Metabolic flux analysis using stoichiometric models for Aspergillus niger: comparison under glucoamylase-producing and non-producing conditions

Aspergillus niger AB1.13 cultures with glucoamylase production (with D-glucose as substrate) and without glucoamylase production (with D-xylose as substrate) were characterized by metabolic flux analysis. Two comprehensive metabolic models for d-glucose- as well as for D-xylose-consumption were used to quantify and compare the metabolic fluxes through the central pathways of carbon metabolism at different pH-values. The models consist of the most relevant metabolic pathways for A. niger including glycolysis, pentose-phosphate pathway, citrate cycle, energy metabolism and anaplerotic reactions comprising two intracellular compartments, the cytoplasm and mitochondrion. When D-xylose was used as the sole carbon source, the relative flux of the substrate through the oxidative pentose-phosphate pathway (PPP) via G6P-dehydrogenase was unaffected by the pH-value of the culture medium. About 30% of D-xylose consumed was routed through the oxidative PPP. In contrast, the flux of D-glucose (i.e., under glucoamylase-producing conditions) through the oxidative PPP was remarkably higher and, in addition was significantly affected by the pH-value of the culture medium (40% at pH 5.5, 56% at pH 3.7, respectively). Summarizing, the flux through the PPP under glucoamylase producing conditions was 30-90% higher than for non-producing conditions.     

Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and ¹³C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest.     

Drosophila Alcohol Dehydrogenase Polymorphism and Carbon-13 Fluxes: Opportunities for Epistasis and Natural Selection

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of (13)C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. α,α-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD(+) balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in Adh(F) larvae. In Adh(S) larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic fluxes may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.     

Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: effects of the oxygen transfer rate.

The metabolic fluxes through the central carbon pathways in the bioprocess for serine alkaline protease (SAP) production by Bacillus licheniformis were calculated by the metabolic flux-based stoichiometric model based on the proposed metabolic network that contains 102 metabolites and 133 reaction fluxes using the time profiles of citrate, dry cell, organic acids, amino acids, and SAP as the constraints. The model was solved by minimizing the SAP accumulation rate in the cell. The effects of the oxygen-transfer rate (OTR) on the metabolic fluxes were investigated in a defined medium where citrate was used as the sole carbon source. The central pathways were active for the growth and the SAP synthesis in all the periods of the bioprocess at low (LOT), medium (MOT), and high (HOT) oxygen-transfer conditions. The flux partitioning in the TCA cycle at alpha-ketoglutarate towards glutamate group and at oxalacetate (OA) toward aspartic acid group amino acids were dependent on the OTR. The flux of the anaplerotic reaction that connects the TCA cycle either from malate or OA to the gluconeogenesis pathway via the main branch point pyruvate (Pyr) was also influenced by the OTR. With the decrease in the OTR, the intracellular flux values after glycerate 3-phosphate (PG3) in the gluconeogenesis pathway and the specific growth rate decreased. The total ATP-generation rate increased with the increase in OTR. The pathway towards the aspartic acid family amino acids which is important for sporulation that precedes the SAP synthesis were all active throughout the bioprocess. Metabolic flux analysis results at LOT, MOT, and HOT conditions encourage the design of an oxygen-transfer strategy in the bioreactor; moreover, asparagine synthetase or aspartate kinase could be the potential metabolic engineering sites due to the low value of the flux from the branch point aspartate toward asparagine.     

Metabolic engineering of pentose phosphate pathway in Ralstoniaeutropha for enhanced biosynthesis of poly-beta-hydroxybutyrate

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Ralstonia eutropha from gluconate as a carbon source is carried out through the Entner-Doudoroff (ED) pathway and the pentose-phosphate (PP) pathway generating NADPH and glyceraldehyde-3-phosphate that flows to acetyl-CoA, actively in the unbalanced PHB accumulation phase. The gnd gene encoding 6-phosphogluconate dehydrogenase (6PGDH) and the tktA gene encoding the transketolase (TK) in PP pathway of E. coli were transformed into R. eutropha H16 to modify the metabolic flux of gluconate to the PHB biosynthesis. Over-generated NADPH by the amplified gnd gene tended to depress the cell growth and PHB concentration. Meanwhile, the amplified tktA gene significantly increased both PHB biosynthesis and cell growth as a result of the effective flow of glyceraldehyde-3-phosphate into acetyl-CoA along with the concomitant supplementation of NADPH. The amplified tktA gene also activated the enzyme activities directly associated with PHB biosynthesis. The transformant R. eutropha harboring tktA gene was cultivated using pH-stat-fed-batch to achieve the overproduction of PHB.     

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Selection of objective function in genome scale flux balance analysis for process feed development in antibiotic production

Using flux variability analysis of a genome scale metabolic network of Streptomyces coelicolor, a series of reactions were identified, from disparate pathways that could be combined into an actinorhodin-generating mini-network. Candidate process feed nutrients that might be expected to influence this network were used in process simulations and in silico predictions compared to experimental findings. Ranking potential process feeds by flux balance analysis optimisation, using either growth or antibiotic production as objective function, did not correlate with experimental actinorhodin yields in fed processes. However, the effect of the feeds on glucose assimilation rate (using glucose uptake as objective function) ranked them in the same order as in vivo antibiotic production efficiency, consistent with results of a robustness analysis of the effect of glucose assimilation on actinorhodin production.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

Parameter estimation and dynamic control analysis of central carbon metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The central carbon metabolic pathway is the most important among metabolic pathways in all microorganisms since it produces energy and precursors for biosynthesis. In this study, a dynamic model for central carbon metabolism in Escherichia coli (E. coli) consisting of the phosphotransferase (PTS) system, glycolysis, pentose-phosphate pathway (PPP), and storage materials was obtained by ameliorating the model proposed by Chassagnole et al. (2002). In order to improve the performance of the model, principal parameters were estimated through the experimental measurements of intracellular concentrations of metabolites under transient conditions. Through dynamic metabolic control analysis (MCA), the tendencies of the metabolic fluxes at branch points were investigated, and the key parameters and enzyme kinetics that most dominantly affected the productivity of the desired metabolites were determined.     

Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. ¹³C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

鸟苷产生菌的代谢途径分析

代谢工程要解决的主要问题就是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径以获得产物的最大转化率。利用途径分析方法对枯草芽孢杆菌生产鸟苷的途径进行了分析,建立了3种基础模型,鸟苷理论摩尔产率分别是0．625、0．75和0．667,确定了枯草芽孢杆菌生产鸟苷的最佳途径的通量分布。     

Solute transporters as connecting elements between cytosol and plastid stroma

Metabolite translocators in the inner membrane of the plastid envelope are the interface between cytosolic and plastidial metabolism. Hence, they integrate plastidial pathways, such as photosynthesis, starch biosynthesis, the oxidative pentose phosphate pathway and the shikimate pathway, into the metabolic network of plant cells. Metabolite transporters not only catalyze the flux of metabolites between compartments but also represent information pathways that communicate the metabolic status of the various compartments within plant cells. Recently, a pentose-phosphate translocator was shown to be a novel member of the phosphate translocator protein family. Furthermore, a protein of previously unknown function was identified as a novel type of maltose transporter, and a glutamate/malate translocator that is involved in photorespiration was discovered. In addition, the pathway for maltose metabolism in the cytosol has been unraveled.     

Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis

Solventogenic clostridia are an important class of microorganisms that can produce various biofuels. One of the bottlenecks in engineering clostridia stems from the fact that central metabolic pathways remain poorly understood. Here, we utilized the power of (13) C-based isotopomer analysis to re-examine central metabolic pathways of Clostridium acetobutylicum ATCC 824. We demonstrate using [1,2-(13) C]glucose, MS analysis of intracellular metabolites, and enzymatic assays that C. acetobutylicum has a split TCA cycle where only Re-citrate synthase (CS) contributes to the production of α-ketoglutarate via citrate. Furthermore, we show that there is no carbon exchange between α-ketoglutarate and fumarate and that the oxidative pentose-phosphate pathway (oxPPP) is inactive. Dynamic gene expression analysis of the putative Re-CS gene (CAC0970), its operon, and all glycolysis, pentose-phosphate pathway, and TCA cycle genes identify genes and their degree of involvement in these core pathways that support the powerful primary metabolism of this industrial organism.     

Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.     

Phosphoketolase pathway dominates in Lactobacillus reuteri ATCC 55730 containing dual pathways for glycolysis

Metabolic flux analysis indicated that the heterofermentative Lactobacillus reuteri strain ATCC 55730 uses both the Embden-Meyerhof pathway (EMP) and phosphoketolase pathway (PKP) when glucose or sucrose is converted into the three-carbon intermediate stage of glycolysis. In all cases studied, the main flux is through the PKP, while the EMP is used as a shunt. In the exponential growth phase, 70%, 73%, and 84% of the flux goes through the PKP in cells metabolizing (i) glucose plus fructose, (ii) glucose alone, and (iii) sucrose alone, respectively. Analysis of the genome of L. reuteri ATCC 55730 confirmed the presence of the genes for both pathways. Further evidence for the simultaneous operation of two central carbon metabolic pathways was found through the detection of fructose-1,6-bisphosphate aldolase, phosphofructokinase, and phosphoglucoisomerase activities and the presence of phosphorylated EMP and PKP intermediates using in vitro ³¹P NMR. The maximum specific growth rate and biomass yield obtained on glucose were twice as low as on sucrose. This was the result of low ATP levels being present in glucose-metabolizing cells, although the ATP production flux was as high as in sucrose-metabolizing cells due to a twofold increase of enzyme activities in both glycolytic pathways. Growth performance on glucose could be improved by adding fructose as an external electron acceptor, suggesting that the observed behavior is due to a redox imbalance causing energy starvation.     

Heterotrophic Metabolism of the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase and the enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase (assayed together), are induced during heterotrophic growth of Thiobacillus ferrooxidans on an iron-glucose-supplemented medium or on glucose alone. By contrast, autotrophic cells (iron-grown) contain low levels of these enzymes. Fructose 1, 6-diphosphate aldolase, an enzyme of the Embden-Meyerhof pathway, is present at low levels irrespective of the growth medium, suggesting that this enzyme is not involved in energy-yielding reactions but merely provides intermediates for biosynthesis. The Entner-Doudoroff and pentose-phosphate pathways are the principle means through which glucose is dissimilated and is presumed to be concerned with energy production. Isotopic studies showed that a high rate of CO(2) formation from specifically labeled glucose came from carbon atoms 1 and 4. An unexpectedly high rate of evolution of CO(2) also came from carbon 6, suggesting that the triose phosphate formed during glucose breakdown and specifically as a result of 2-keto-3-deoxy-6-phosphogluconate aldolase activity, was metabolized via some unorthodox metabolic route. Cells grown in the iron-supplemented and glucose-salts media have a complete tricarboxylic acid cycle, whereas autotrophically grown T. ferrooxidans lacked both alpha-ketoglutarate dehydrogenase and reduced nicotinamide adenine dinucleotide oxidase. Two isocitrate dehydrogenases [nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) specific] were present. NAD-linked enzyme was constitutive, whereas the NADP-linked enzyme was induced upon adaptation of autotrophic cells to heterotrophic growth.