SYM-23-01

Looking to the past to evolve enzymes for the future: thermostable yet promiscuous cytochrome P450 enzymes for synthetic biology

Y Gumulya1, R Thomson1, J Baek1, KL Harris1, G Foley1, M Boden1 and EMJ Gillam1,2

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
  2. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

Cytochrome P450 enzymes are of great interest for synthetic biology and biotechnology, due to their ability to interact with, and introduce modifications into, a diverse variety of chemical substrates in a regio- and stereo-selective manner. P450s from the CYP1-3 families are of particular interest since they show broad but defined ranges of substrates, including many drugs and drug-like molecules, a characteristic that is due to conformational flexibility in the P450 fold. However for industrial applications, it is essential that P450s used as biocatalysts or biosensors are stable for longer times and at higher temperatures than those for which naturally occurring enzymes have evolved. Recent studies in our laboratory indicate that the CYP3 and other families of P450s can be engineered for a high degree of thermostability, with 60T50 values (i.e. the temperature at which 50% of the protein remains intact after an incubation of one hour) of ~66 °C, an increase of ~30 °C over the native forms. Surprisingly, this stabilization of the P450 fold failed to significantly affect the substrate range of the enzyme, suggesting that the flexibility of the P450 was not impaired by the changes required for thermostability. CYP3 expression yield and solvent tolerance were also improved, and translated to a significant improvement in product yield in incubations conducted at 50–60 °C. Limited site-saturation mutagenesis of the initial thermostable P450 revealed mutants with even higher stability and diverse catalytic profiles. These results challenge the expectation that thermostability reduces flexibility in P450s, and extend our understanding of the evolution of the P450 fold. Thermostable yet promiscuous P450s will provide versatile starting materials for the further engineering of novel "P450 biobricks" for biocatalysis and other synthetic biology applications.