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Research Interests: Microbiology, Technology, Computational Biology, Biology, Industrial Biotechnology, and 14 moreResearch, Drug, Biological Sciences, Saccharomyces cerevisiae, Discriminant Analysis, Drug Screening, Microbial Fuel Cell, Growth rate, Yeast Metabolite Profiling, Drug Targeting, Metabolite, Partial Least Square, Production Rate, and metabolite profiling
Bassem Ben Yahia, Biochemical Engineering Institute, Saarland University, Germany, Upstream Process Sciences Biotech Sciences, UCB Pharma S.A., Avenue de l’Industrie, Belgium bassem.benyahia@ucb.com Boris Gourevitch, Institut de... more
Bassem Ben Yahia, Biochemical Engineering Institute, Saarland University, Germany, Upstream Process Sciences Biotech Sciences, UCB Pharma S.A., Avenue de l’Industrie, Belgium bassem.benyahia@ucb.com Boris Gourevitch, Institut de NeuroScience Paris-Saclay (NeuroPSI), France, Université Paris-Sud, France Elmar Heinzle, Biochemical Engineering Institute, Saarland University, Saarbrücken, Germany Laetitia Malphettes, Upstream Process Sciences Biotech Sciences, UCB Pharma S.A., Avenue de l’Industrie, Belgium
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Bassem Ben Yahia, Biochemical Engineering Institute, Saarland University, Germany ; Upstream Process Sciences Biotech Sciences, UCB Pharma S.A., Belgium bassem.benyahia@ucb.com Boris Gourevitch, Institut de NeuroScience Paris-Saclay... more
Bassem Ben Yahia, Biochemical Engineering Institute, Saarland University, Germany ; Upstream Process Sciences Biotech Sciences, UCB Pharma S.A., Belgium bassem.benyahia@ucb.com Boris Gourevitch, Institut de NeuroScience Paris-Saclay (NeuroPSI) ; Université Paris-Sud, France Laetitia Malphettes, Upstream Process Sciences Biotech Sciences, UCB Pharma S.A., Belgium Elmar Heinzle, Biochemical Engineering Institute, Saarland University, Germany
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The analysis of the intracellular metabolic fluxes in mammalian cells can be used for optimizing culture media, for tracing metabolic boUle-necks and for toxicological and metabolic studies. It is desirable to detennine metabolic fluxes... more
The analysis of the intracellular metabolic fluxes in mammalian cells can be used for optimizing culture media, for tracing metabolic boUle-necks and for toxicological and metabolic studies. It is desirable to detennine metabolic fluxes without using Isotopic tracers. This could potentialy be achieved by using the material balances of the relevant metabolites. For eaeh metabolite a mass balance is set up using the known biochemical stoichiomeby and ills measured uptake or production rate. Together, these mass balances form a linear set of equations, containing the unknown fluxes. However, such a set of linear equations is undercletennined due to the presence of cyclic pathways in mammalian cell metabolism. Fluxes In such pathways have to be quantitated using isotopic tracers. Instead of detennining fluxes in cyclic pathways experimentally using isotopic tracers, additional theoretical assumptions can be applied to obtain an estimate for these fluxes. In this work, both methods have ...
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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... more
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. Ksapp values ranged from 0.17 mM for leucine to 0.57 mM for phenylalanine, and rmaxapp 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.
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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... more
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 ...
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The analysis of the intracellular metabolic fluxes in mammalian cells can be used for optimizing culture media, for tracing metabolic bottle-necks and for toxicological and metabolic studies. It is desirable to determine metabolic fluxes... more
The analysis of the intracellular metabolic fluxes in mammalian cells can be used for optimizing culture media, for tracing metabolic bottle-necks and for toxicological and metabolic studies. It is desirable to determine metabolic fluxes without using isotopic tracers. This could potentially be achieved by using the material balances of the relevant metabolites. For each metabolite a mass balance is set up using the known biochemical stoichiometry and its measured uptake or production rate. Together, these mass balances form a linear set of equations, containing the unknown fluxes. However, such a set of linear equations is underdeterwined due to the presence of cyclic pathways in mammalian cell metabolism. Fluxes in such pathways have to be quantitated using isotopic tracers.
