Fluxes In Central Carbon Metabolism Research Articles

Switching carbon metabolic flux for enhancing the production of sesquiterpene-based high-density biofuel precursor in Saccharomyces cerevisiae

BackgroundSesquiterpenes are designated as a large class of plant-derived natural active compounds, which have wide applications in industries of energy, food, cosmetics, medicine and agriculture. Neither plant extraction nor chemical synthesis can meet the massive market demands and sustainable development goals. Biosynthesis in microbial cell factories represents an eco-friendly and high-efficient way. Among several microorganisms, Saccharomyces cerevisiae exhibited the potential as a chassis for bioproduction of various sesquiterpenes due to its native mevalonate pathway. However, its inefficient nature limits biosynthesis of diverse sesquiterpenes at industrial grade.ResultsHerein, we exploited an artificial synthetic malonic acid-acetoacetyl-CoA (MAAC) metabolic pathway to switch central carbon metabolic flux for stable and efficient biosynthesis of sesquiterpene-based high-density biofuel precursor in S. cerevisiae. Through investigations at transcription and metabolism levels, we revealed that strains with rewired central metabolism can devote more sugars to β-caryophyllene production. By optimizing the MVA pathway, the yield of β-caryophyllene from YQ-4 was 25.8 mg/L, which was 3 times higher than that of the initial strain YQ-1. Strain YQ-7 was obtained by introducing malonic acid metabolic pathway. Combing the optimized flask fermentation process, the target production boosted by about 13-fold, to 328 mg/L compared to that in the strain YQ-4 without malonic acid metabolic pathway.ConclusionThis designed MAAC pathway for sesquiterpene-based high-density biofuel precursor synthesis can provide an impressive cornerstone for achieving a sustainable production of renewable fuels.

Complex regulation in a Comamonas platform for diverse aromatic carbon metabolism

Critical to a sustainable energy future are microbial platforms that can process aromatic carbons from the largely untapped reservoir of lignin and plastic feedstocks. Comamonas species present promising bacterial candidates for such platforms because they can use a range of natural and xenobiotic aromatic compounds and often possess innate genetic constraints that avoid competition with sugars. However, the metabolic reactions of these species are underexplored, and the regulatory mechanisms are unknown. Here we identify multilevel regulation in the conversion of lignin-related natural aromatic compounds, 4-hydroxybenzoate and vanillate, and the plastics-related xenobiotic aromatic compound, terephthalate, in Comamonas testosteroni KF-1. Transcription-level regulation controls initial catabolism and cleavage, but metabolite-level thermodynamic regulation governs fluxes in central carbon metabolism. Quantitative 13C mapping of tricarboxylic acid cycle and cataplerotic reactions elucidates key carbon routing not evident from enzyme abundance changes. This scheme of transcriptional activation coupled with metabolic fine-tuning challenges outcome predictions during metabolic manipulations.

1366-P: Examining Central Carbon Metabolic Flux in the Perfused Exocrine Pancreas Using Dissolution Dynamic Nuclear Polarization

The pancreas functions as both an endocrine and exocrine gland. The endocrine component consists of the islets of Langerhans, whereas the exocrine tissue produces digestive enzymes through the ductal system. Although the majority of Type I Diabetes research has focused on the islets, the exocrine tissue is likely implicated in T1D as well. This research utilizes a novel method to investigate exocrine pancreas metabolism with a pancreas perfusion system. I hypothesized that dissolution dynamic nuclear polarization (dDNP) combined with the pancreas perfusion system would provide real-time measurements of central carbon metabolic turnover in the exocrine pancreas. DNP is a phenomenon in which a transfer of high spin polarization greatly enhances the signal in the resulting nuclear magnetic resonance (NMR) spectra. This research compares dDNP measurements in the exocrine pancreas in C57BL/6 male mice under varying glucose conditions. It also examines central carbon metabolism using this system in the T1D mouse model NOD.Rag1-/-.AI4α/β and the NOD.Rag1-/- control. After cannulation of the common bile duct, the exocrine pancreas was perfused for 30 minutes with perfusate containing either 3mM or 16.7mM glucose. Tissue viability was confirmed throughout the perfusion with oxygen consumption measurements. dDNP was performed using the trityl radical and 4mM [1-13C]pyruvate. NMR spectra were collected on a 600MHz instrument with a 10mm [13C]-optimized cryoprobe and analyzed with Bruker TopSpin. Several metabolites were identified and quantified in all measured spectra, demonstrating the detection of real-time metabolic turnover. Ultimately, this work demonstrates that dissolution dynamic nuclear polarization can be used to evaluate metabolic flux in the pancreas in multiple mouse models. Future will investigate the real-time metabolic response of the exocrine pancreas to insulin concentrations in the T1D disease model. Disclosure A.Rushin: None. M.Mcleod: None. M.Ragavan: None. M.Merritt: None. Funding National Institutes of Health (T32 DK10876) National Science Foundation (DMR-1644779)

