Bitte benutzen Sie diese Referenz, um auf diese Ressource zu verweisen: 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 GrößeFormat 
s12934-015-0209-5.pdf950,59 kBAdobe PDFÖffnen/Anzeigen


Diese Ressource wurde unter folgender Copyright-Bestimmung veröffentlicht: Lizenz von Creative Commons Creative Commons