The University of Queensland, St Lucia, QLD, 4072
Ancestral reconstruction is a growing field of bioinformatics, involving the inference of an ancestral protein based on the available extant sequences of its descendants. This approach has been used previously to generate proteins significantly more thermostable than the extant forms, including cytochrome P450 enzymes belonging to the CYP3 and CYP2 families. Presented here is the use of a maximum likelihood, joint reconstruction approach to generate the ancestor of the CYP4ABXZ clade containing the CYP4A, CYP4B, CYP4X and CYP4Z subfamilies. These proteins consist primarily of fatty acid omega-hydroxylases with potential applications in the production of biopolymers, but currently lack the thermostability required of industrial biocatalysts. CYP4ABXZ was successfully synthesised and expressed in E. coli, along with corresponding extant forms (human CYP4A11 and rabbit CYP4B1), and was shown to demonstrate a significant increase of >25 °C in thermostability over the extant forms, as measured by 10T50 (the temperature at which half the protein remained intact after ten minutes). CYP4ABXZ will provide a robust scaffold for the future engineering of thermostable omega-hydroxylases for industrial applications.