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We describe a systematic approach to establish predictive models of CHO cell growth, cell metabolism and monoclonal antibody (mAb) formation during biopharmaceutical production. The prediction is based on a combination of an empirical metabolic model connecting extracellular metabolic fluxes with cellular growth and product formation with mixed Monod-inhibition type kinetics that we generalized to every possible external metabolite. We describe the maximum specific growth rate as a function of the integral viable cell density (IVCD). Moreover, we also take into account the accumulation of metabolites in intracellular pools that can influence cell growth. This is possible even without identification and quantification of these metabolites as illustrated with fed-batch cultures of Chinese Hamster Ovary (CHO) cells producing a mAb. The impact of cysteine and tryptophan on cell growth and cell productivity was assessed, and the resulting macroscopic model was successfully used to predict the impact of new, untested feeding strategies on cell growth and mAb production. This model combining piecewise linear relationships between metabolic rates, growth rate and production rate together with Monod-inhibition type models for cell growth did well in predicting cell culture performance in fed-batch cultures even outside the range of experimental data used for establishing the model. It could therefore also successfully be applied for in silico prediction of optimal operating conditions.

Cell-free systems containing multiple enzymes are becoming an increasingly interesting tool for one-pot syntheses of biochemical compounds. To extensively explore the enormous wealth of enzymes in the biological space, we present methods for assembling and curing data from databases to apply them for the prediction of pathway candidates for directed enzymatic synthesis. We use Kyoto Encyclopedia of Genes and Genomes to establish single organism models and a pan-organism model that is combining the available data from all organisms listed there. We introduce a filtering scheme to remove data that are not suitable, for example, generic metabolites and general reactions. In addition, a valid stoichiometry of reactions is required for acceptance. The networks created are analyzed by graph theoretical methods to identify a set of metabolites that are potentially reachable from a defined set of starting metabolites. Thus, metabolites not connected to such starting metabolites cannot be produced unless new starting metabolites or reactions are introduced. The network models also comprise stoichiometric and thermodynamic data that allow the definition of constraints to identify potential pathways. The resulting data can be directly applied using existing or future pathway finding tools.

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.

Introduction The switch from quiescence (G0) into G1 and cell cycle progression critically depends on specific nutrients and metabolic capabilities. Conversely, metabolic networks are regulated by enzyme–metabolite interaction and transcriptional regulation that lead to flux modifications to support cell growth. How cells process and integrate environmental information into coordinated responses is challenging to analyse and not yet described quantitatively. Objectives To quantitatively monitor the central carbon metabolism during G0 exit and the first 2 h after reentering the cell cycle from synchronized Saccharomyces cerevisiae. Methods Dynamic tailored 13C metabolic flux analysis was used to observe the intracellular metabolite flux changes, and the metabolome and proteome were observed to identify regulatory mechanisms. Results G0 cells responded immediately to an extracellular increase of glucose. The intracellular metabolic flux changed in time and specific events were observed. High fluxes into trehalose and glycogen synthesis were observed during the G0 exit. Both fluxes then decreased, reaching a minimum at t = 65 min. Here, storage degradation contributed significantly (i.e. 21%) to the glycolytic flux. In contrast to these changes, the glucose uptake rate remained constant after the G0 exit. The flux into the oxidative pentose phosphate pathway was highest (29-fold increase, 36.4% of the glucose uptake) at t = 65 min, while it was very low at other time points. The maximum flux seems to correlate with a late G1 state preparing for the S phase transition. In the G1/S phase (t = 87 min), anaplerotic reactions such as glyoxylate shunt increased. Protein results show that during this transition, proteins belonging to clusters related with ribosome biogenesis and assembly, and initiation transcription factors clusters were continuously synthetised. Conclusion The intracellular flux distribution changes dynamically and these major rearrangements highlight the coordinate reorganization of metabolic flux to meet requirements for growth during different cell state. Electronic supplementary material The online version of this article (10.1007/s11306-019-1584-4) contains supplementary material, which is available to authorized users.

