Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

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 h...

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Published in:Chembiochem : a European journal of chemical biology 2010-03, Vol.11 (5), p.713-721
Main Authors: Zehentgruber, Daniela, Hannemann, Frank, Bleif, Sabrina, Bernhardt, Rita, Lütz, Stephan
Format: Article
Language:English
Subjects:
Adrenodoxin - genetics
Adrenodoxin - metabolism
Alcohol Dehydrogenase - metabolism
Animals
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
biocatalysis
Cattle
CYP106A2
Cytochrome P-450 Enzyme System - chemistry
Cytochrome P-450 Enzyme System - metabolism
cytochrome P450 monooxygenase
cytochromes
Ferredoxin-NADP Reductase - genetics
Ferredoxin-NADP Reductase - metabolism
Hydroxylation
Oxidation-Reduction
Progesterone - biosynthesis
Solubility
Stereoisomerism
steroid hydroxylation
Testosterone - biosynthesis
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Summary: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 15β-OH-progesterone and 15β-OH-testosterone and also the reuse of the biocatalyst.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.200900706