Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C--H bonds mainly in a stereo- and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3-keto-4-ene steroids mainly at position 15. We expressed this enzyme together with the electron-transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme-coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450-catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2-catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18-fold to yield an absolute productivity up to 5.5 g L(-1) d(-1). Product extraction with absorber resins allowed the recovery of quantitative amounts of 15beta-OH-progesterone and 15beta-OH-testosterone and also the reuse of the biocatalyst.

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