Application of a Novel Pancreas Perfusion Technique to Characterize Exocrine Pancreas Metabolism

The pancreas functions as both an endocrine and exocrine gland. The endocrine component consists of the islets of Langerhans, which contain several pancreatic cell types that produce hormones. In the islets, α‐cells produce glucagon and β‐cells produce insulin, both of which serve to regulate glucose metabolism. The exocrine tissue produces digestive enzymes such as chymotrypsin and is connected through the ductal system. Type I Diabetes (T1D) is an autoimmune disease in which immune cells attack the pancreatic β‐cells, resulting in the pancreas producing little to no insulin. Although the vast majority of research has focused on the islets, the exocrine tissue may be implicated in T1D as well. This research introduces a novel method to investigate exocrine pancreas metabolism specifically using a pancreas perfusion system through the common bile duct. I hypothesized that the exocrine pancreas would demonstrate metabolic activity through uptake and utilization of [2H7]glucose during a perfusion.Using C57BL/6 male mice, the surgical procedure consisted of clamping the ampulla of Vater with a John Hopkins bulldog clamp and cannulation of the common bile duct. The pancreas was then perfused for 20 minutes with perfusate containing Krebs electrolytes, free fatty acids, and 16.7mM of either unlabeled or [2H7]glucose. The tissue was flash frozen in liquid nitrogen and subjected to an acetonitrile isopropanol water extraction. The extracts were derivatized using methoxyamine and MTBSTFA before gas chromatography‐mass spectrometry (GC‐MS) analysis.GC‐MS measurements confirmed the pancreas exocrine tissue metabolized [2H7]glucose, as shown by an increased enrichment in lactate mass isotopologues in the tissue perfused with isotopically labeled glucose compared to the unlabeled control. Additional central carbon metabolites also demonstrated increased enrichment, namely malate and fumarate. The observed isotopologue labeling patterns are indicative of anaplerosis. This research demonstrates metabolism specific to the exocrine pancreas can be measured using a pancreas perfusion system and isotopic labeling. Future work will examine real‐time central carbon metabolic flux using dissolution dynamic nuclear polarization (dDNP) combined with the pancreas perfusion system. This research will use the T1D mouse model NOD. Rag1‐/‐.AI4α/β and compare the results to the NOD.Rag1‐/‐ control mouse model to examine how exocrine pancreas central carbon metabolism is altered in T1D models in a more physiologically relevant manner than previously possible.

Valine feeding reduces ammonia production through rearrangement of metabolic fluxes in central carbon metabolism of CHO cells.

Ammonia is a toxic byproduct of CHO cell metabolism, which inhibits cell growth, reduces cell viability, alters glycosylation, and decreases recombinant protein productivity. In an attempt to minimize the ammonium accumulation in cell culture media, different amino acids were added individually to the culture medium before the production phase to alleviate the negative effects of ammonium on cell culture performance. Among all the amino acids examined in this study, valine showed the most positive impact on CHO cell culture performance. When the cultured CHO cells were fed with 5mM valine, EPO titer was increased by 25% compared to the control medium, and ammonium and lactate production were decreased by 23 and 26%, respectively, relative to the control culture. Moreover, the sialic acid content of the EPO protein in valine-fed culture was higher than in the control culture, most likely because of the lower ammonium concentration. Flux balance analysis (FBA) results demonstrated that the citric acid cycle was enriched by valine feeding. The measurement of TCA cycle activity supported this finding. The analysis revealed that there might be a link between promoting tricarboxylic acid (TCA) cycle metabolism in valine-fed culture and reduction in lactate and ammonia accumulation. Furthermore, in valine-fed culture, FBA outcomes showed that alanine was excreted into the medium as the primary mechanism for reducing ammonium concentration. It was predicted that the elevated TCA cycle metabolism was concurrent with an increment in recombinant protein production. Taken together, our data demonstrate that valine addition could be an effective strategy for mitigating the negative impacts of ammonium and enhancing glycoprotein production in both quality and quantity. KEY POINTS: • Valine feeding can mitigate the negative impacts of ammonia on CHO cell growth. • Valine addition assists the ammonia removal mechanism by enriching the TCA cycle. • Ammonia is removed from the media through alanine excretion in valine-fed culture.

