Oxygenation cascade analysis in conversion of n-octane catalyzed by cytochrome P450 CYP102A3 mutants at the P331 site.

To improve the hydroxylation efficiency of cytochrome P450 CYP102A3 to substrate n-octane, a previously reported triple-mutant F88V/S188Q/A330V with altered regioselectivity was selected and site-saturation mutagenesis was performed at its P331 site, which is adjacent to one of the active sites A330. Using whole-cell biotransformations to analyze the created mutants, four better mutants P331A, P331T, P331S, and P331V were achieved and exhibited significantly improved conversion rates toward n-octane, which are 33%, 33%, 22% and 28%, respectively, whereas the activity of P331R was greatly reduced and P331K gave almost zero conversion of n-octane. Besides the main product octanols, different octanones and 1,7-octanediol were also detected for some of the mutants. The above results demonstrated that the P331 site of CYP102A3 also plays an important role in the n-octane oxidation and CYP102A3 is a functionally flexible biocatalyst that can be optimized for a variety of industrial applications.