Saarbrücken, Germany
Saarbrücken, Germany

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Zollner A.,PomBioTech GmbH | Parr M.K.,German Sport University Cologne | Dragan C.-A.,PomBioTech GmbH | Dras S.,PomBioTech GmbH | And 6 more authors.
Biological Chemistry | Year: 2010

Anabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13- trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeast Schizosaccharomyces pombe that express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite via S. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays. © by Walter de Gruyter.


Zehentgruber D.,Jülich Research Center | Dragan C.-A.,PomBioTech GmbH | Bureik M.,PomBioTech GmbH | Lutz S.,Jülich Research Center
Journal of Biotechnology | Year: 2010

Since cytochrome P450 monooxygenases enable the regio- and stereo-selective hydroxylation of C-H bonds, they are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals. Nevertheless, for industrial applications of such enzymes, e.g., steroid hydroxylation, several challenges like cofactor and oxygen supply, limited stability and activity, or low substrate solubility have to be overcome. To identify the limiting factors in a P450 catalyzed whole cell biotransformation, 21-hydroxylation of 17-α-hydroxyprogesterone in Schizosacharomyces pombe expressing human CYP21 was chosen as model reaction. We report here that resting cells of this recombinant yeast strain can be used for efficient biotransformation. In the present study, we analyzed the intracellular redox cofactor pool of S. pombe by LC-MS/MS measurements and report the first quantification of the intracellular cofactor pool during P450 hydroxylation. Thereby a limitation caused by the redox cofactor could be excluded for resting cells. In contrary, low substrate solubility and its transport into the cell affect activity. Screening for an appropriate cosolvent identified methanol as the most promising candidate, since it showed the lowest inactivation effect on the biocatalyst. Through permeabilization of the membrane with the detergent tween 80 steroid hydroxylation activity increases, leading to a productivity of 540μM d -1 in a final batch experiment under optimized reaction conditions. © 2010 Elsevier B.V.


Zollner A.,PomBioTech GmbH | Buchheit D.,PomBioTech GmbH | Meyer M.R.,Saarland University | Maurer H.H.,Saarland University | And 2 more authors.
Bioanalysis | Year: 2010

Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs and poisons in humans, while UDP glycosyltransferases catalyze the majority of phase II reactions. In addition, a number of other enzymes or enzyme families contribute to the metabolism of xenobiotica, including alcohol dehydrogenase, aldehyde dehydrogenase, ester and amide hydrolases, epoxide hydrolase and flavine monooxygenases, as well as sulfotransferases, catechol-O-methyltransferase and N-acetyltransferase. A thorough understanding of their activity and of the properties of the metabolites they form is an essential prerequisite for the assessment of drug-caused side effects or toxicity. In this context of MIST, efficient production systems are needed to permit the large-scale production of human drug metabolites. As classical chemical synthesis cannot always provide these metabolites, biotechnological approaches have been developed that typically employ the recombinant expression of human drug-metabolizing enzymes. This review summarizes the current knowledge regarding whole-cell biotransformation processes that make use of such an approach. © 2010 Future Science Ltd.


Naumann J.M.,PomBioTech GmbH | Messinger J.,Solvay Group | Bureik M.,PomBioTech GmbH
Journal of Biotechnology | Year: 2010

While phase I and phase II drug metabolites are important for drug development and toxicity studies, e.g. in the context of metabolites in safety testing (MIST), they are often not commercially available and their classical chemical synthesis can be cumbersome. Therefore, a biotechnological production of drug metabolites using microorganisms that recombinantly express human enzymes has been established in recent years. However, no whole-cell biotransformations that make use of human aldo-keto reductases (AKRs) have yet been reported. In this study, we have functionally expressed human AKR1C1 (20α-hydroxysteroid dehydrogenase) in the fission yeast Schizosaccharomyces pombe and demonstrate the ability of the resulting yeast strain to efficiently catalyze the reduction of progesterone or dydrogesterone to 20α-dihydroprogesterone (20α-DHP) and 20α-dihydrodydrogesterone (20α-DHD), respectively. The formation of any by-products or the occurrence of a back reaction were not detected. Seven other steroids with a 20-keto group (pregnenolone, 17α-hydroxyprogesterone, 11-deoxycortisol, cortisol, 11-deoxycorticosterone, corticosterone, and aldosterone) were not reduced by this system. At shaking flask scale we obtained conversion rates of 90 (±26) μM/d 20α-DHP and 244 (±93) μM/d 20α-dihydrodydrogesterone (20α-DHD), respectively. In a fed-batch fermentation under optimized reaction conditions an average 20α-DHP production rate of 300 μM/d was determined for a total biotransformation time of 72. h. We thus established an AKR-dependent whole-cell biotransformation process that can be used for production of human AKR metabolites on a large scale. © 2010 Elsevier B.V.


Naumann J.M.,PomBioTech GmbH | Kuttner G.,Humboldt University of Berlin | Bureik M.,PomBioTech GmbH
Applied Biochemistry and Biotechnology | Year: 2011

There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion. © 2010 Springer Science+Business Media, LLC.


Klein T.,Saarland University | Lange S.,Saarland University | Wilhelm N.,PomBioTech GmbH | Bureik M.,PomBioTech GmbH | And 3 more authors.
Metabolic Engineering | Year: 2014

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying 13C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations. © 2013 International Metabolic Engineering Society.