Nitrate reduction by Paracoccus thiophilus strain LSL 251 under aerobic condition: Performance and intracellular central carbon flux pathways

The intracellular carbon metabolic flux pathways of denitrifying bacteria under aerobic conditions remain unclear. Here, a newly strain LSL251 was identified as Paracoccus thiophilus. Strain LSL251 removed 94.79% and 98.78% of total organic carbon and nitrate. 74.66% of nitrogen in culture system was lost as gaseous nitrogen. Moreover, 13C stable isotopic labeling and metabolic flux analyses revealed that the primary intracellular carbon metabolic pathways were the Entner-Doudoroff pathway and the tricarboxylic acid (TCA) cycle. Electrons are primarily donated as direct electron donor-NADH through the TCA cycle. Furthermore, response surface methodology modeled that the highest total nitrogen removal efficiency was 98.43%, where the optimal parameters were C/N ratio of 8.00, 32.98 °C, 50.18 rpm, and initial pH of 7.73. All together, these results have shed new lights on intracellular central carbon metabolic distribution and flux pathways of aerobic denitrifying bacteria.

Transcriptional control of central carbon metabolic flux in Bifidobacteria by two functionally similar, yet distinct LacI-type regulators

Bifidobacteria resident in the gastrointestinal tract (GIT) are subject to constantly changing environmental conditions, which require rapid adjustments in gene expression. Here, we show that two predicted LacI-type transcription factors (TFs), designated AraQ and MalR1, are involved in regulating the central, carbohydrate-associated metabolic pathway (the so-called phosphoketolase pathway or bifid shunt) of the gut commensal Bifidobacterium breve UCC2003. These TFs appear to not only control transcription of genes involved in the bifid shunt and each other, but also seem to commonly and directly affect transcription of other TF-encoding genes, as well as genes related to uptake and metabolism of various carbohydrates. This complex and interactive network of AraQ/MalR1-mediated gene regulation provides previously unknown insights into the governance of carbon metabolism in bifidobacteria.

Genome-Scale Model-Based Identification of Metabolite Indicators for Early Detection of Kidney Toxicity.

Identifying early indicators of toxicant-induced organ damage is critical to provide effective treatment. To discover such indicators and the underlying mechanisms of toxicity, we used gentamicin as an exemplar kidney toxicant and performed systematic perturbation studies in Sprague Dawley rats. We obtained high-throughput data 7 and 13 h after administration of a single dose of gentamicin (0.5 g/kg) and identified global changes in genes in the liver and kidneys, metabolites in the plasma and urine, and absolute fluxes in central carbon metabolism. We used these measured changes in genes in the liver and kidney as constraints to a rat multitissue genome-scale metabolic network model to investigate the mechanism of gentamicin-induced kidney toxicity and identify metabolites associated with changes in tissue gene expression. Our experimental analysis revealed that gentamicin-induced metabolic perturbations could be detected as early as 7 h postexposure. Our integrated systems-level analyses suggest that changes in kidney gene expression drive most of the significant metabolite alterations in the urine. The analyses thus allowed us to identify several significantly enriched injury-specific pathways in the kidney underlying gentamicin-induced toxicity, as well as metabolites in these pathways that could serve as potential early indicators of kidney damage.

Central carbon metabolism influences cellulase production in Bacillus licheniformis.

Cellulase is very important in the field of agriculture and bioenergy because of its degrading effect on cellulosic biomass. This study presented the effect of central carbon metabolism on cellulase production in Bacillus licheniformis. The study also provided a holistic view of the physiological status in B. licheniformis. The shifted metabolism provided a quantitative evaluation of the biosynthesis of cellulase and a priority ranked target list for further strain engineering.