Efforts are underway to transform regulatory toxicology and chemical safety assessment from a largely empirical science based on direct observation of apical toxicity outcomes in whole organism toxicity tests to a predictive one in which outcomes and risk are inferred from accumulated mechanistic understanding. The adverse outcome pathway (AOP) framework provides a systematic approach for organizing knowledge that may support such inference. Likewise, computational models of biological systems at various scales provide another means and platform to integrate current biological understanding to facilitate inference and extrapolation. We argue that the systematic organization of knowledge into AOP frameworks can inform and help direct the design and development of computational prediction models that can further enhance the utility of mechanistic and in silico data for chemical safety assessment. This concept was explored as part of a workshop on AOP-Informed Predictive Modeling Approaches for Regulatory Toxicology held September 24–25, 2015. Examples of AOP-informed model development and its application to the assessment of chemicals for skin sensitization and multiple modes of endocrine disruption are provided. The role of problem formulation, not only as a critical phase of risk assessment, but also as guide for both AOP and complementary model development is described. Finally, a proposal for actively engaging the modeling community in AOP-informed computational model development is made. The contents serve as a vision for how AOPs can be leveraged to facilitate development of computational prediction models needed to support the next generation of chemical safety assessment.

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed-batch cultures. Using the model structure and parameter values from a small-scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed-batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

We review major modeling strategies and methods to understand and simulate the macroscopic behavior of mammalian cells. These strategies comprise two important steps: the first step is to identify stoichiometric relationships for the cultured cells connecting the extracellular inputs and outputs. In a second step, macroscopic kinetic models are introduced. These relationships together with bioreactor and metabolite balances provide a complete description of a system in the form of a set of differential equations. These can be used for the simulation of cell culture performance and further for optimization of production.

Recombinant protein secretion in yeasts poses a burden to the metabolism of the host cell. Consequently, unfavorable cultivation conditions during strain screening or process development can lead to limitations in the energy and carbon metabolism of the cell, constraining the cell's ability to secrete the protein of interest. Recently, we demonstrated that improving cultivation conditions by using substrate mixtures of glycerol and acetate strongly elevated secretion of the homologous model protein maltase in the fission yeast Schizosaccharomyces pombe. In this work, we investigated if these previous findings were also applicable to the expression of recombinant proteins. Strains were constructed secreting either green fluorescent protein or a fluorescent single-chain antibody fragment. These strains were cultivated under fermentative and respiratory growth conditions on glucose as sole carbon source and on mixtures of glucose/acetate and glycerol/acetate. We observed an increase in the specific secretion of both recombinant proteins by 1.8- and 3.8-fold, respectively. This clearly demonstrates that the proper choice of process conditions and the applied carbon sources have a significant impact on the secretion of at least two recombinant proteins in S. pombe allowing an improved production of the protein of interest.

Respiration analysis using isolated mitochondria and electrochemical oxygen sensing has contributed significantly to the knowledge about mitochondrial metabolism, which is involved in energy generation but also in ageing and numerous diseases. Here, we present a high-throughput respiration screening for functional in situ mitochondrial studies in permeabilized Chinese hamster ovary cells. The determination of oxygen uptake rates allowed a quantitative comparison between different conditions and a distinction of substrates into three groups providing an insight into tricarboxylic acid (TCA) cycle regulation. The mitochondrial metabolization of citrate, isocitrate, glutamine, and glutamate was highly stimulated by ADP supply. In contrast, the metabolization of α-ketoglutarate, succinate, fumarate, and malate was little controlled by the energy and redox state. Metabolization of pyruvate was very strictly regulated by several independent mechanisms: phosphorylation, feedback inhibition, but also by the availability of CoA. A moderate stimulation of pyruvate metabolization was accomplished by feeding both pyruvate and aspartate simultaneously. The presented high-throughput respiration screening provides comprehensive information about the effect of single or mixed substrates on mitochondrial metabolic activities, including transport and TCA cycle regulation, and metabolic bottlenecks. This supports the design of efficient mammalian producer strains or feeding strategies, but also the investigation of pathological and toxicological effects related to mitochondrial metabolism.

