Directed OmniChange Evolution Converts P450 BM3 into an Alkyltrimethylammonium Hydroxylase

Cetyl-trimethylammonium bromide (CTAB) is a widely used cationic surfactant that is biodegradable in nature. CTAB biodegradation requires hydroxylation in the first step, which is rate-limiting and crucial for solubility in water. In this study, the OmniChange multi-site mutagenesis method was applied to reengineer the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four previously reported positions (R47, Y51, F87, and L188). 1740 clones from the P450 BM3 OmniChange library were screened with the NADPH depletion assay. A total of 696 clones were rescreened with the NADPH depletion and an Ampliflu™ Red/ horseradish peroxidase based H₂ O₂ detection assay. Several improved P450 BM3 variants were identified and finally four were kinetically characterized with respect to CTAB hydroxylation, based on both performance and coupling efficiency. Based on NADPH consumption, the P450 BM3 variant P3A8 (R47E/Y51M/F87V/L188E) displayed an initial activity (64.9±4.8 s^-1 , 13.5-fold increased activity compared with wild-type P450 BM3), which nearly matches the specific activity for its natural fatty acid substrate (palmitic acid (32-122 s^-1 )). Variant P3A8 showed high coupling efficiency (92.5 %), whereas wild-type P450 BM3 displayed a low coupling efficiency (0.5 %). HPLC-MS/MS detection confirmed that P3A8 and P2E7 (R47D/Y51L/F87V/L188A) form 13 and 35 times more 2-hydroxylated CTAB than P450 BM3. In addition, di-hydroxylated CTAB products were detected for all four investigated P450 BM3 variants (up to a yield of 77 %; P3A8). Di-hydroxylated quaternary amines are highly interesting bolaform surfactants with a high hydrophilicity (surface contact angle: θ=16.7°).