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Background  Synthetic glucocorticoids like methylprednisolone (medrol) are of high pharmaceutical interest and represent powerful drugs due to their anti-inflammatory and immunosuppressive effects. Since the chemical hydroxylation of carbon atom 21, a crucial step in the synthesis of the medrol precursor premedrol, exhibits a low overall yield because of a poor stereo- and regioselectivity, there is high interest in a more sustainable and efficient biocatalytic process. One promising candidate is the mammalian cytochrome P450 CYP21A2 which is involved in steroid hormone biosynthesis and performs a selective oxyfunctionalization of C21 to provide the precursors of aldosterone, the main mineralocorticoid, and cortisol, the most important glucocorticoid. In this work, we demonstrate the high potential of CYP21A2 for a biotechnological production of premedrol, an important precursor of medrol.    Results  We successfully developed a CYP21A2-based whole-cell system in  Escherichia coli  by coexpressing the cDNAs of bovine  CYP21A2  and its redox partner, the NADPH-dependent cytochrome P450 reductase ( CPR ), via a bicistronic vector. The synthetic substrate medrane was selectively 21-hydroxylated to premedrol with a max. yield of 90 mg L −1  d −1 . To further improve the biocatalytic activity of the system by a more effective electron supply, we exchanged the CPR with constructs containing five alternative redox systems. A comparison of the constructs revealed that the redox system with the highest endpoint yield converted 70 % of the substrate within the first 2 h showing a doubled initial reaction rate compared with the other constructs. Using the best system we could increase the overall yield of premedrol to a maximum of 320 mg L −1  d −1  in shaking flasks. Optimization of the biotransformation in a bioreactor could further improve the premedrol gain to a maximum of 0.65 g L −1  d −1 .    Conclusions  We successfully established a CYP21-based whole-cell system for the biotechnological production of premedrol, a pharmaceutically relevant glucocorticoid, in  E. coli  and could improve the system by optimizing the redox system concerning reaction velocity and endpoint yield. This is the first step for a sustainable replacement of a complicated chemical low-yield hydroxylation by a biocatalytic cytochrome P450-based whole-cell system.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-015-0333-2) contains supplementary material, which is available to authorized users.

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