title: A CYP21A2 based whole-cell system in Escherichia coli for the biotechnological production of premedrol.
abstract: 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.
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. s.anderko@mx.uni-saarland.de.
authors.forename: Simone
authors.initials: S
authors.lastname: Brixius-Anderko
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. lina.schiffer@uni-saarland.de.
authors.forename: Lina
authors.initials: L
authors.lastname: Schiffer
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. f.hannemann@mx.uni-saarland.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.affiliation: Sanofi-Aventis Deutschland GmbH, C&BD Frankfurt Biotechnology, 65926, Frankfurt-Höchst, Germany. Bernd.Janocha@sanofi.com.
authors.forename: Bernd
authors.initials: B
authors.lastname: Janocha
authors.affiliation: Department of Biochemistry, Saarland University, 66123, Saarbrücken, Germany. ritabern@mx.uni-saarland.de.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000818:Animals; D055162:Biocatalysis; D019149:Bioreactors; D001711:Biotransformation; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D060847:Metabolic Engineering; D008775:Methylprednisolone / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D013255:Steroid 21-Hydroxylase / Q000235:genetics* / Q000378:metabolism
chemical_list: D013255:Steroid 21-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D008775:Methylprednisolone