Figure 4.

Influence of copy number and type of reductase on CYP activity. CYP2D6 (A) and CYP3A4 (B) activity of and CPR activities of Y. lipolytica single-copy and multicopy transformants. Strains coexpressed under pICL1-control the codon-optimized P450 2D6syn (A) or P450 3A4syn (B) in combination with the wild-type human or Y. lipolytica NADPH-P450 reductases (hCPRwt, YlCPR) from integrated single-copies or multiple vector copies as indicated, except the control strain YL23 on the left in A and B, containing only one endogenous chromosomal YlCPR copy, which is present as background in all strains. Cultivation at 28°C in YNBG on glucose, induction by 1% ethanol, microsomes isolation, whole-cell CYP activity and cytochrome P450 reductase (CPR) activity (determined as NADPH-cytochrome c reductase, NCR) measurements were performed as described in Materials and Methods. Filled bars, CPR activity; hatched bars, CYP activities; -, no genes; + genes in single copy; ++ genes in multicopy.