ASR was employed as a tool to explore the sequence space of the CYP116B family, which enabled us to analyse a comparatively small number of potential biocatalysts with a greater chance of successfully identifying catalytically active enzymes with the desired properties of thermostability, self-sufficiency, and selective fatty-acid hydroxylation. ASR allows the generation of new proteins based upon a sequence alignment and inferred evolutionary tree that link a set of extant sequences; the most probable common ancestor and other evolutionary intermediates are inferred from the initial input . The method has previously been used to identify and engineer enzymes with improved properties: greater thermal stability, enhanced expression, broader pH, and solvent tolerances . This technique can identify novel sequences leading to functional diversity that otherwise may not have been investigated via conventional enzyme engineering and, unlike mutagenesis, most ancestors are functional . ASR has also been used to improve the thermostability and solvent tolerance of animal P450s , which can improve the heterologous expression and durability of biocatalyts ; typically, inferred ancestors have been found to be more thermostable than their extant counterparts. However, the evolution of thermostable enzymes within a largely mesophilic family had not been previously explored. As the extant CYP116B family has both mesophilic and thermophilic branches, and several previously characterised CYP116B sequences (e.g., P450-TT) originate from thermophilic (rather than mesophilic) bacteria we were interested in what evolutionary trends would emerge in the ASR of the CYP116B enzymes. ) positions, and their properties. The previously identified self-sufficient extant CYP116B enzymes displayed activity towards fatty acids with a range of midchain and sub-terminal regioselectivities . P450-TT uniquely favoured mid-chain hydroxylation of decanoic acid. Therefore, the CYP116B family was further explored through ancestral sequence reconstruction, with particular focus on the lineage leading to P450-TT. Extant sequences with a sequence similarity >45% were gathered to generate a phylogenetic tree for the CYP116B family using IQ-TREE 2 (v2.1.2). Ancestral sequences from within the tree were then resurrected to create a panel of ancestral self-sufficient biocatalysts, where the nodes shown depict a simplified representation of the differing regioselective of decanoic acid hydroxylation from mid-chain (w-5; orange) to different sub-terminal positions (w-1, dark blue; w-2, cyan). A shift in regioselectivity was identified in the oldest ancestor (w-2), differing from both the mid-chain selectivity of P450-TT, and the predominant sub-terminal w-1 regioselectivity observed for other members of the CYP116B family. This highlights the usefulness of ancestral sequence reconstruction to enable the exploration of the sequence space within the CYP116B family and identify different clades with differing regioselectivity.