title: Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes.
abstract: BACKGROUND
  Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase.
  
  
  RESULTS
  For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains.
  
  
  CONCLUSIONS
  Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, ACIB GmbH, Petersgasse 14, Graz, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Braun
authors.forename: Martina
authors.initials: M
authors.lastname: Geier
authors.forename: Bruno
authors.initials: B
authors.lastname: Bühler
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.forename: Stephan
authors.initials: S
authors.lastname: Mauersberger
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
mesh_terms: D018533:Biomass; D001711:Biotransformation; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009829:Oleic Acids / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013256:Steroids / Q000378:metabolism*; D025062:Yarrowia / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D009829:Oleic Acids; D011994:Recombinant Proteins; D013256:Steroids; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D009251:NADPH-Ferrihemoprotein Reductase; C033180:ethyl oleate