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Sicherheit und Umweltschutz in der chemischen Produktion werden masgeblich durch die Entwicklung der chemischen Prozesse bestimmt. Dabei gilt es, den Schutz von Mensch und Umwelt zusammen mit Produktqualitat und Wirtschaftlichkeit schon... more
Sicherheit und Umweltschutz in der chemischen Produktion werden masgeblich durch die Entwicklung der chemischen Prozesse bestimmt. Dabei gilt es, den Schutz von Mensch und Umwelt zusammen mit Produktqualitat und Wirtschaftlichkeit schon in den fruhesten Entwicklungsstufen als Leitkriterien zu nutzen [1–5] (Abb.l l.l).
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Chlorinated bis(bibenzyls) of the bazzanine type were detected in crude bryophyte plant extracts of Bazzania trilobata from different locations using laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry without addition... more
Chlorinated bis(bibenzyls) of the bazzanine type were detected in crude bryophyte plant extracts of Bazzania trilobata from different locations using laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry without addition of an additional chemical matrix. The degree of chlorination could be identified by the characteristic isotope patterns. These results suggest that these chlorinated compounds are not artefacts of an incidental occurrence, or of the sample preparation, but are genuine natural products produced by the liverwort or by an endosymbiotic metabolism.
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improved the mTORC2-mediated phosphorylation of AKT at Ser in the ethanol-fed, PFT injected animals. Conclusions: Inhibition of p53 improved liver injury, apoptosis, oxidative and reductive stress as well as insulin/PI3K/AKT signaling in... more
improved the mTORC2-mediated phosphorylation of AKT at Ser in the ethanol-fed, PFT injected animals. Conclusions: Inhibition of p53 improved liver injury, apoptosis, oxidative and reductive stress as well as insulin/PI3K/AKT signaling in the livers of ethanol-fed LE rats. This observation underlines the importance of p53 in the pathogenesis of ALD. This study also identifies TIGAR that may mediate ethanol-induced hepatic insulin resistance.
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Systematic procedures for reducing wastes in complex chemical plants are needed to allow efficient optimisation. A hierarchical procedure was applied to the optimisation of a real industrial plant to reduce wastes as well as energy and... more
Systematic procedures for reducing wastes in complex chemical plants are needed to allow efficient optimisation. A hierarchical procedure was applied to the optimisation of a real industrial plant to reduce wastes as well as energy and raw material consumption. In the case studied, the continuous production of methyl-butynol (MBI), acetylene reacts with acetone. The solvent ammonia and acetylene are recycled to the reactor. Unreacted substrates and by-products are separated from the product stream by distillation. Part of the unreacted acetone can be reused for other purposes after distillation. A substantial part of the unreacted substrates and by-products is delivered to a wastewater treatment plant. These waste streams constitute a substantial problem for the operation of this plant. First, waste streams were characterised and tracked back to their origin. Following the hierarchical design procedure, the overall input-output structure was fixed. The entrainer in the present process was critically examined and options were suggested. Then various recycle schemes were considered for later detailed study. The existing plant was simulated using ASPENPLUS. After adjusting the model to all important aspects of the real process scheme, excellent agreement between actual process performance data and simulation was obtained. The various process schemes were simulated and assessed for their economic and ecological performance. The objective functions used included utility, substrate and catalyst costs, as well as costs for wastewater treatment. Additionally, the environmental burden related to energy supply was accounted for by a carbon dioxide tax as suggested by the Nordic countries. The process changes included separation of unreacted acetone from the product stream and recycling to the reactor. By-products were converted back to substrates in an additional reactor separation system and recycled. In various simulated process configurations and operational schemes substantial economic and ecologic improvements were achieved. This study demonstrates the usefulness of hierarchical approaches combined with process simulation for plant optimisation.