Metabolic bottlenecks play an increasing role in yeasts and mammalian cells applied for high-performance production of proteins, particularly of pharmaceutical ones that require complex posttranslational modifications. We review the present status and developments focusing on the rational metabolic engineering of such cells to optimize the supply chain for building blocks and energy. Methods comprise selection of beneficial genetic modifications, rational design of media and feeding strategies. Design of better producer cells based on whole genome-wide metabolic network analysis becomes increasingly possible. High-resolution methods of metabolic flux analysis for the complex networks in these compartmented cells are increasingly available. We discuss phenomena that are common to both types of organisms but also those that are different with respect to the supply chain for the production and secretion of pharmaceutical proteins.

We constructed and applied a recombinant, permeabilized Escherichia coli strain for the multistep synthesis of UDP-glucose. Sucrose phosphorylase (E.C. 2.4.1.7) of Leuconostoc mesenteroides was over expressed and the pgm gene encoding for phosphoglucomutase (E.C. 5.4.2.2) was deleted in E. coli to yield the E. coli JW 0675-1 SP strain. The cells were permeabilized with the detergent Triton X-100 at 0.05 % v/v. The synthesis of UDP-glucose with permeabilized cells was then optimized with regard to pH, cell density during the synthesis and growth phase during cell harvest, metal cofactor, other media components, and temperature. In one configuration sucrose, phosphate, UMP, and ATP were used as substrates. At pH 7.8, 27 mg/ml cell dry weight, cell harvest during the early stationary phase of growth and Mn(2+) as cofactor a yield of 37 % with respect to UMP was achieved at 33 °C. In a second step, ATP was regenerated by feeding glucose and using only catalytic amounts of ATP and NAD(+). A UDP-glucose yield of 60 % with respect to UMP was obtained using this setup. With the same setup but without addition of external ATP, the yield was 54%.

We report on tailored regeneration modules for the essential cofactors ATP and NADPH as well as for SAM supply, adapted to support biocatalytic multistep synthesis of oxytetracycline (OTC) in permeabilized cells of E. coli. ATP is regenerated from ADP and AMP by the use of polyphosphate. The efficiency of this system was proven with permeabilized cells producing flaviolin. NADPH is regenerated from NADP+ by oxidation of phosphonate. A constant level of 0.7 mg/L of SAM is provided by the ligation of ATP and methionine. OTC is synthesized at a rate of 2.05 ng/(h (g of the combined biocatalyst)), reaching a final concentration of 0.9 mg/L.

The physiology of animal cells is characterized by constantly changing environmental conditions and adapting cellular responses. Applied dynamic metabolic flux analysis captures metabolic dynamics and can be applied to industrially relevant cultivation conditions. We investigated the impact of glutamine availability or limitation on the physiology of CHO K1 cells in eight different batch and fed-batch cultivations. Varying glutamine availability resulted in global metabolic changes. We observed dose-dependent effects of glutamine in batch cultivation. Identifying metabolic links from the glutamine metabolism to specific metabolic pathways, we show that glutamine feeding results in its coupling to tricarboxylic acid cycle fluxes and in its decoupling from metabolic waste production. We provide a mechanistic explanation of the cellular responses upon mild or severe glutamine limitation and ammonia stress. The growth rate of CHO K1 decreased with increasing ammonia levels in the supernatant. On the other hand, growth, especially culture longevity, was stimulated at mild glutamine-limiting conditions. Flux rearrangements in the pyruvate and amino acid metabolism compensate glutamine limitation by consumption of alternative carbon sources and facilitating glutamine synthesis and mitigate ammonia stress as result of glutamine abundance.

