The ancestors were all catalytically active, i.e., maintained electron transfer across the inferred domains. In multi-domain enzymes such as the self-sufficient CYP116B forms, loop and linker variation across domains, such as seen here among in the ancestors (Supplementary Fig. ), can influence the dynamics and interactions between the domains. In particular, efficient electron transfer across the domains to the haem domain is vital for hydroxylation activity. The ability of the ancestors to hydroxylate fatty acids was analysed using purified enzyme (10 and 50 μM; Supplementary Figures ), with decanoic acid (0.5 mM) as the substrate. The biotransformations utilised glucose dehydrogenase for concomitant regeneration of NADPH, the preferred cofactor of the reductase domain of P450-TT . At the lower enzyme concentration (10 μM), N70 displayed 69% conversion of decanoic acid, whereas the conversion with the other ancestral enzymes was low or negligible (0-37%, Supplementary Table ). Therefore, the ancestral regioselectivity fingerprints (Fig. ) were examined using a five-fold higher enzyme loading. Under these conditions, all self-sufficient ancestors were catalytically active (Supplementary Tables and), implying that electron transfer was maintained in all the ancestors, and attesting to the ability of ASR to facilitate the engineering of active electron transport chains. Interestingly, the highest turnover among ancestral forms at both P450 loadings was achieved with N70, N106, and N115, forms which also showed the best expression. ) was determined from the residual P450 peak after incubating the enzyme at 25-80 °C for 15 min. A full sigmoidal denaturation curve was not obtained for 6 of the enzymes at 60 min, so the 60 T50 values are not shown (see Table ); however, 15 T50 correlated with 60 T50 where both values could be determined (R 2 = 0.874). P450-TT and -AX were the most stable extant forms whereas N3 was the most thermostable ancestral enzyme. The regioselectivity of the ancestral and previously characterised extant P450s are shown in (c). The regioselectivity product distribution of the ancestral enzymes (50 μM) towards the hydroxylation of decanoic acid (0.5 mM) was investigated in biotransformations carried out with a glucose dehydrogenase recycling system, in 0.1 M potassium phosphate buffer, pH 8 and at 30 °C, with agitation at 200 rpm for 20 h. The ancestral enzymes in the pathway to P450-TT showed a change from the mid-chain selectivity in P450-TT towards sub-terminal selectivity in the older ancestors. In addition, there was a change in the ancestral nodes from ω-1 hydroxylation in the majority of the ancestral enzymes to ω-2 hydroxylation in N0 and N374. Data for the extant forms are from Manning et al. . Evolutionary shift in regioselectivity. It was previously shown that P450-TT was highly selective for mid-chain hydroxylation of decanoic acid . Across the ancestral panel, hydroxylation of decanoic acid at each position between w-5 and w-1 was detected; yet there was no observation of any mid-chain w-6 hydroxylation or hydroxylation closer to the carboxylate. In N0 and N374, there was an unexpected shift in selectivity to favour the w-2 position over the w-1 position. This novel sub-terminal preference for the w-2 position was not seen in any of the wild-type product distributions. By contrast, the other deep ancestors, N1, N104, N3 and N23, showed a preference for w-1 hydroxylation (Fig. ). The deeper ancestral enzymes tended to be more regioselective than the more recent ancestors or the extant enzymes that were examined, with the exception of P450-TT and -TB. Mid-chain hydroxylation arose independently in two different lineages (N70 and N106), from ancestors that were predominantly w-1 hydroxylases (N1, N3 and N104). In the N70 lineage, a non-selective ancestor (N70: 38% mid-chain and 32% sub-terminal), gave rise to two non-selective wild-type enzymes P450-JT and -AX which displayed slight preferences for the w-5 (51%) and w-1 (48%) positions, respectively. In contrast, the P450-TT lineage developed a preference for mid-chain hydroxylation much earlier, in the N106 ancestor, and resulted in a switch to exclusively mid-chain hydroxylation in P450-TT. Indeed, the major product was hydroxylated at the w-5 position, with 99% conversion, for both N115 and N106, although these ancestors also showed a greater tendency towards hydroxylation at the w-3 and w-4 positions, compared to P450-TT. Therefore, this lineage shows a prominent shift in the regioselectivity, compared with the CYP116B bacterial ancestors and suggests an evolutionary pathway towards the unusual regioselectivity of P450-TT. These results highlight the different ways in which selectivity might have evolved and provide biocatalysts for hydroxylation at alternative C-H positions in fatty acids.