The distinctive pathway to the unique mid-chain selectivity of wild-type, P450-TT, highlights the utility of ASR to provide insight into the determinants of fatty acid hydroxylation regioselectivity and other properties. The shifts in regioselectivity seen here appear to occur steadily along specific evolutionary lineages, with the most dramatic being from the poor overall conversion and subterminal hydroxylation in N104 to the higher conversion and mid-chain w-5 selectivity of N106, ancestors that are separated by a single node (N105) in the tree (which contains the AF motif at residues 205-206). Inspection of the sequence differences in this lineage compared to the rest of the ancestors may reveal sequence elements related to the enhanced overall turnover, thermostability or expression yield, not only regioselectivity, an investigation that could also be accelerated by a directed evolution approach .