Humane steroidogene Cytochrome P450 : Biotransformation von Arzneistoffen und biotechnologische Anwendung

In humans, monooxygenases from the cytochrome P450 superfamily contribute to the biosynthesis of steroid hormones by catalyzing the side-chain cleavage of cholesterol and subsequent regio- and stereoselective hydroxylations, which determine the bioactivity of the hormones. In this work, the additional contribution of these steroidogenic cytochromes P450s to the metabolism of xenobiotics was established and their employment as highly selective and efficient biocatalysts in recombinant bacteria for the production of commercially relevant glucocorticoids, which are important anti-inflammatory andimmunosuppressive drugs, was realized. The capability of CYP11A1, CYP11B1 and CYP11B2 to metabolize synthetic anabolic steroids, mineralocorticoid receptor antagonists and synthetic gestangens was demonstrated. Products were identified and analyzed for an alteration of their pharmacodynamic properties. For the biotechnological application of CYP11B1 in the production of cortisol from 11-deoxycortisol, a whole-cell biocatalyst was developed by reconstituting the functional CYP11B1 system, including its redox partners adrenodoxin reductase and adrenodoxin, in Escherichia coli. It was optimized by molecular evolution of CYP11B1 and by increasing the synthesis of the redox partner adrenodoxin. Additionally, the author contributed to the establishment of an Escherichia coli-based system for the application of CYP21A2 in the production of the synthetic glucocorticoid premedrol.Beim Menschen tragen Monooxygenasen der Cytochrom P450 Superfamilie zur Steroidhormonbiosynthese bei, indem sie die Seitenkettenspaltung von Cholesterin sowie sich anschließende regio- und stereoselektive Hydroxylierungen katalysieren, die die Bioaktivität der Hormone bestimmen. In dieser Arbeit wurde die zusätzliche Beteiligung dieser Steroidhormon synthetisierenden Cytochrome P450 am Metabolismus von Xenobiotika festgestellt und ihre Anwendung als hoch selektive und effiziente Biokatalysatoren in rekombinanten Bakterien für die Produktion von kommerziell relevanten Glucocorticoiden, die wichtige anti-inflammatorische und immunsuppressive Medikamente darstellen, realisiert. Das Potential von CYP11A1, CYP11B1 und CYP11B2, synthetische anabole Steroide, Mineralocorticoidrezeptor Antagonisten und Gestagene zu metabolisieren, wurde aufgezeigt. Produkte wurden identifiziert und auf Veränderungen ihrer pharmakodynamischen Eigenschaften hin untersucht. Für die biotechnologische Anwendung von CYP11B1 bei der Produktion von Cortisol aus 11-Desoxycortisol wurde ein Ganzzellbiokatalysator in Escherichia coli durch die Widerherstellung des funktionellen CYP11B1 Systems mit seinen Redoxpartnern, Adrenodoxin Reduktase und Adrenodoxin, entwickelt. Das System wurde durch molekulare Evolution von CYP11B1 und eine Erhöhung der Synthese des Redoxpartners Adrenodoxin optimiert. Zusätzlich wirkte die Autorin an der Etablierung eines Escherichia coli-basierten Systems für die Anwendung von CYP21A2 für die Produktion des synthetischen Glucocorticoids Premedrol mit. system for the application of CYP21A2 in the production of the synthetic glucocorticoid premedrol

5α-reduction of epitestosterone is catalysed by human SRD5A1 and SRD5A2 and increases androgen receptor transactivation

Epitestosterone is a stereoisomer of the active androgen testosterone and its circulating concentrations are similar to those of testosterone in women and children. However, its biological function and pathways of metabolism remain unknown. The structural similarity to testosterone suggests a potential function in the modulation of androgen receptor signalling. It is well established that the conversion of testosterone to 5α-dihydrotestosterone enhances local androgen receptor signalling. In this study, we show that epitestosterone is metabolized to 5α-dihydroepitestosterone by both human steroid 5α-reductase isoforms, SRD5A1 and SRD5A2. Using two different variations of a reporter assay for transactivation of the human androgen receptor, we show that epitestosterone is a partial AR agonist and that the 5α-reduction of epitestosterone increases its androgenic activity. In line with this, we show that 5α-reduction of epitestosterone reduces its ability to antagonize 5α-dihydrotestosterone-induced androgen receptor transactivation. In conclusion, we provide evidence that steroid 5α-reductases regulate the modulatory effect of epitestosterone on androgen receptor signalling.</p

A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol

Additional file 4: Fig. S4. In vitro conversion of medrane with the redox systems AdR/Adx/CYP21A2 or arh1/Adx/CYP21A2 with either NADH or NADPH. 400 ÎźM Medrane was converted in a reconstituted in vitro assay with Adx based redox systems containing AdR or arh1 as reductase. To each system either NADH or NADPH was added to verify the ability of arh1 to receive electrons from NADH. AdR served as a negative control. All values represent the mean of triplicates with the respective standard deviation

A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale

Background: Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). Results: We established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L−1 by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library. Conclusions: Under optimized conditions a maximum productivity of 0.84 g cortisol∗L−1∗d−1 was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry

A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale

Background: Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). Results: We established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L−1 by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library. Conclusions: Under optimized conditions a maximum productivity of 0.84 g cortisol∗L−1∗d−1 was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry

Human steroidogenic cytochromes P450 : biotransformation of drugs and biotechnological application

In humans, monooxygenases from the cytochrome P450 superfamily contribute to the biosynthesis of steroid hormones by catalyzing the side-chain cleavage of cholesterol and subsequent regio- and stereoselective hydroxylations, which determine the bioactivity of the hormones. In this work, the additional contribution of these steroidogenic cytochromes P450s to the metabolism of xenobiotics was established and their employment as highly selective and efficient biocatalysts in recombinant bacteria for the production of commercially relevant glucocorticoids, which are important anti-inflammatory andimmunosuppressive drugs, was realized. The capability of CYP11A1, CYP11B1 and CYP11B2 to metabolize synthetic anabolic steroids, mineralocorticoid receptor antagonists and synthetic gestangens was demonstrated. Products were identified and analyzed for an alteration of their pharmacodynamic properties. For the biotechnological application of CYP11B1 in the production of cortisol from 11-deoxycortisol, a whole-cell biocatalyst was developed by reconstituting the functional CYP11B1 system, including its redox partners adrenodoxin reductase and adrenodoxin, in Escherichia coli. It was optimized by molecular evolution of CYP11B1 and by increasing the synthesis of the redox partner adrenodoxin. Additionally, the author contributed to the establishment of an Escherichia coli-based system for the application of CYP21A2 in the production of the synthetic glucocorticoid premedrol.Beim Menschen tragen Monooxygenasen der Cytochrom P450 Superfamilie zur Steroidhormonbiosynthese bei, indem sie die Seitenkettenspaltung von Cholesterin sowie sich anschließende regio- und stereoselektive Hydroxylierungen katalysieren, die die Bioaktivität der Hormone bestimmen. In dieser Arbeit wurde die zusätzliche Beteiligung dieser Steroidhormon synthetisierenden Cytochrome P450 am Metabolismus von Xenobiotika festgestellt und ihre Anwendung als hoch selektive und effiziente Biokatalysatoren in rekombinanten Bakterien für die Produktion von kommerziell relevanten Glucocorticoiden, die wichtige anti-inflammatorische und immunsuppressive Medikamente darstellen, realisiert. Das Potential von CYP11A1, CYP11B1 und CYP11B2, synthetische anabole Steroide, Mineralocorticoidrezeptor Antagonisten und Gestagene zu metabolisieren, wurde aufgezeigt. Produkte wurden identifiziert und auf Veränderungen ihrer pharmakodynamischen Eigenschaften hin untersucht. Für die biotechnologische Anwendung von CYP11B1 bei der Produktion von Cortisol aus 11-Desoxycortisol wurde ein Ganzzellbiokatalysator in Escherichia coli durch die Widerherstellung des funktionellen CYP11B1 Systems mit seinen Redoxpartnern, Adrenodoxin Reduktase und Adrenodoxin, entwickelt. Das System wurde durch molekulare Evolution von CYP11B1 und eine Erhöhung der Synthese des Redoxpartners Adrenodoxin optimiert. Zusätzlich wirkte die Autorin an der Etablierung eines Escherichia coli-basierten Systems für die Anwendung von CYP21A2 für die Produktion des synthetischen Glucocorticoids Premedrol mit. system for the application of CYP21A2 in the production of the synthetic glucocorticoid premedrol