Abstract 439: Metabolic flux analysis reveals targets to sensitize chemoresistance in acute myeloid leukemia induced by mesenchymal stromal cell-derived exosomes

Abstract The tumor microenvironment has a pleiotropic role in supporting cancer cell growth, metastasis and drug resistance. Exosomes from mesenchymal stromal cells (MSC) were found to regulate metabolism of acute myeloid leukemia (AML) cells that led to a chemoresistant phenotype. Exosomes carry a host of proteins, nucleotides and metabolites that can induce metabolic reprogramming recipient cells via direct supply of metabolite cargo or through signaling. We utilized 13C tracer techniques and two metabolic flux analysis techniques to reveal the mechanism of metabolic reprogramming induced by MSC-derived exosomes. First, 13C metabolic flux analysis was used to quantify intracellular fluxes of central carbon, amino acids and fatty acid metabolism in AML cells cultured with and without exosomes in media with 13C substrates. Second, we employed a novel technique, exosome-mediated metabolic flux analysis (Exo-MFA), to estimate direct support provided by metabolite supply from exosome cargo by introducing 13C-labeled exosomes. The combined analysis dissected the metabolite supply from exosome cargo from the overall reprogramming of metabolic pathways due to exosomes. Our results revealed key metabolic pathways that could be targeted to inhibit exosome-induced reprogramming to reverse the chemoresistant phenotype. Citation Format: Abhinav Achreja, Hongyun Zhao, Ziwen Zhu, Jonathan Gerszberg, Marina Y. Konopleva, Michael Andreeff, Deepak Nagrath. Metabolic flux analysis reveals targets to sensitize chemoresistance in acute myeloid leukemia induced by mesenchymal stromal cell-derived exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 439. doi:10.1158/1538-7445.AM2017-439

13C metabolite profiling to compare the central metabolic flux in two yeast strains

13C metabolite profiling to quantify the dynamic changes of central carbon metabolites was attempted using mass isotopomer distribution analysis in two yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus. Mass and isotopomer balances of the intermediates were examined and calculated in both yeast species and central carbon metabolic fluxes were successfully determined. Metabolic fluxes of pentose phosphate pathway in K. marxianus were 1.66 times higher than S. cerevisiae. The flux difference was also supported by relatively high abundance of partially labeled fructose 6-phosphate and 3-phosphoglycerate as well as an increased concentration of labeled L-valine in K. marxianus. Metabolic flux analysis combined with dynamic metabolite profiling has provided better understanding in the central carbon metabolic pathways of two model organisms and can be applied as a method to analyze more complicated metabolic networks in other organisms.

Difference in C3-C4 metabolism underlies tradeoff between growth rate and biomass yield in Methylobacterium extorquens AM1.

BackgroundTwo variants of Methylobacterium extorquens AM1 demonstrated a trade-off between growth rate and biomass yield. In addition, growth rate and biomass yield were also affected by supplementation of growth medium with different amounts of cobalt. The metabolism changes relating to these growth phenomena as well as the trade-off were investigated in this study. 13C metabolic flux analysis was used to generate a detailed central carbon metabolic flux map with both absolute and normalized flux values.ResultsThe major differences between the two variants occurred at the formate node as well as within C3–C4 inter-conversion pathways. Higher relative fluxes through formyltetrahydrofolate ligase, phosphoenolpyruvate carboxylase, and malic enzyme led to higher biomass yield, while higher relative fluxes through pyruvate kinase and pyruvate dehydrogenase led to higher growth rate. These results were then tested by phenotypic studies on three mutants (null pyk, null pck mutant and null dme mutant) in both variants, which agreed with the model prediction.ConclusionsIn this study, 13C metabolic flux analysis for two strain variants of M. extorquens AM1 successfully identified metabolic pathways contributing to the trade-off between cell growth and biomass yield. Phenotypic analysis of mutants deficient in corresponding genes supported the conclusion that C3–C4 inter-conversion strategies were the major response to the trade-off.Electronic supplementary materialsThe online version of this article (doi:10.1186/s12866-016-0778-4) contains supplementary material, which is available to authorized users.

Flux Balance Analysis of Plant Metabolism: The Effect of Biomass Composition and Model Structure on Model Predictions.

The biomass composition represented in constraint-based metabolic models is a key component for predicting cellular metabolism using flux balance analysis (FBA). Despite major advances in analytical technologies, it is often challenging to obtain a detailed composition of all major biomass components experimentally. Studies examining the influence of the biomass composition on the predictions of metabolic models have so far mostly been done on models of microorganisms. Little is known about the impact of varying biomass composition on flux prediction in FBA models of plants, whose metabolism is very versatile and complex because of the presence of multiple subcellular compartments. Also, the published metabolic models of plants differ in size and complexity. In this study, we examined the sensitivity of the predicted fluxes of plant metabolic models to biomass composition and model structure. These questions were addressed by evaluating the sensitivity of predictions of growth rates and central carbon metabolic fluxes to varying biomass compositions in three different genome-/large-scale metabolic models of Arabidopsis thaliana. Our results showed that fluxes through the central carbon metabolism were robust to changes in biomass composition. Nevertheless, comparisons between the predictions from three models using identical modeling constraints and objective function showed that model predictions were sensitive to the structure of the models, highlighting large discrepancies between the published models.

