Thermostable fatty acid hydroxylases from ancestral reconstruction of cytochrome P450 family 4 enzymes

Biopolymers produced from plant sources offer sustainable alternatives to plastics derived from petrochemicals. Hydroxylated fatty acids (OHFAs) can be readily polymerized to form polyesters. The CYP4 family of cytochrome P450 monooxygenases are potential biocatalysts for fatty acid (FA) omega-hydroxylation, but existing CYP4 enzymes are limited by their inherently low thermostability. Here we resurrected ancestral FA hydroxylases belonging to the CYP4ABTXZ clade, that were significantly more thermostable than the corresponding extant forms with 10T50 values up to 68 degrees C. All ancestors were active towards C12-C20 FAs, but differed in chain length preference and hydroxylation regioselectivity. While extant CYP4A and CYP4B forms preferentially hydroxylate the terminal (omega) carbon, the ancestors showed less specificity for the omega-position for C12-C18 FAs overall. All ancestors were more active towards arachidonic acid (C20) than the extant forms tested, preferring hydroxylation at the omega-position. Reactions could be made more cost effective by using O2 surrogates, obviating the need for O2, NADPH or a co-expressed redox partner. These enzymes will serve as robust templates for the further engineering of stable FA hydroxlases with activity towards different FAs and alkyl chain positions to produce precursors for synthetic fibres with desired properties. Ancestral sequence reconstruction can be used to obtain highly thermostable catalysts of fatty acid hydroxylation.