Inference of the phylogenetic and ancestral intermediates. The CYP116B family is highly diverse and CYP116B enzymes vary substantially in their thermostability, fatty acid hydroxylation regioselectivity , and sequence identity (Supplementary Tables and). To generate an enzyme panel with diversified catalytic range, CYP116B sequences were collected by database searches and curated. We obtained 467 CYP116B sequences that included all three domains (P450/haem, iron-sulfur, and reductase). The sequences were mostly derived from two main phyla, Actinobacteria and Proteobacteria (Supplementary Fig. ) and covered twenty different bacterial orders, encompassing sequences that were >45% identical to P450-TT. In contrast to the previously characterised thermophilic panel of extant CYP116B sequences, most of the sequences were isolated from mesophiles (Supplementary Fig. ) , although specific lineages appeared to be thermophilic, such as P450-TT and its closest homologues from Caldimonas taiwanensis and Schlegelella thermodepolymerans, which have been isolated from hot springs and activated sludge, respectively . To explore the sampled sequence space, we inferred the ancestors from each major phylogenetic group (N374, N1, N213, N104, N118, N23, N70, and N3), as well as specific ancestors of the P450-TT lineage (N106 and N115) and the node (N0) connecting the major clade (Proteobacteria) with the outgroup (Actinobacteria). A total of eleven full-length multi-domain CYP116B ancestors (Fig. , the highlighted nodes within the tree) were inferred using the inference program Graphical Representation of Ancestral Sequence Predictions (GRASP), via a joint reconstruction maximum likelihood approach . These self-sufficient ancestral enzymes were selected to explore the thermostability and fatty acid hydroxylation regioselectivity trends in the P450-TT lineage and to investigate nodes on divergent CYP116B branches corresponding to mesophilic wild-type sequences. All ancestors were expressed as holoenzymes with characteristic P450 spectra, with two forms reaching micromolar concentrations in culture (Supplementary Table ). N106 and N115 showed higher expression at 25 °C than all other CYP116B enzymes including their direct descendant, P450-TT, which was the most highly expressed extant form.