To better understand the switch in selectivity from the w-5 to w-2 or w-1 products associated with the Ala205Thr and Phe206Trp conserved motif, Ala205 and Phe206 in P450-TT were mutated to obtain the corresponding single and double mutants. The double mutant was more active than P450-TT (Supplementary Figure ), which may explain the prominence of these residues in other wild-type sequences. Although w-5 hydroxylation was still the most favoured product, an increase in the proportion of w-4 and w-3 hydroxylated alkyl chains was observed (Fig. ), suggesting that the increased polarity in the active site has an impact on regioselectivity. Overall, ASR has enabled the identification of active site residues influencing the differing C-H regioselectivity in a panel of ancestral and wild-type CYP116B biocatalysts without the need for a large-scale mutagenic screen. Predominantly, across all the domains, most of the non-conserved amino acids were located on the enzyme surface. The cartoon representation of P450-TT (PDB ID: 6laa) shows the locations of all the residues that are not conserved between one or more of the ancestors and P450-TT (grey cartoon). The conserved haem domain residues are in green, conserved reductase residues in pink, and conserved ferredoxin residues in cyan. However, when comparing the haem domain active sites the only differences are A205 and F206 (orange sticks) between P450-TT and the ancestral sequences. b. The multiple sequence alignment of the active site residues in the ancestral sequences compared to P450-TT, where the order of residues corresponds to the chronological order in the active site of P450-TT. Almost all the residues were conserved, apart from A205 and F206, which were only in P450-TT and its closest ancestors, N115 and N106. c. Phylogeny highlighting the locations of the A205 and F206 diversity in the wild-type sequences, where only 17 sequences were not the consensus TW residues in the active site. Also, the branch containing P450-TT was the main region of divergence. d. Regioselectivity observed after introducing the A205T and F206W mutations separately and together into P450-TT; the product distribution was altered while the preference for mid-chain hydroxylation was retained.