The aim of this study was to evaluate carbon flux in Azotobacter vinelandii using metabolic flux analysis (MFA) under high and low aeration conditions to achieve an improved understanding of how these changes could be related to alginate acetylation and PHB production. Changes in oxygen availability had a considerable impact on the metabolic fluxes and were reflected in the growth rate, the specific glucose consumption rate, and the alginate and PHB yields. The main differences at the metabolic flux level were observed in three important pathways. The first important difference was consistent with respiratory protection; an increase in the flux generated through the tricarboxylic acid (TCA) cycle for cultures grown under high aeration conditions (up to 2.61 times higher) was observed. In the second important difference, the fluxes generated through pyruvate dehydrogenase, phosphoenol pyruvate carboxykinase and pyruvate kinase, all of which are involved in acetyl-CoA metabolism, increased by 10, 43.9 and 17.5%, respectively, in cultures grown under low aeration conditions compared with those grown under high aeration conditions. These changes were related to alginate acetylation, which was 2.6 times higher in the cultures with limited oxygen, and the changes were also related to a drastic increase in PHB production. Finally, the glyoxylate shunt was active under both of the conditions that were tested, and a 2.79-fold increase was observed in cultures that were grown under the low aeration condition.

Systems biology approaches involve collaboration of a larger number of research groups. Experiments are being performed in different laboratories dealing with different aspects of the topic of interest. Therefore, comparability of data collected for further analysis and modeling needs critical assessment. Here, growth and product formation of two human designer cell lines (AGE1.HN and its 1-antitrypsin producing clone AGE1.HN.AAT) were investigated by four research laboratories. Cell lines were cultivated in shake flasks and stirred tank bioreactors operated at standardized conditions using a chemically defined medium, and a simple mathematical model was used to estimate characteristic process parameters. Results obtained for 35 batches showed that neither the initial viable cell concentration nor the initial concentration of glucose and glutamine showed significant differences between laboratories. For these measurements with low variations, specific growth rate and yields varied between 8.5 and 26% (relative standard error), indicating comparability of cultivations between laboratories for exponential growth. Higher variations of fitted parameters related to measurements with high initial variation. Comparing the nonproducing with the producing cell line, no significant differences were found regarding growth dynamics and metabolism. Overall, it seems justified to draw conclusions based on the entire experimental dataset of this systems biology project.

Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysC(fbr)dld(Psod)pyc(Psod)malE(Psod)fbp(Psod)gapX(Psod) was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of D-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35 h⁻¹ and lysine carbon yields of approximately 90 C-mmol (C-mol)⁻¹. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.

Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.

We used a recombinant, permeabilized E. coli Nissle strain harbouring the plu3263 gene cluster from Photorhabdus luminescens for the synthesis of luminmide type cyclic pentapeptides belonging to the class of nonribosomally biosynthesized peptides (NRP). Cells could be fully permeabilized using 1 % v/v toluene. Synthesis of luminmides was increased fivefold when 0.3 mM EDTA was added to the substrate mixture acting as an inhibitor of metal proteases. Luminmide formation was studied applying different amino acid concentrations. Apparent kinetic parameters for the synthesis of the main product luminmide A from leucine, phenylalanine and valine were calculated from the collected data. K sapp values ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and r maxapp was about 3 × 10-8 mmol min-1(g CDW)-1). By removing phenylalanine from the substrate mixture, the formation of luminmide A was reduced tenfold while luminmide B was increased from 50 to 500 μg/l becoming the main product. Two new luminmides were synthesized in this study. Luminmide H incorporates tryptophan replacing phenylalanine in luminmide A. In luminmide I, leucine was replaced with 4,5-dehydro-leucine, a non-proteinogenic amino acid fed to the incubation mixture. Our study shows new opportunities for increasing the spectrum of luminmide variants produced, for improving production selectivity and for kinetic in vitro studies of the megasynthetases.

