Genetically
engineered yeast cells and their applications.
Pompon D, Perret A, Bellamine A, Laine R, Gautier JC, Urban P.
Centre de Genetique Moleculaire du CNRS, UPR 2420, Gifu-sur-Yvette, France.
The first generation of yeast expression systems relies on inducible expression cassettes
borne by multicopy plasmids for production of unmodified human P450s and on the endogenous
NADPH-P450 reductase to support activities. A second generation of engineered yeast
involved targeted genomic modifications allowing overexpression of the yeast reductase and
coexpression of human cytochrome b5 and of a phase II enzyme such as epoxide hydrolase.
These features allow improved P450 turnover numbers and simulation of some phase I-phase
II couplings. In the third generation, the human reductase was substituted for the yeast
reductase by genome engineering. Simultaneously, induction procedures were optimized to
reach high P450 specific contents. Dramatic improvements (1000-fold) of yeast-expressed
P450 activities have thus been obtained. To get more insight into complex metabolic
events, such as that of a typical pollutant: benzo[a]pyrene, an approach was designed
which involves a complementary use of yeast expression and computer simulations.
