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doi:10.22028/D291-38154
Titel: | A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale |
VerfasserIn: | Schiffer, Lina Anderko, Simone Hobler, Anna Hannemann, Frank Kagawa, Norio Bernhardt, Rita |
Sprache: | Englisch |
Titel: | Microbial Cell Factories |
Bandnummer: | 14 |
Verlag/Plattform: | BMC |
Erscheinungsjahr: | 2015 |
Freie Schlagwörter: | Cortisol Human CYP11B1 Steroid biotransformation Whole-cell biocatalysis E. coli |
DDC-Sachgruppe: | 500 Naturwissenschaften |
Dokumenttyp: | Journalartikel / Zeitschriftenartikel |
Abstract: | 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. |
DOI der Erstveröffentlichung: | 10.1186/s12934-015-0209-5 |
URL der Erstveröffentlichung: | https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-015-0209-5 |
Link zu diesem Datensatz: | urn:nbn:de:bsz:291--ds-381547 hdl:20.500.11880/34456 http://dx.doi.org/10.22028/D291-38154 |
ISSN: | 1475-2859 |
Datum des Eintrags: | 23-Nov-2022 |
Fakultät: | NT - Naturwissenschaftlich- Technische Fakultät |
Fachrichtung: | NT - Systems Engineering |
Professur: | NT - Prof. Dr. Bruce Morgan |
Sammlung: | SciDok - Der Wissenschaftsserver der Universität des Saarlandes |
Dateien zu diesem Datensatz:
Datei | Beschreibung | Größe | Format | |
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s12934-015-0209-5.pdf | 950,59 kB | Adobe PDF | Öffnen/Anzeigen |
Diese Ressource wurde unter folgender Copyright-Bestimmung veröffentlicht: Lizenz von Creative Commons