The identification of fur origins from the 5300-year-old Tyrolean Iceman's accoutrement is not yet complete, although definite identification is essential for the socio-cultural context of his epoch. Neither have all potential samples been identified so far, nor there has a consensus been reached on the species identified using the classical methods. Archaeological hair often lacks analyzable hair scale patterns in microscopic analyses and polymer chain reaction (PCR)-based techniques are often inapplicable due to the lack of amplifiable ancient DNA. To overcome these drawbacks, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method was used exclusively based on hair keratins. Thirteen fur specimens from his accoutrement were analyzed after tryptic digest of native hair. Peptide mass fingerprints (pmfs) from ancient samples and from reference species mostly occurring in the Alpine surroundings at his lifetime were compared to each other using multidimensional scaling and binary hierarchical cluster tree analysis. Both statistical methods highly reflect spectral similarities among pmfs as close zoological relationships. While multidimensional scaling was useful to discriminate specimens on the zoological order level, binary hierarchical cluster tree reached the family or subfamily level. Additionally, the presence and/or absence of order, family and/or species-specific diagnostic masses in their pmfs allowed the identification of mammals mostly down to single species level. Red deer was found in his shoe vamp, goat in the leggings, cattle in his shoe sole and at his quiver's closing flap as well as sheep and chamois in his coat. Canid species, like grey wolf, domestic dog or European red fox, were discovered in his leggings for the first time, but could not be differentiated to species level. This is widening the spectrum of processed fur-bearing species to at least one member of the Canidae family. His fur cap was allocated to a carnivore species, but differentiation between brown bear and a canid species could not be made with certainty.

This study focused on metabolic changes in the neuronal human cell line AGE1.HN upon increased ammonia stress. Batch cultivations of α(1)-antitrypsin (A1AT) producing AGE1.HN cells were carried out in media with initial ammonia concentrations ranging from 0mM to 5mM. Growth, A1AT production, metabolite dynamics and finally metabolic fluxes calculated by metabolite balancing were compared. Growth and A1AT production decreased with increasing ammonia concentration. The maximum A1AT concentration decreased from 0.63g/l to 0.51g/l. Central energy metabolism remained relatively unaffected exhibiting only slightly increased glycolytic flux at high initial ammonia concentration in the medium. However, the amino acid metabolism was significantly changed. Fluxes through transaminases involved in amino acid degradation were reduced concurrently with a reduced uptake of amino acids. On the other hand fluxes through transaminases working in the direction of amino acid synthesis, i.e., alanine and phosphoserine, were increased leading to increased storage of excess nitrogen in extracellular alanine and serine. Glutamate dehydrogenase flux was reversed increasingly fixing free ammonia with increasing ammonia concentration. Urea production additionally observed was associated with arginine uptake by the cells and did not increase at high ammonia stress. It was therefore not used as nitrogen sink to remove excess ammonia. The results indicate that the AGE1.HN cell line can adapt to ammonia concentrations usually present during the cultivation process to a large extent by changing metabolism but with slightly reduced A1AT production and growth.

Addition of the flavonoid quercetin to cultivations of the α1-antitrypsin (A1AT) producing human AGE1.HN.AAT cell line resulted in alterations of the cellular physiology and a remarkable improvement of the overall performance of these cells. In a first screening in 96-well plate format, toxicity and the effect of quercetin on the lactate/glucose ratio was analyzed. It was found that quercetin treatment reduced the lactate/glucose ratio dose dependently. An increase in culture longevity, viable cell density (160% of control), and A1AT concentration (from 0.39 g/L in the control to 0.76 g/L with quercetin, i.e., 195% of the control) was observed in batch cultivation with 10 μM quercetin compared to the control. A detailed analysis of quercetin effects on primary metabolism revealed dose-dependent alterations in metabolic fluxes. Quercetin addition resulted in an improved channeling of pyruvate into the mitochondria accompanied by reduced waste product formation and stimulation of TCA cycle activity. The observed changes in cellular physiology can be explained by different properties of quercetin and its metabolites, e.g., inhibition of specific enzymes, stimulation of oxidation of cytoplasmic, and mitochondrial NADH resulting in reduced NADH/NAD+ ratio, and cytoprotective activity. The present study shows that the addition of specific effectors to the culture medium represents a promising strategy to improve the cellular metabolic phenotype and the production of biopharmaceuticals. The provided results contribute, additionally, to an improved understanding of quercetin action on the metabolism of human cells in a general physiological context.

