Biotransformation of Steroids by a Recombinant Yeast Strain Expressing Bovine Cytochrome P-45017alpha

V. M. Shkumatov^1*, E. V. Usova¹, Y. S. Poljakov¹, N. S. Frolova¹, V. G. Radyuk¹, S. Mauersberger², A. A. Chernogolov^1,3, H. Honeck³, and W.-H. Schunck³

¹Research Institute for Physical Chemical Problems, Belarussian State University, ul. Leningradskaya 14, Minsk, 220050 Belarus; fax: (375-17) 209-5461; E-mail: biopharm@bsu.by

²Institute of Microbiology, Dresden University of Technology, Mommsenstr. 13, 01062 Dresden, Germany; fax: (49-351) 463-7715; E-mail: smau@rcs.urz.tu-dresden.de

³Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, D-13122 Berlin, Germany; fax: (49-30) 9406-3329; E-mail: schunck@mdc-berlin.de

^* To whom correspondence should be addressed.

Received January 23, 2001; Revision received May 4, 2001

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The cDNA encoding cytochrome P-45017alpha from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactose-induced yeast cells showed expression of about 240 nmol of P-45017alpha per liter of the culture. Binding of progesterone to the cytochrome P-45017alpha was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17alpha-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017alpha and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17alpha-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017alpha-dependent hydroxylation reaction. The reduction of the 20-keto group of 17alpha-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017alpha expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11beta- > 11alpha- > 19-hydroxyprogesterone. In conclusion, the present results show that the host-vector system used is suitable for high-level functional expression of P-45017alpha and further application of enzymatic properties of this protein to perform specific steroid biotransformations.

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KEY WORDS: cytochrome P-45017alpha, recombinant yeast S. cerevisiae, 17alpha-hydroxyprogesterone, 17alpha-hydroxy-20-dihydroprogesterone, 20-ketosteroid reductase

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