A CYP21A2 based whole-cell system inEscherichia coli for the biotechnologicalproduction of premedrol

Microbial Cell Factories

Research

Simone Brixius-Anderko¹  Lina Schiffer¹  Frank Hannemann¹  Rita Bernhardt¹  Bernd Janocha² 

[1] Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany;Sanofi-Aventis Deutschland GmbH, C&BD Frankfurt Biotechnology, 65926, Frankfurt-Höchst, Germany;

关键词: Methylprednisolone;    Medrane;    CYP21A2;    Cytochrome P450;    Whole-cell biocatalysis;    E. coli;    Etp1;    Arh1;    CPR;    Adx;    Steroid;   

DOI  :  10.1186/s12934-015-0333-2

 received in 2015-07-14, accepted in 2015-08-31,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundSynthetic glucocorticoids like methylprednisolone (medrol) are ofhigh pharmaceutical interest and represent powerful drugs due to theiranti-inflammatory and immunosuppressive effects. Since the chemicalhydroxylation of carbon atom 21, a crucial step in the synthesis of the medrolprecursor premedrol, exhibits a low overall yield because of a poor stereo- andregioselectivity, there is high interest in a more sustainable and efficientbiocatalytic process. One promising candidate is the mammalian cytochrome P450CYP21A2 which is involved in steroid hormone biosynthesis and performs aselective oxyfunctionalization of C21 to provide the precursors of aldosterone,the main mineralocorticoid, and cortisol, the most important glucocorticoid. Inthis work, we demonstrate the high potential of CYP21A2 for a biotechnologicalproduction of premedrol, an important precursor of medrol.ResultsWe successfully developed a CYP21A2-based whole-cell system inEscherichia coli by coexpressing thecDNAs of bovine CYP21A2 and its redoxpartner, the NADPH-dependent cytochrome P450 reductase (CPR), via a bicistronic vector. The synthetic substrate medranewas selectively 21-hydroxylated to premedrol with a max. yield of90 mg L−1 d−1. Tofurther improve the biocatalytic activity of the system by a more effectiveelectron supply, we exchanged the CPR with constructs containing fivealternative redox systems. A comparison of the constructs revealed that theredox system with the highest endpoint yield converted 70 % of the substratewithin the first 2 h showing a doubled initial reaction rate compared with theother constructs. Using the best system we could increase the overall yield ofpremedrol to a maximum of320 mg L−1 d−1 inshaking flasks. Optimization of the biotransformation in a bioreactor couldfurther improve the premedrol gain to a maximum of0.65 g L−1 d−1.ConclusionsWe successfully established a CYP21-based whole-cell system for thebiotechnological production of premedrol, a pharmaceutically relevantglucocorticoid, in E. coli and could improvethe system by optimizing the redox system concerning reaction velocity andendpoint yield. This is the first step for a sustainable replacement of acomplicated chemical low-yield hydroxylation by a biocatalytic cytochromeP450-based whole-cell system.

【 授权许可】

CC BY   
© Brixius-Anderko et al. 2015