Templates for rapid directed evolution of biocatalysts

Enzyme biocatalysts are now involved in lots of industrial processes and are particularly useful in drug synthesis and conversion of biomass to renewable fuels This is because naturally occurring enzymes can be highly active and very selective (enantio-, regio- and chemoselectivity). The potential of biocatalysts has become increasingly accessible largely due to directed evolution and advances in generating libraries of quality mutants i.e effectively exploring sequence space, as well as high throughput screening (HTP) for functional variants. Often directed evolution has been employed to improve low enzyme activity, increase thermostability or tolerance to industrial conditions, as well as expanding activity towards substrates which an enzyme has low activity. There are also good examples of directed evolution used to develop novel enzyme function. However, biocatalysts for processes that have no biological context still remains a challenge for protein engineering. De novo design has been used in attempt to bridge this gap but the time taken to achieve novel biocatalysts in this way is less than ideal. Phylogeny based creation of ancestral proteins suggests that these were more promiscuous and had broader substrate specifity than their current day counterparts. With that in mind a rational place to start any directed evolution towards a truly novel biocatalyst ought to be an enzyme with low activity towards several substrates. Folding units of proteins can be traced to common ancestors. Commonly seen protein folds include but are not limited to; the Rossman fold, alpha/beta plait, TIM barrels and the immunoglobulin fold. This project aims to explore the idea of a de-evolved enzyme as a template for evolvability towards new functions. The focus of this will be on generating a protein scaffold with minimal functional decoration, ancestral sequence reconstruction and capitalising on neutral drift combined with HTP screening methods.