title: Towards preparative scale steroid hydroxylation with cytochrome P450 monooxygenase CYP106A2.
abstract: 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.
authors.affiliation: Institute of Biotechnology 2, Forschungszentrum Jülich GmbH, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Zehentgruber
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.forename: Sabrina
authors.initials: S
authors.lastname: Bleif
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
authors.forename: Stephan
authors.initials: S
authors.lastname: Lütz
mesh_terms: D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism; D006900:Hydroxylation; D010084:Oxidation-Reduction; D011374:Progesterone / Q000096:biosynthesis*; D012995:Solubility; D013237:Stereoisomerism; D013739:Testosterone / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D000325:Adrenodoxin; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D000426:Alcohol Dehydrogenase; C517094:15beta-hydroxylase CYP106A2, Bacillus megaterium; D005287:Ferredoxin-NADP Reductase