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Microb Cell Fact. 2015 Feb 25;14:25. doi: 10.1186/s12934-015-0209-5.

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

Author information

1
Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. lina.schiffer@uni-saarland.de.
2
Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. s.anderko@mx.uni-saarland.de.
3
Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. anna.hobler@gmx.de.
4
Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. f.hannemann@mx.uni-saarland.de.
5
Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. norio.kagawa@med.nagoya-u.ac.jp.
6
Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. ritabern@mx.uni-saarland.de.

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.

PMID:
25880059
PMCID:
PMC4347555
DOI:
10.1186/s12934-015-0209-5
[Indexed for MEDLINE]
Free PMC Article

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