Jens O. Krömer

Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol

Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 degrees C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells.

In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum.

OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

BackgroundThe quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic engineering. It is based on a comprehensive approach combining (i) tracer cultivation on 13C substrates, (ii) 13C labelling analysis by mass spectrometry and (iii) mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis.ResultsWe have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU) framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing its user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly (<20 sec).ConclusionWe have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and enhance the design, calculation and interpretation of metabolic flux studies. By providing the software open source, we hope it will evolve with the rapidly growing field of fluxomics.

Metabolic flux analysis in mammalian cell culture

Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while (13)C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale (13)C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.

Microbial electron transport and energy conservation – the foundation for optimizing bioelectrochemical systems

Microbial electrochemical techniques describe a variety of emerging technologies that use electrode–bacteria interactions for biotechnology applications including the production of electricity, waste and wastewater treatment, bioremediation and the production of valuable products. Central in each application is the ability of the microbial catalyst to interact with external electron acceptors and/or donors and its metabolic properties that enable the combination of electron transport and carbon metabolism. And here also lies the key challenge. A wide range of microbes has been discovered to be able to exchange electrons with solid surfaces or mediators but only a few have been studied in depth. Especially electron transfer mechanisms from cathodes towards the microbial organism are poorly understood but are essential for many applications such as microbial electrosynthesis. We analyze the different electron transport chains that nature offers for organisms such as metal respiring bacteria and acetogens, but also standard biotechnological organisms currently used in bio-production. Special focus lies on the essential connection of redox and energy metabolism, which is often ignored when studying bioelectrochemical systems. The possibility of extracellular electron exchange at different points in each organism is discussed regarding required redox potentials and effect on cellular redox and energy levels. Key compounds such as electron carriers (e.g., cytochromes, ferredoxin, quinones, flavins) are identified and analyzed regarding their possible role in electrode–microbe interactions. This work summarizes our current knowledge on electron transport processes and uses a theoretical approach to predict the impact of different modes of transfer on the energy metabolism. As such it adds an important piece of fundamental understanding of microbial electron transport possibilities to the research community and will help to optimize and advance bioelectrochemical techniques.

Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae†

Monoterpenes are a diverse class of compounds with applications as flavors and fragrances, pharmaceuticals and more recently, jet fuels. Engineering biosynthetic pathways for monoterpene production in microbial hosts has received increasing attention. However, monoterpenes are highly toxic to many microorganisms including Saccharomyces cerevisiae, a widely used industrial biocatalyst. In this work, the minimum inhibitory concentration (MIC) for S. cerevisiae was determined for five monoterpenes: β‐pinene, limonene, myrcene, γ‐terpinene, and terpinolene (1.52, 0.44, 2.12, 0.70, 0.53 mM, respectively). Given the low MIC for all compounds tested, a liquid two‐phase solvent extraction system to alleviate toxicity during fermentation was evaluated. Ten solvents were tested for biocompatibility, monoterpene distribution, phase separation, and price. The solvents dioctyl phthalate, dibutyl phthalate, isopropyl myristate, and farnesene showed greater than 100‐fold increase in the MIC compared to the monoterpenes in a solvent‐free system. In particular, the MIC for limonene in dibutyl phthalate showed a 702‐fold (308 mM, 42.1 g L−1 of limonene) improvement while cell viability was maintained above 90%, demonstrating that extractive fermentation is a suitable tool for the reduction of monoterpene toxicity. Finally, we estimated that a limonane to farnesane ratio of 1:9 has physicochemical properties similar to traditional Jet‐A aviation fuel. Since farnesene is currently produced in S. cerevisiae, its use as a co‐product and extractant for microbial terpene‐based jet fuel production in a two‐phase system offers an attractive bioprocessing option. Biotechnol. Bioeng. 2012; 109: 2513–2522.