Metabolic compartmentation represents a major characteristic of eukaryotic cells. The analysis of compartmented metabolic networks is complicated by separation and parallelization of pathways, intracellular transport, and the need for regulatory systems to mediate communication between interdependent compartments. Metabolic flux analysis (MFA) has the potential to reveal compartmented metabolic events, although it is a challenging task requiring demanding experimental techniques and sophisticated modeling. At present no ready-made solution can be provided to cope with the complexity of compartmented metabolic networks, but new powerful tools are emerging. This review gives an overview of different strategies to approach this issue, focusing on different MFA methods and highlighting the additional information that should be included to improve the outcome of an experiment and associate estimation procedures.

Grass silage provides a great potential as renewable feedstock. Two fractions of the grass silage, a press juice and the fiber fraction, were evaluated for their possible use for bioethanol production. Direct production of ethanol from press juice is not possible due to high concentrations of organic acids. For the fiber fraction, alkaline peroxide or enzymatic pretreatment was used, which removes the phenolic acids in the cell wall. In this study, we demonstrate the possibility to integrate the enzymatic pretreatment with a simultaneous saccharification and fermentation to achieve ethanol production from grass silage in a one-process step. Achieved yields were about 53 g ethanol per kg silage with the alkaline peroxide pretreatment and 91 g/kg with the enzymatic pretreatment at concentrations of 8.5 and 14.6 g/L, respectively. Furthermore, it was shown that additional supplementation of the fermentation medium with vitamins, trace elements and nutrient salts is not necessary when the press juice is directly used in the fermentation step.

Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening comprising of not only electrophysiological characterization but also cellular molecular alterations are expected to improve the cardiotoxicity prediction potential. Metabolomic approaches recently have become an important focus of research in pharmacological testing and prediction. In this study, the culture medium supernatants from HL-1 cardiomyocytes after exposure to drugs from different classes (analgesics, antimetabolites, anthracyclines, antihistamines, channel blockers) were analyzed to determine specific metabolic footprints in response to the tested drugs. Since most drugs influence energy metabolism in cardiac cells, the metabolite "sub-profile" consisting of glucose, lactate, pyruvate and amino acids was considered. These metabolites were quantified using HPLC in samples after exposure of cells to test compounds of the respective drug groups. The studied drug concentrations were selected from concentration response curves for each drug. The metabolite profiles were randomly split into training/validation and test set; and then analysed using multivariate statistics (principal component analysis and discriminant analysis). Discriminant analysis resulted in clustering of drugs according to their modes of action. After cross validation and cross model validation, the underlying training data were able to predict 50%-80% of conditions to the correct classification group. We show that HPLC based characterisation of known cell culture medium components is sufficient to predict a drug's potential classification according to its mode of action.

The Corynebacterium glutamicum ATCC 13032 lysC(fbr) strain was engineered to grow fast on racemic mixtures of lactate and to secrete lysine during growth on lactate as well as on mixtures of lactate and glucose. The wild-type C. glutamicum only grows well on L-lactate. Overexpression of D-lactate dehydrogenase (dld) achieved by exchanging the native promoter of the dld gene for the stronger promoter of the sod gene encoding superoxide dismutase in C. glutamicum resulted in a duplication of biomass yield and faster growth without any secretion of lysine. Elementary mode analysis was applied to identify potential targets for lysine production from lactate as well as from mixtures of lactate and glucose. Two targets for overexpression were pyruvate carboxylase and malic enzyme. The overexpression of these genes using again the sod promoter resulted in growth-associated production of lysine with lactate as sole carbon source with a carbon yield of 9% and a yield of 15% during growth on a lactate-glucose mixture. Both substrates were taken up simultaneously with a slight preference for lactate. As surmised from the elementary mode analysis, deletion of glucose-6-phosphate isomerase resulted in a decreased production of lysine on the mixed substrate. Elementary mode analysis together with suitable objective functions has been found a very useful tool guiding the design of strains producing lysine on mixed substrates.

