Looking back to the future of biocatalysis: exploring the natural and artificial evolution of cytochrome P450 enzymes to explain protein evolution and develop novel bio-bricks for synthetic biology

Cytochrome P450 enzymes are catalysts of unrivalled versatility, able to mediate over 60 different monooxygenation and other reactions, such as aromatic and aliphatic hydroxylation at unactivated C-H bonds, epoxidation of olefins and aromatic rings, heteroatom oxidation and dealkylation, C-C bond cleavage, ring rearrangements, isomerizations and reductions. This catalytic versatility has led to the exploitation of P450s across the biosphere, for diverse functions such as the mobilization of carbon sources, chemical communication (e.g. hormonal signalling), and inter-organismal chemical warfare, such as between plants and the herbivores that feed on them. They were pivotal for the transition from water to land by plants and animals and play central roles in natural product biosynthesis in microbes and secondary metabolism in plants. This versatility also makes them attractive for catalyzing industrially important reactions in pharmaceutical and other fine chemical syntheses. However, P450 enzymes from natural sources are limited by poor stability and the need for accessory enzymes and a reducing cofactor. Moreover, expression of P450s in recombinant systems typically requires rich media, all of which increases the cost of using P450s for industrial biocatalysis. We have used ancestral sequence reconstruction as a technique for both engineering P450s as biocatalysts and exploring their evolution. This presentation will look both backwards and forwards. Using examples drawn from reconstructions of plant, animal and microbial P450 families, I will discuss what we have learned about the natural evolution of these enzymes. Then, I will show how ancestral P450s can be used as cost-effective, modular bio-bricks for synthetic biology, to create biocatalytic systems powered by photosynthesis and nanobioreactors based on P450s in virus-like particles. Cytochrome P450 enzymes are catalysts of unrivalled versatility, able to mediate over 60 different monooxygenation and other reactions, such as aromatic and aliphatic hydroxylation at unactivated C-H bonds, epoxidation of olefins and aromatic rings, heteroatom oxidation and dealkylation, C-C bond cleavage, ring rearrangements, isomerizations and reductions. This catalytic versatility has led to the exploitation of P450s across the biosphere, for diverse functions such as the mobilization of carbon sources, chemical communication (e.g. hormonal signalling), and inter-organismal chemical warfare, such as between plants and the herbivores that feed on them. They were pivotal for the transition from water to land by plants and animals and play central roles in natural product biosynthesis in microbes and secondary metabolism in plants. This versatility also makes them attractive for catalyzing industrially important reactions in pharmaceutical and other fine chemical syntheses. However, P450 enzymes from natural sources are limited by poor stability and the need for accessory enzymes and a reducing cofactor. Moreover, expression of P450s in recombinant systems typically requires rich media, all of which increases the cost of using P450s for industrial biocatalysis. We have used ancestral sequence reconstruction as a technique for both engineering P450s as biocatalysts and exploring their evolution. This presentation will look both backwards and forwards. Using examples drawn from reconstructions of plant, animal and microbial P450 families, I will discuss what we have learned about the natural evolution of these enzymes. Then, I will show how ancestral P450s can be used as cost-effective, modular bio-bricks for synthetic biology, to create biocatalytic systems powered by photosynthesis and nanobioreactors based on P450s in virus-like particles.