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 of individual Candida maltosa cytochrome P450 monooxygenases, the NADPH-cytochrome P450 reductase from this yeast species was co-expressed in Saccharomyces cerevisiae with each of the following cytochrome P450 forms; P450Cm1 (CYP52 A3), P450Cm2 (CYP52 A4), and P450AlK2A (CYP52 A5). For this purpose, 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 galactose 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 the reductase component was obtained resulting in P450/reductase molar ratios of about 1:3 in the microsomal fractions prepared from the co-expression strains. At the same time, the rates of cytochrome P450-dependent lauric acid hydroxylation increased more than 10-fold, showing a proper reconstitution of the C. maltosa monooxygenase systems in S. cerevisiae. Using intact cells, an efficient biotransformation of lauric acid to omega-hydroxylauric acid and dodecanedioic acid was found. S. cerevisiae cells coexpressing cytochrome P450 and NADPH-cytochrome P450 reductase were characterized by a marked proliferation of the endoplasmic reticulum. Immunoelectron microscopy revealed a colocalization of the monooxygenase components produced to these newly formed membrane structures.
Zimmer
T
T
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Kaminski
K
K
Scheller
U
U
Vogel
F
F
Schunck
W H
WH
D001483:Base Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D015870:Gene Expression; D005800:Genes, Fungal; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase