Using ASR to identify important mutational paths that lead to changes in enzymatic activity can provide insight into the mechanisms that control substrate specificity and activity , as demonstrated in the recent work on stereoselective C-H activation by flavin-dependent monooxygenases . We were interested in how the regioselectivity towards fatty acid hydroxylation arose within the ancestral CYP116B family, especially in the pathway toward the unique mid-chain selectivity of P450-TT. In particular, we wondered whether some of these ancestors would allow us to access new regioselectivities, such as w-2, which is not commonly observed in other biocatalysts.