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Biological degradation of chloroguaiacols contained in sulphite pulp chlorine bleaching wastewater was studied in four parallel biological fluidised bed reactor systems—one single aerobic, one single anaerobic and two combined... more
Biological degradation of chloroguaiacols contained in sulphite pulp chlorine bleaching wastewater was studied in four parallel biological fluidised bed reactor systems—one single aerobic, one single anaerobic and two combined anaerobic–aerobic reactors. At low loading rates, trichloroguaiacols were removed nearly quantitatively. 4,5‐Dichloroguaiacol was only partly removed. At high loading rates the anaerobic–aerobic recycle reactor removed individual guaiacols more than the other reactors. Only 4,5,6‐trichloroguaiacol was removed best by the anaerobic–aerobic reactors in series. Even mixed culture biofilms adapted during several years of continuous operation did not satisfactorily remove these compounds. Synthetic wastewater, containing chlorinated guaiacols, treated with ozone produced formate and oxalate and quantitatively inorganic chloride. Combined ozonation–biotreatment in two reactors in series as well as in a recycle system allowed complete removal of all individual chlori...
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ABSTRACT Bio-based processes are commonly accepted to be environmentally preferable to chemical alternatives. Reasons include high selectivities, the avoidance of heavy metals, and mild reaction conditions. However, ecological benefits... more
ABSTRACT Bio-based processes are commonly accepted to be environmentally preferable to chemical alternatives. Reasons include high selectivities, the avoidance of heavy metals, and mild reaction conditions. However, ecological benefits and economic viability have to be verified in each case. Oxygenases are a very attractive enzyme class, allowing selective oxyfunctionalization by introduction of molecular oxygen into hydrocarbons at optical purities unparalleled by traditional chemical methods. Here, styrene monooxygenase from Pseudomonas sp. strain VLB120 was used in recombinant Escherichia coli for the production of enantiopure (S)-styrene oxide from styrene. Substrate and product toxicity was attenuated using a two-liquid phase system with bis(2-ethylhexyl)phthalate as organic carriersolvent. By maintaining previously reported productivities for a longer time period, product concentrations were doubled to 36.3 g Ltot−1 making it one of the most productive biocatalytic oxyfunctionalization processes. This biotransformation was incorporated along with an appropriate upstream and downstream processing into a complete process scheme and compared with chemical process alternatives. Ecological assessment showed the bioprocess to be superior to two chemical process alternatives and slightly inferior to the third one, with solvent use being the most critical factor. However, the bioprocess performed best in terms of production costs ($10.2 kg−1). This study underlines the importance of a detailed ecological and economic assessment of bioprocesses to verify their sustainability and to identify weak points with respect to environmental and/or economical sustainability.
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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... more
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 t...
Research Interests: Engineering, Chemistry, Technology, Food Science, Ethanol Fuel, and 4 moreEthanol, Fermentation, Hydrolysis, and Silage
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Background The authors have developed a new clinical bymixer that bypasses a constant fraction of gas flow through a mixing arm. A separate bymixer was interposed in the expiratory and inspiratory limbs of the ventilation circuit to... more
Background The authors have developed a new clinical bymixer that bypasses a constant fraction of gas flow through a mixing arm. A separate bymixer was interposed in the expiratory and inspiratory limbs of the ventilation circuit to measure mixed gas fractions. By utilizing nitrogen conservation, the clinical bymixer allows the determination of airway carbon dioxide elimination (VCO2) and oxygen uptake (VO2), whenever basic expired flow and gas monitoring measurements are used for the patient. Neither an expiratory exhaust gas collection bag nor expensive, complex equipment are needed. This study tested the accuracy of airway bymixer-flow measurements of VCO2 and VO2 in a new bench apparatus. Methods The authors compared airway bymixer-flow measurements of VCO2 and VO2 over a range of reference values generated by ethanol combustion in a new metabolic lung simulator, which was ventilated by a volume-cycled respirator. An airway humidity and temperature sensor permitted standard temp...
