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In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo\u2010 and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant <jats:italic>E. coli<\/jats:italic> system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone\u2010precursor premedrol by optimizing the CYP21A2\u2010based whole\u2010cell system on a laboratory scale. We successfully improved the whole\u2010cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N\u2010terminally truncated version of the bovine NADPH\u2010dependent cytochrome P450 reductase (bCPR<jats:sub>\u221227<\/jats:sub>) and coexpression of microsomal cytochrome <jats:italic>b<\/jats:italic><jats:sub>5<\/jats:sub>; (b)\u00a0enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c)\u00a0circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed\u2010batch protocol.\u00a0By overcoming the presented limitations in whole\u2010cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691\u2009mg\u00b7L<jats:sup>\u22121<\/jats:sup>\u00b7d<jats:sup>\u22121<\/jats:sup> premedrol.<\/jats:p>","DOI":"10.1002\/bit.27246","type":"journal-article","created":{"date-parts":[[2019,12,9]],"date-time":"2019-12-09T00:52:25Z","timestamp":1575852745000},"page":"901-911","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Identification and circumvention of bottlenecks in CYP21A2\u2010mediated premedrol production using recombinant <i>Escherichia coli<\/i>"],"prefix":"10.1002","volume":"117","author":[{"given":"Lisa","family":"K\u00f6nig","sequence":"first","affiliation":[{"name":"Department of Biochemistry Saarland University  Saarbr\u00fccken Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3036-9285","authenticated-orcid":false,"given":"Simone","family":"Brixius\u2010Anderko","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Saarland University  Saarbr\u00fccken 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