In this article, recent progress in cardiotoxicity testing based on the use of immortalized cell lines or human embryonic stem cell (hESC) derived cardiomyocytes in combination with state-of-the-art bioanalytical methods and sensors is reviewed. The focus is on hESC-derived cells and their refinement into competent testing cells, but the access and utility of other relevant cell types are also discussed. Recent developments in sensor techniques and bioanalytical approaches for measuring critical cardiotoxicity parameters are highlighted, together with aspects of data evaluation and validation. Finally, recommendations for further research are given.

Grassilage stellt einen nachwachsenden Rohstoff mit grosem Potenzial dar. Neben Cellulose und Hemicellulose enthalt sie auch organische Sauren, insbesondere Milchsaure. In einem Bioraffinerie-Projekt wird die Milchsaure aus der Silage isoliert und mit gentechnisch optimierten Stammen zu L-Lysin weiterverarbeitet. Die Lignocellulose wird hydrolysiert und zu Ethanol fermentiert. Ein besonderes Augenmerk liegt auf der Integration der unterschiedlichen Prozesse sowie der einzelnen Prozessschritte zu einem Gesamtprozess, der samtliche Inhaltsstoffe der Silage verwertet.

Report and recommendation of a workshop on education and training for measurement, monitoring, modelling and control ((MC)-C-3) in biochemical engineering

Metabolic flux analysis (MFA) represents a powerful tool for systems biology research on eukaryotic cells. This review describes recent advances, the challenges as well as applications of metabolic flux analysis comprising fungi, mammalian cells and plants. While MFA is widely established and applied in microorganisms, it remains still a challenge to adapt these methods to eukaryotic cell systems having a higher complexity particularly concerning compartmentation or media composition. In fungi MFA was used in the past few years to analyze a variety of conditions and factors and their effects on cellular metabolism. In mammalian cells MFA was applied mainly in cell culture technology and in medical and toxicological research. (13)C metabolic studies on native whole plants are additionally challenging by the fact that CO(2) is usually the only carbon source.

Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC(50) values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs.

Permeabilization of Corynebacterium glutamicum cells permits direct determination of enzyme activity measuring NAD(P)H after appropriate calibration and correction for cell density. The optimized conditions found were the treatment of 10 mg cells/ml (dry weight) by Triton X-100 at a final concentration of 0.05% for 5 min at room temperature. Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were very close to those determined in the cell-free extract. In cell-free extracts malic enzyme activity was about double and that of isocitrate dehydrogenase about triple the values in permeabilized cells. K m values were similar in cell-free extract and permeabilized cells, but larger than literature values obtained with purified enzymes.

We describe a new device with parallel optical measurement of dissolved oxygen (DO) and pH in up to nine shake flasks applicable in any conventional shaking incubator. Measurement ranges are 0–500% of air saturation for oxygen and 5.5–8.5 for pH. It was used to characterize growth profiles of different l-lysine producing strains of Corynebacterium glutamicum, of Saccharomyces cerevisiae and of Escherichia coli. Cultures in unbaffled flasks were highly reproducible. Oxygen limitation was indicated online which is particularly important when cultivating fast growing cells as E. coli. C. glutamicum strains showed distinct characteristic patterns of DO and pH indicating biological events. During the cultivation of S. cerevisiae on glucose, fructose and galactose, oxygen uptake rate was determined using the predetermined value of k L a. pH measurement was used to determine the minimum buffer requirement for a culture of C. glutamicum.

