ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.
Keywords: Cytochrome CYP153A; Electron transfer; Iterative saturation mutagenesis; Oleic acid; Site-directed saturation mutagenesis.