[6] Yeast expression of animal and plant P450s in optimized redox environments

Yeast Saccharomyces cerevisiae offers a low-cost and efficient way to express heterologous P450s. The limiting amounts of endogenous P450-reductase (CPR) present in this organism and the limited sequence similarity of yeast redox enzymes (CPR and cytochrome b5) with human liver or plant equivalents are severe limitations when high specific activities of expressed P450s are required. The construction of artificial gene fusions encoding chimeric proteins composed of the heterologous P450 to express fused in frame to a yeast, rat, or human CPR. This approach improves the turnover numbers of several P450s, but does not permit to easily include a third component such as cyt. b5 or to modulate relative enzyme stoichiometries. Protein engineering required may have some unpredictable consequences on P450 functions, thus making this system questionable for toxicological predictions in human drug development. Alternatively, coexpression systems involving multiple plasmids or multiple expression cassettes on a single plasmid have been developed. These approaches suffer from genetic instabilities of strains when several or large plasmids are used. To avoid the limitations, multiple genomic modifications yeast featuring redox environment optimized for P450 functions was developed.