The correlation between the central carbon metabolic flux distribution and the number of shared enzyme regulators in Saccharomyces cerevisiae

The central carbon metabolic system is the upstream energy source for microbial fermentation. In addition, it is a master switch for increasing the production of metabolites and an important part of the microbial metabolic network. Investigation into the relationship between genes, environmental factors, and metabolic networks is a main focus of systems biology, which significantly impacts research in biochemistry, metabolic engineering, and synthetic biology. To this end, the central carbon metabolic flux under a variety of growth conditions or using strains with various genetic modifications was previously measured in Saccharomyces cerevisiae using 13C tracer technology. However, the measured values were not integrated and investigated further. In this study, we collected and analyzed the metabolic flux rates of the central carbon metabolic system in S. cerevisiae measured in recent studies. We carried out preliminary analyses of flux values of each pathway, performed regression analyses on relationship between different fluxes, and extracted principal component factors of the flux variables. Based on the results, the general characteristics of pathway flux distribution were clustered and explored, and the effects of environmental and genetic factors on the flux distribution were analyzed. Furthermore, this study explored the relationship between similarity in the enzyme's transcriptional regulation and the correlations in the enzyme's reaction flux. Our results provide a foundation for further studies on the control of the central carbon metabolic flux and facilitate the search for targets in metabolic engineering research.

Fluxes through plant metabolic networks: measurements, predictions, insights and challenges.

Although the flows of material through metabolic networks are central to cell function, they are not easy to measure other than at the level of inputs and outputs. This is particularly true in plant cells, where the network spans multiple subcellular compartments and where the network may function either heterotrophically or photoautotrophically. For many years, kinetic modelling of pathways provided the only method for describing the operation of fragments of the network. However, more recently, it has become possible to map the fluxes in central carbon metabolism using the stable isotope labelling techniques of metabolic flux analysis (MFA), and to predict intracellular fluxes using constraints-based modelling procedures such as flux balance analysis (FBA). These approaches were originally developed for the analysis of microbial metabolism, but over the last decade, they have been adapted for the more demanding analysis of plant metabolic networks. Here, the principal features of MFA and FBA as applied to plants are outlined, followed by a discussion of the insights that have been gained into plant metabolic networks through the application of these time-consuming and non-trivial methods. The discussion focuses on how a system-wide view of plant metabolism has increased our understanding of network structure, metabolic perturbations and the provision of reducing power and energy for cell function. Current methodological challenges that limit the scope of plant MFA are discussed and particular emphasis is placed on the importance of developing methods for cell-specific MFA.

Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

CeCaFDB: a curated database for the documentation, visualization and comparative analysis of central carbon metabolic flux distributions explored by 13C-fluxomics.

The Central Carbon Metabolic Flux Database (CeCaFDB, available at http://www.cecafdb.org) is a manually curated, multipurpose and open-access database for the documentation, visualization and comparative analysis of the quantitative flux results of central carbon metabolism among microbes and animal cells. It encompasses records for more than 500 flux distributions among 36 organisms and includes information regarding the genotype, culture medium, growth conditions and other specific information gathered from hundreds of journal articles. In addition to its comprehensive literature-derived data, the CeCaFDB supports a common text search function among the data and interactive visualization of the curated flux distributions with compartmentation information based on the Cytoscape Web API, which facilitates data interpretation. The CeCaFDB offers four modules to calculate a similarity score or to perform an alignment between the flux distributions. One of the modules was built using an inter programming algorithm for flux distribution alignment that was specifically designed for this study. Based on these modules, the CeCaFDB also supports an extensive flux distribution comparison function among the curated data. The CeCaFDB is strenuously designed to address the broad demands of biochemists, metabolic engineers, systems biologists and members of the -omics community.

The role of flexibility and optimality in the prediction of intracellular fluxes of microbial central carbon metabolism.

Prediction of intracellular metabolic fluxes based on optimal biomass assumption is a well-known computational approach. While there has been a significant emphasis on the optimality, cellular flexibility, the co-occurrence of suboptimal flux distributions in a microbial population, has hardly been considered in the related computational methods. We have implemented a flexibility-incorporated optimization framework to calculate intracellular fluxes based on a few extracellular measurement constraints. Taking into account slightly suboptimal flux distributions together with a dual-optimality framework (maximization of the growth rate followed by the minimization of the total enzyme amount) we were able to show the positive effect of incorporating flexibility and minimal enzyme consumption on the better prediction of intracellular fluxes of central carbon metabolism of two microorganisms: E. coli and S. cerevisiae.

Non-stationary 13C metabolic flux analysis of Chinese hamster ovary cells in batch culture using extracellular labeling highlights metabolic reversibility and compartmentation.

BackgroundMapping the intracellular fluxes for established mammalian cell lines becomes increasingly important for scientific and economic reasons. However, this is being hampered by the high complexity of metabolic networks, particularly concerning compartmentation.ResultsIntracellular fluxes of the CHO-K1 cell line central carbon metabolism were successfully determined for a complex network using non-stationary 13C metabolic flux analysis. Mass isotopomers of extracellular metabolites were determined using [U-13C6] glucose as labeled substrate. Metabolic compartmentation and extracellular transport reversibility proved essential to successfully reproduce the dynamics of the labeling patterns. Alanine and pyruvate reversibility changed dynamically even if their net production fluxes remained constant. Cataplerotic fluxes of cytosolic phosphoenolpyruvate carboxykinase and mitochondrial malic enzyme and pyruvate carboxylase were successfully determined. Glycolytic pyruvate channeling to lactate was modeled by including a separate pyruvate pool. In the exponential growth phase, alanine, glycine and glutamate were excreted, and glutamine, aspartate, asparagine and serine were taken up; however, all these amino acids except asparagine were exchanged reversibly with the media. High fluxes were determined in the pentose phosphate pathway and the TCA cycle. The latter was fueled mainly by glucose but also by amino acid catabolism.ConclusionsThe CHO-K1 central metabolism in controlled batch culture proves to be robust. It has the main purpose to ensure fast growth on a mixture of substrates and also to mitigate oxidative stress. It achieves this by using compartmentation to control NADPH and NADH availability and by simultaneous synthesis and catabolism of amino acids.

Assessment of metabolic flux distribution in the thermophilic hydrogen producer Caloramator celer as affected by external pH and hydrogen partial pressure.

BackgroundCaloramator celer is a strict anaerobic, alkalitolerant, thermophilic bacterium capable of converting glucose to hydrogen (H2), carbon dioxide, acetate, ethanol and formate by a mixed acid fermentation. Depending on the growth conditions C. celer can produce H2 at high yields. For a biotechnological exploitation of this bacterium for H2 production it is crucial to understand the factors that regulate carbon and electron fluxes and therefore the final distribution of metabolites to channel the metabolic flux towards the desired product.ResultsCombining experimental results from batch fermentations with genome analysis, reconstruction of central carbon metabolism and metabolic flux analysis (MFA), this study shed light on glucose catabolism of the thermophilic alkalitolerant bacterium C. celer. Two innate factors pertaining to culture conditions have been identified to significantly affect the metabolic flux distribution: culture pH and partial pressures of H2 (PH2). Overall, at alkaline to neutral pH the rate of biomass synthesis was maximized, whereas at acidic pH the lower growth rate and the less efficient biomass formation are accompanied with more efficient energy recovery from the substrate indicating high cell maintenance possibly to sustain intracellular pH homeostasis. Higher H2 yields were associated with fermentation at acidic pH as a consequence of the lower synthesis of other reduced by-products such as formate and ethanol. In contrast, PH2 did not affect the growth of C. celer on glucose. At high PH2 the cellular redox state was balanced by rerouting the flow of carbon and electrons to ethanol and formate production allowing unaltered glycolytic flux and growth rate, but resulting in a decreased H2 synthesis.ConclusionC. celer possesses a flexible fermentative metabolism that allows redistribution of fluxes at key metabolic nodes to simultaneously control redox state and efficiently harvest energy from substrate even under unfavorable conditions (i.e. low pH and high PH2). With the H2 production in mind, acidic pH and low PH2 should be preferred for a high yield-oriented process, while a high productivity-oriented process can be achieved at alkaline pH and high PH2.