The reversible unfolding of metallo-beta-lactamase from Chryseobacterium meningosepticum (BlaB) by guanidinium hydrochloride is best described by a three-state model including folded, intermediate, and unfolded states. The transformation of the folded apoenzyme into the intermediate state requires only very low denaturant concentrations, in contrast to the Zn2-enzyme. Similarly, circular dichroism spectra of both BlaB and metallo-beta-lactamase from Bacillus cereus 569/H/9 (BcII) display distinct differences between metal-free and Zn2-enzymes, indicating that the zinc ions affect the folding of the proteins, giving a larger alpha-helix content. To identify the regions of the protein involved in this zinc ion-induced change, a hydrogen deuterium exchange study with matrix-assisted laser desorption ionization tandem time of flight mass spectrometry on metal-free and Zn1- and Zn2-BcII was carried out. The region spanning the metal binding metallo-beta-lactamases (MBL) superfamily consensus sequence His-X-His-X-Asp motif and the loop connecting the N- and C-terminal domains of the protein undergoes a zinc ion-dependent structural change between intrinsically disordered and ordered states. The inherent flexibility even appears to allow for the formation of metal ion-bridged protein-protein complexes which may account for both electrospray ionization-mass spectroscopy results obtained upon variation of the zinc/protein ratio and stoichiometry-dependent variations of 199mHg-perturbed angular correlation of gamma-rays spectroscopic data. We suggest that this flexible "zinc arm" motif, present in all the MBL subclasses, is disordered in metal-free MBLs and may be involved in metal ion acquisition from zinc-carrying molecules different from MBL in an "activation on demand" regulation of enzyme activity.

Attaining metabolic and isotopic balanced growth is one critical condition for physiological studies using isotope-labeled tracers, but is very difficult to obtain in batch culture due to the extensive metabolite exchange with the surrounding medium and related physiological changes. In the present study, we investigated metabolic and isotopic behavior of CHO cells in differently designed media. We observed that the assumption of balanced cell growth cannot be justified in batch culture of CHO cells directly using conventional, commercially available media. By systematically redesigning media composition and characterizing metabolic steady state based on mass balances and measurement of labeling dynamics, we achieved balanced cell growth for the main cellular substrates in CHO cells. This was done in a step-by-step analysis of growth and primary metabolism of CHO cells with the use of [U-13C]glucose feeding and adjusting concentrations of amino acids in the growth medium. The optimized media obtained at the end of the study provide balanced growth and isotopic steady state or at least asymptotic steady state. As a result, we established a platform to conduct isotope-based physiological studies of mammalian systems more reliably and therefore well suited for later use in metabolic profiling of mammalian systems such as 13C-labeled metabolic flux analysis.

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.

The applicability of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of 13C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the 13C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-13C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the 13C enrichment. The approach to correct for these effects in metabolic flux studies using delta13C measurement by GC-C-IRMS uses two parallel experiments applying substrate with natural abundance and 13C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of 13C in proteinogenic amino acids over the whole range of applied concentrations of [1-13C]glucose. The current findings indicate a great potential of GC-C-IRMS for labeling quantification in 13C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.

Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE from Salmonella enterica serovar Typhimurium, a member of this group, has been expressed in Escherichia coli and was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope of E. coli and was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.

Strains belonging to the yeast species Kluyveromyces marxianus have been isolated from a great variety of habitats, which results in a high metabolic diversity and a substantial degree of intraspecific polymorphism. As a consequence, several different biotechnological applications have been investigated with this yeast: production of enzymes (beta-galactosidase, beta-glucosidase, inulinase, and polygalacturonases, among others), of single-cell protein, of aroma compounds, and of ethanol (including high-temperature and simultaneous saccharification-fermentation processes); reduction of lactose content in food products; production of bioingredients from cheese-whey; bioremediation; as an anticholesterolemic agent; and as a host for heterologous protein production. Compared to its congener and model organism, Kluyveromyces lactis, the accumulated knowledge on K. marxianus is much smaller and spread over a number of different strains. Although there is no publicly available genome sequence for this species, 20% of the CBS 712 strain genome was randomly sequenced (Llorente et al. in FEBS Lett 487:71-75, 2000). In spite of these facts, K. marxianus can envisage a great biotechnological future because of some of its qualities, such as a broad substrate spectrum, thermotolerance, high growth rates, and less tendency to ferment when exposed to sugar excess, when compared to K. lactis. To increase our knowledge on the biology of this species and to enable the potential applications to be converted into industrial practice, a more systematic approach, including the careful choice of (a) reference strain(s) by the scientific community, would certainly be of great value.