We recently identified CYP116B46 (P450-TT) from a panel of self-sufficient thermostable wild-type, extant CYP116B enzymes as being capable of selective mid-chain hydroxylation of decanoic acid to (S)-5-hydroxydecanoic acid . While related enzymes from the same family (P450-AT, -TB, -AX, and -JT, Fig. ) were either not regioselective or favoured ω-1 hydroxylation . To further understand what made P450-TT selective for mid-chain hydroxylation, and to develop other P450s with similar capacities for midchain hydroxylation, we performed ancestral sequence reconstruction (ASR) on the CYP116B family.