Neunzig I.,PomBioTech GmbH | Dragan C.-A.,PomBioTech GmbH | Widjaja M.,PomBioTech GmbH | Schwaninger A.E.,Saarland University | And 4 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2011

The cytochrome P450 isoform CYP3A7 (wildtype) is the major form of CYP in human fetal liver. Since it is not exclusively expressed in the fetus but also in a significant number of adults, CYP3A7 has been moving into the focus of investigation on adverse drug reactions and interindividual differences in drug metabolism in the last few years. In addition, CYP3A7 is overexpressed in hepatocellular carcinoma (HCC), where it contributes to the elimination of drugs. We here report the development of a convenient and reliable whole-cell system for testing CYP3A7 activity using recombinant fission yeast. As expected, catalytic properties of wild type CYP3A7.1 and its polymorphic form CYP3A7.2 towards DHEA and testosterone resembled those reported previously. Interestingly, both isoforms of CYP3A7 did not metabolize the anti-cancer drug sorafenib (which is approved for the treatment of HCC), while CYP3A4 produced the N-oxide in our system, as expected. This finding suggests that CYP3A7 activity does not influence the effectiveness of this anti-cancer drug against HCC. Furthermore, CYP3A7-expressing fission yeast cells specifically converted a luciferin-derivate (luciferin-PFBE) to a luminescent product and this activity can conveniently be monitored by spectrometry, which allowed the determination of IC50-values for the broad-range P450 inhibitors econazole and miconazole, respectively. We believe that these new tools for a fast and easy investigation of substrates and inhibitors of human CYP3A7 will contribute to the gain of important insights for drug metabolism, efficacy and safety. © 2010 Elsevier B.V. All rights reserved.


Neunzig I.,Pom BioTech GmbH | Gohring A.,Pom BioTech GmbH | Dragan C.-A.,Pom BioTech GmbH | Zapp J.,Saarland University | And 3 more authors.
Journal of Biotechnology | Year: 2012

The anti-inflammatory drug ibuprofen (Ibu) is metabolized in the human liver to a number of metabolites including 1-hydroxyibuprofen (1-OH-Ibu), 2-OH-Ibu, and 3-OH-Ibu, respectively. The only human CYP known to produce relevant amounts of 3-OH-Ibu is CYP2C9 and as genetic polymorphisms of CYP2C9 influence the metabolization of numerous drugs, the availability of reference standards for CYP2C9-specific metabolites is of considerable interest. The aim of this study was to develop a biological production process for 3-OH-Ibu and to affirm its NMR characteristics. The recombinant fission yeast strain CAD68 coexpressing human CYP2C9 and CPR was used for the whole-cell biotransformation of Ibu to 3-OH-Ibu in 1. L batch-scale for 75. h. The average space-time yield for the bioproduction of 3-OH-Ibu (125± 34μmol/L. d) considerably exceeded that of 2-OH-Ibu (44 ± 10μmol/L. d). Accordingly, average biotransformation activities normalized to dry biomass weight were 5.0 ± 0.8μmol/g. d (3-OH-Ibu) and 1.9 ± 0.7μmol/g. d (2-OH-Ibu). The metabolite was prepurified on preparative TLC-plates, isolated by HPLC fractionation, and characterized by LC-MS and NMR. As expected, differential fragmentation patterns of 2-OH-Ibu and 3-OH-Ibu were detected in ESI-LC-MS analysis. 44. mg of 3-OH-Ibu was efficiently purified from four 1. L batch cultures and its structure was clearly confirmed by one- and two-dimensional NMR. © 2012 Elsevier B.V..


Naumann J.M.,PomBioTech GmbH | Zollner A.,PomBioTech GmbH | Dragan C.-A.,PomBioTech GmbH | Messinger J.,Abbott Laboratories | And 2 more authors.
Applied Biochemistry and Biotechnology | Year: 2011

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α- dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 μm day.1 was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3±1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale. © Springer Science+Business Media, LLC 2011.


Buchheit D.,PomBioTech GmbH | Schmitt E.I.,PomBioTech GmbH | Bischoff D.,Boehringer Ingelheim | Ebner T.,Boehringer Ingelheim | Bureik M.,PomBioTech GmbH
Biological Chemistry | Year: 2011

Human UDP glycosyltransferases (UGTs) play an important role in xenobiotic detoxification. They increase the solubility of their substrates by adding a sugar moiety (such as glucuronic acid) to different functional entities (such as hydroxyl groups). The aim of this study was to investigate how glucuronidation of a standard substrate is affected by a change of the hetero-atom at the conjugation site. For this purpose, we compared the in vitro glucuronidation rates of 4-methylumbelliferone and 7-mercapto-4-methylcoumarin, respectively. Human liver microsomes catalyzed the S-glucuronidation of 7-mercapto-4- methylcoumarin almost as efficient as the O-glucuronidation of 4-methylumbelliferone. When testing isoenzyme specificity by whole cell biotransformation with fission yeast strains that recombinantly express all 19 human members of the UGT1 and UGT2 families, it was found that 13 isoenzymes were able to glucuronidate 7-mercapto-4-methylcoumarin, with five of them being specific for this substrate and the other eight also converting 4-methylumbelliferone under these conditions. The remaining six UGTs did not accept either substrate. Out of the eight isoenzymes that glucuronidated both substrates, four catalyzed both reactions approximately to the same extent, while three displayed higher conversion rates towards 4-methylumbelliferone and one preferred 7-mercapto-4-methylcoumarin. These data suggest that 7-mercapto-4-methylcoumarin is a convenient new standard substrate for monitoring S-glucuronidation. © 2011 by Walter de Gruyter Berlin Boston 2011.

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