Isotopomer Profiling of Leishmania mexicana Promastigotes Reveals Important Roles for Succinate Fermentation and Aspartate Uptake in Tricarboxylic Acid Cycle (TCA) Anaplerosis, Glutamate Synthesis, and Growth

Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains poorly defined. In this study, we have followed the incorporation of various 13C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and 13C NMR. [U-13C]Glucose was rapidly incorporated into intermediates in glycolysis, the pentose phosphate pathway, and the cytoplasmic carbohydrate reserve material, mannogen. Enzymes involved in the upper glycolytic pathway are sequestered within glycosomes, and the ATP and NAD+ consumed by these reactions were primarily regenerated by the fermentation of phosphoenolpyruvate to succinate (glycosomal succinate fermentation). The initiating enzyme in this pathway, phosphoenolpyruvate carboxykinase, was exclusively localized to the glycosome. Although some of the glycosomal succinate was secreted, most of the C4 dicarboxylic acids generated during succinate fermentation were further catabolized in the TCA cycle. A high rate of TCA cycle anaplerosis was further suggested by measurement of [U-13C]aspartate and [U-13C]alanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.

Response of the central metabolism of Escherichia coli to modified expression of the gene encoding the glucose-6-phosphate dehydrogenase

The deletion of the zwf gene encoding G6PDH activity led to restructuring of the carbon flux through central metabolism in Escherichia coli, though over‐expression of this gene had only minor consequences for overall carbon flux. The modified carbon flux seen in the zwf deletion mutant enabled alternative routes of anabolic precursor formation and an adequate supply of NADPH synthesis via a modified TCA cycle to be generated so as to sustain growth rates comparable to the WT.

Accumulation of Homolanthionine and Activation of a Novel Pathway for Isoleucine Biosynthesis in Corynebacterium glutamicum McbR Deletion Strains

In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) in Corynebacterium glutamicum, which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol. 103:51-65, 2003), were studied. C. glutamicum ATCC 13032 DeltamcbR showed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry as L-homolanthionine {S-[(3S)-3-amino-3-carboxypropyl]-L-homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the DeltamcbR strain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-gamma-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine (Km = 260 microM) than for homocysteine (Km = 540 microM). The cell is able to cleave homolanthionine at low rates via cystathionine-beta-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis of C. glutamicum DeltamcbR revealed that only 24% of the O-acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis; the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion of metB completely prevents homolanthionine accumulation, which is regarded as an important step in the development of C. glutamicum strains for biotechnological methionine production.

Physiological response of Corynebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR

In the present work the metabolic response of Corynebacterium glutamicum to deletion of the global transcriptional regulator McbR, which controls, e.g. the expression of enzymes of L-methionine and L-cysteine biosynthesis and sulfur assimilation, was studied. Several oxidative stress proteins were significantly upregulated among about 40 proteins in response to deletion of McbR. Linked to this oxidative stress, the mutant exhibited a 50 % reduced growth rate, a 30 % reduced glucose uptake rate and a 30 % reduced biomass yield. It also showed metabolic flux rerouting in response to the deletion. NADPH metabolism was strongly altered. In contrast to the wild-type, the deletion strain supplied significantly more NADPH than required for anabolism, indicating the activity of additional NADPH-consuming reactions. These involved enzymes of oxidative stress protection. Through redirection of metabolic carbon flux in the central catabolism, including a 40 % increased tricarboxylic acid (TCA) cycle flux, the mutant revealed an enhanced NADPH supply to provide redox power for the antioxidant systems. This, however, was not sufficient to compensate for the oxidative stress, as indicated by the drastically disturbed redox equilibrium. The NADPH/NADP+ ratio in C. glutamicum DeltamcbR was only 0.29, and thus much lower than that of the wild-type (2.35). Similarly, the NADH/NAD+ ratio was substantially reduced from 0.18 in the wild-type to 0.08 in the mutant. Deletion of McbR is regarded as a key step towards biotechnological L-methionine overproduction in C. glutamicum. C. glutamicum DeltamcbR, however, did not overproduce L-methionine; this was very likely linked to the low availability of NADPH. Since oxidative stress is often observed in industrial production processes, engineering of NADPH metabolism could be a general strategy for improvement of production strains. Unlike the wild-type, C. glutamicum DeltamcbR contained large granules with high phosphorus content. The storage of these energy-rich polyphosphates is probably the result of a large excess of formation of ATP, as revealed by estimation of the underlying fluxes linked to energy metabolism.