IN-VIVO RECONSTITUTION OF HIGHLY-ACTIVE CANDIDA-MALTOSA CYTOCHROME-P450 MONOOXYGENASE SYSTEMS IN INDUCIBLE MEMBRANES OF SACCHAROMYCES-CEREVISIAE

To establish a system for functional characterization ofindividual Can dida maltosa cytochrome P450 monooxygenases, the NADPH-cytochrome P450 reductase from this yeast species was co-expressed in Saccharomyces c erevisiae with each of the following cytochrome P450 forms: P450Cm1 (C YP52 A3), P450Cm2 (CYP52 A4), and P450Alk2A (CYP52 A5). For this purpo se, a multicopy plasmid was constructed that contained two independent expression units controlled by the galactose-inducible GAL10 promoter . As shown by spectral and immunological methods, large amounts of the desired monooxygenase components could be simultaneously produced in the respective S. cerevisiae transformants. It was important, however, to adjust semi-anaerobic cultivation conditions during induction by g alactose to minimize a mutual impairment of cytochrome P450 and NADPH- cytochrome P450 reductase formation. Compared to the specific cellular content of the host-own enzyme, a 75- to 100-fold overproduction of t he reductase component was obtained resulting in P450/reductase molar ratios of about 1:3 in the microsomal fractions prepared from the co-e xpression strains. At the same time, the rates of cytochrome P450-depe ndent lauric acid hydroxylation increased more than 10-fold, showing a proper reconstitution of the C. maltosa monooxygenase systems in S. c erevisiae. Using intact cells, an efficient biotransformation of lauri c acid to omega-hydroxylauric acid and dodecanedioic acid was found. S . cerevisiae cells coexpressing cytochrome P450 and NADPH-cytochrome P 450 reductase were characterized by a marked proliferation of the endo plasmic reticulum. Immunoelectron microscopy revealed a colocalization of the monooxygenase components produced to these newly formed membra ne structures.