A Reconstructed Common Ancestor of the Fatty Acid Photo‐decarboxylase Clade Shows Photo‐decarboxylation Activity and Increased Thermostability

Light‐dependent enzymes are a rare type of biocatalyst with high potential for research and biotechnology. A recently discovered fatty acid photo‐decarboxylase from Chlorella variabilis NC64A (CvFAP) converts fatty acids to the corresponding hydrocarbons only when irradiated with blue light (400 to 520 nm). To expand the available catalytic diversity for fatty acid decarboxylation, we reconstructed possible ancestral decarboxylases from a set of 12 extant sequences that were classified under the fatty acid decarboxylases clade within the glucose‐methanol choline (GMC) oxidoreductase family. One of the resurrected enzymes (ANC1) showed activity in the decarboxylation of fatty acids, showing that the clade indeed contains several photo‐decarboxylases. ANC1 has a 15 °C higher melting temperature (T (m)) than the extant CvFAP. Its production yielded 12‐fold more protein than this wild type decarboxylase, which offers practical advantages for the biochemical investigation of this photoenzyme. Homology modelling revealed amino acid substitutions to more hydrophilic residues at the surface and shorter flexible loops compared to the wild type. Using ancestral sequence reconstruction, we have expanded the existing pool of confirmed fatty acid photo‐decarboxylases, providing access to a more robust catalyst for further development via directed evolution.