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Wistar and Sprague-Dawley (SD) rats are the most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and... more
Wistar and Sprague-Dawley (SD) rats are the most commonly used experimental rats. They have similar genetic background and are therefore, not discriminated in practical research. In this study, we compared metabolic profiles of Wistar and SD rat hepatocytes from middle (6 months) and old (23 months) age groups. Specific uptake and production rates of amino acids, glucose, lactate and urea showed clear differences between Wistar and SD rats as shown by principle component analysis (PCA). SD rat hepatocytes showed higher uptake rates of various essential and non-essential amino acids, particularly in early culture phases (0-12 h) compared to later phases (12-24 h). SD hepatocytes seem to be more sensitive to isolation procedure and in vitro culture requiring more amino acids for cellular maintenance and repair. Major differences between Wistar and SD rat hepatocytes were observed for glucose and branched chain amino acid metabolism. We conclude that the observed differences in the cen...
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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... more
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 an...
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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... more
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 Appro...
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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... more
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 upregulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared to the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-f...
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Matrix-assisted laser desorption/ionization mass spectrometry has become an indispensable tool for identification of proteins by peptide mass-fingerprint analysis. Selection of the matrix, addition of matrix additives, and... more
Matrix-assisted laser desorption/ionization mass spectrometry has become an indispensable tool for identification of proteins by peptide mass-fingerprint analysis. Selection of the matrix, addition of matrix additives, and sample-preparation techniques are known to affect the quality of the spectra and hence protein identification. We investigated the effect of pyridine as matrix additive for the commonly used crystalline matrix alpha-cyano-4-hydroxycinnamic acid (CCA), forming a pyridinium based ionic liquid matrix, on the mass spectra of synthetic peptides and tryptic protein digests. Beside the equimolar mixture of CCA and pyridine, the effect of addition of substoichiometric amounts of the base to the acid was tested. Optimum results in terms of signal-to-noise ratios, reduction of chemical noise, and reduced formation of alkali adducts and matrix clusters were observed for the matrix CCA-pyridine in the molar ratio 2:1. The optimized ionic liquid matrix was used for identification of tryptic digests of six model proteins and for identification of a protein extracted from a two-dimensional gel with the proteome of the bacterium Corynebacterium glutamicum, and shown to facilitate protein identification, yielding higher scores and increased sequence coverage compared with pure CCA. Thus CCA-Py 2:1 is a potential alternative for identification and characterization of proteins by peptide mass-fingerprint analysis.
Research Interests: Engineering, Mass Spectrometry, Proteomics, Ionic Liquid, Sample Preparation, and 13 moreBiological Sciences, Noise reduction, Analytical, Peptides, Proteins, CHEMICAL SCIENCES, Analytical and Bioanalytical Chemistry, Signal to Noise Ratio, Hydrogen-Ion Concentration, Ions, MALDI, Adduct, and limit of detection
A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated.... more
A two-dimensional separation scheme for shotgun proteome analysis employing high-pH reversed-phase HPLC in the first and low-pH ion-pair reversed-phase HPLC in the second dimension (RP x IP-RP-HPLC) has been developed and evaluated. Compared to the classical strong cation exchange x ion-pair reversed-phase (SCX x IP-RP-HPLC) approach, the RP x IP-RP-HPLC system was characterized by a lower degree of orthogonality, which was, however, more than counterbalanced by higher separation efficiency, more homogeneous distribution of peptide elution, and easier experimental handling. Peptide fragment fingerprinting by electrospray-ionization tandem mass spectrometry (ESI-MS/MS) was employed for peptide detection and identification. About 13% more peptides and 7% more proteins could be identified with the alternative approach in 30% less analysis time, enabling the analysis of the proteome of Corynebacterium glutamicum with a coverage of 24.9% (745 proteins). Combining the identification results both of the SCX- x IP-RP-HPLC-ESI-MS/MS and RP- x IP-RP-HPLC-ESI-MS/MS methods, a total of 871 proteins were identified in a cytosolic protein preparation, which represented 29.1% of all proteins annotated in the genome of C. glutamicum.
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Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption.... more
Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly-ß-hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate