glucose consumption, citrate and isocitrate productions, carbon dioxide evolution, and cellular syntheses of protein and carbohydrate were observed in an NH₄⁺-limited chemostat culture. These data permitted one to assess the carbon flux in vivo by solving simultaneous carbon balance equations with respect to intermediary metabolite pools in the steady State. Among the three models considered here, model I (which coordinates the pyruvate carboxylation with the tricarboxylic acid cycle, but disregards the glyoxylate cycle) was considered plausible because the carbon flux calculated so far was acceptable. On the other hand, models II and III (which overlook the pyruvate carboxylation and the 2-oxoglutarate dehydrogenation, respectively) were found to be most unlikely because of the unusual flux assessed from these models.     

Aerobic glucose metabolism of Saccharomyces kluyveri: growth, metabolite production, and quantification of metabolic fluxes.

The growth and product formation of Saccharomyces kluyveri was characterized in aerobic batch cultivation on glucose. At these conditions it was found that ethyl acetate was a major overflow metabolite in S. kluyveri. During the exponential-growth phase on glucose ethyl acetate was produced at a constant specific rate of 0.12 g ethyl acetate per g dry weight per hour. The aerobic glucose metabolism in S. kluyveri was found to be less fermentative than in S. cerevisiae, as illustrated by the comparably low yield of ethanol on glucose (0.08 +/- 0.02 g/g), and high yield of biomass on glucose (0.29 +/- 0.01 g/g). The glucose metabolism of S. kluyveri was further characterized by the new and powerful techniques of metabolic network analysis. Flux distributions in the central carbon metabolism were estimated for respiro-fermentative growth in aerobic batch cultivation on glucose and respiratory growth in aerobic glucose-limited continuous cultivation. It was found that in S. kluyveri the flux into the pentose phosphate pathway was 18.8 mmole per 100 mmole glucose consumed during respiratory growth in aerobic glucose-limited continuous cultivation. Such a low flux into the pentose phosphate pathway cannot provide the cell with enough NADPH for biomass formation which is why the remaining NADPH will have to be provided by another pathway. During batch cultivation of S. kluyveri the tricarboxylic acid cycle was working as a cycle with a considerable flux, that is in sharp contrast to what has previously been observed in S. cerevisiae at the same growth conditions, where the tricarboxylic acid cycle operates as two branches. This indicates that the respiratory system was not significantly repressed in S. kluyveri during batch cultivation on glucose.     

Sustained and constitutive high levels of protein production in continuous cultures of bacillus subtilis

The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h(-1) production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h(-1) were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. (c) 1995 John Wiley & Sons Inc.     

Ensemble Modeling of Hepatic Fatty Acid Metabolism with a Synthetic Glyoxylate Shunt

The liver plays a central role in maintaining whole body metabolic and energy homeostasis by consuming and producing glucose and fatty acids. Glucose and fatty acids compete for hepatic substrate oxidation with regulation ensuring glucose is oxidized preferentially. Increasing fatty acid oxidation is expected to decrease lipid storage in the liver and avoid lipid-induced insulin-resistance. To increase hepatic lipid oxidation in the presence of glucose, we previously engineered a synthetic glyoxylate shunt into human hepatocyte cultures and a mouse model and showed that this synthetic pathway increases free fatty acid β-oxidation and confers resistance to diet-induced obesity in the mouse model. Here we used ensemble modeling to decipher the effects of perturbations to the hepatic metabolic network on fatty acid oxidation and glucose uptake. Despite sampling of kinetic parameters using the most fundamental elementary reaction models, the models based on current metabolic regulation did not readily describe the phenotype generated by glyoxylate shunt expression. Although not conclusive, this initial negative result prompted us to probe unknown regulations, and malate was identified as inhibitor of hexokinase 2 expression either through direct or indirect actions. This regulation allows the explanation of observed phenotypes (increased fatty acid degradation and decreased glucose consumption). Moreover, the result is a function of pyruvate-carboxylase, mitochondrial pyruvate transporter, citrate transporter protein, and citrate synthase activities. Some subsets of these flux ratios predict increases in fatty acid and decreases in glucose uptake after glyoxylate expression, whereas others predict no change. Altogether, this work defines the possible biochemical space where the synthetic shunt will produce the desired phenotype and demonstrates the efficacy of ensemble modeling for synthetic pathway design.     

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.     

Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study

(sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism.     

Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated over a broad range with an isopropyl-beta-D-thiogalactopyranoside- (IPTG-) inducible plasmid, and the consequences on microaerobic Escherichia coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygen-limited conditions was most visible during late fed-batch phase where the final cell density increased initially linearly with increasing VHb concentrations, ultimately saturating at a 2.7-fold increase over the VHb-negative (Vhb(-)) control. During the same growth phase, the specific excretions of fermentation by-products, acetate, ethanol, formate, lactate, and succinate from the culture expressing the highest amount of VHb were reduced by 25%, 49%, 68%, 72%, and 50%, respectively, relative to the VHb(-) control. During the exponential growth phase, VHb exerted a positive but smaller control on growth rate, growth yield, and respiration. Varying the amount of VHb from 0 to 3.8 mumol/g dry cell weight (DCW) increased the specific growth rate, the growth yield, and the oxygen consumption rate by 33%, 35%, and 60%, respectively. Increasing VHb concentration to 3.8 mumol/g DCW suppressed the rate of carbon dioxide evolution in the exponential phase by 30%. A metabolic flux distribution analysis incorporating data from these cultivations discloses that VHb(+) cells direct a larger fraction of glucose toward the pentose phosphate pathway and a smaller fraction of carbon through the tricarboxylic acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cells generate a net NADH flux by the NADH/NADPH transhydrogenase while the VHb(-) cells yield a net NADPH flux under the same growth conditions. Flux distribution analysis also reveals that VHb(+) cells have a smaller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microaerobic conditions. The thermodynamic efficiency of growth, based on reducing equivalents generated per unit of biomass produced, is greater for VHb(+) cells. (c) 1996 John Wiley & Sons, Inc.     

The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger

Summary The influence of various carbon sources and their concentration on the production of citrate by Aspergillus niger has been investigated. The sugars maltose, sucrose, glucose, mannose and fructose (in the given order) were carbon sources giving high yields of citric acid. Optimal yields were observed at sugar concentrations of 10% (w/v), with the exception of glucose (7.5%). No citric acid was produced on media containing less than 2.5% sugar. Precultivation of A. niger on 1% sucrose and transference to a 14% concentration of various other sugars induced citrate accumulation. This could be blocked by the addition of cycloheximide, an inhibitor of de novo protein synthesis. This induction was achieved using maltose, sucrose, glucose, mannose and fructose, and also by some other carbon sources (e.g. glycerol) that gave no citric acid accumulation in direct fermentation. Precultivation of A. niger at high (14%) sucrose concentrations and subsequent transfer to the same concentrations of various other carbohydrates, normally not leading to citric acid production, led to formation of citrate. Endogenous carbon sources were also converted to citrate under these conditions. A 14%-sucrose precultivated mycelium continued producing some citrate upon transfer to 1% sugar. These results indicate that high concentrations of certain carbon sources are required for high citrate yields, because they induce the appropriate metabolic imbalance required for acidogenesis.     

Metabolic flux in glucose/citrate co-fermentation by lactic acid bacteria as measured by isotopic ratio analysis

The flux of carbon into lactic acid, diacetyl and acetoin during the co-metabolism of glucose and citrate by Lactococcus lactis subsp. lactis biovar. diacetylactis has been determined using natural abundance isotopic ratio analysis. During fermentation in the conditions used (glucose, 27.8 mM; citric acid, 13.9 mM; initial pH 6.2-6.4, anaerobic) it is shown that approximately 65% of the carbon source used for the aroma compounds is derived from the carbohydrate. Equally, citrate contributes approximately 30% of the carbon recovered in lactic acid. Thus, there is no evidence for a metabolic separation of the catabolism of these two carbon sources.     

13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis

When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.     

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha-amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose (gamma=4.0), acetate (gamma=4.0), and the TCA cycle organic-acids succinate (gamma=3.5), malate (gamma=3.0), and citrate (gamma=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates (&mgr;) were calculated as 1.142 and 0.766h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191mmolg(-1)DWh(-1) when glucose uptake rate was 10mmolg(-1)DWh(-1) and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.