Design and Evolution of Enzymes with Non-Canonical Catalytic Mechanisms

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The ability to rationally design an enzyme for any desired transformation would have major impacts across the pharmaceutical and wider chemical sectors, leading to more efficient and sustainable synthetic routes to high value chemicals. The combination of computational enzyme design and directed evolution is currently the most attractive strategy to achieve this ambition. However, the limited range of functional groups presented by the genetic code (i.e. 20 amino acids) restricts the range of catalytic mechanisms which can be installed into designed active sites, thus severely limiting the repertoire of chemical transformations accessible. Advanced protein engineering technologies available in our laboratory now allow us to install non-canonical 'chemically inspired' amino acids into enzyme active sites, thus greatly expanding upon the limited range of functionality accessible with Nature's genetically encoded residues. We have recently combined this genetic code expansion technology with computational enzyme design and laboratory evolution to create the first enzyme that operates via a non-canonical organocatalytic mechanism (Nature 2019, 570, 219). In this PhD studentship, we will now expand ambitiously upon this early success, to demonstrate that the integration of non-canonical amino acids into computational design and directed evolution workflows can lead to the creation of enzymes with novel catalytic mechanisms and new activities. The design of new functional amino acids will be inspired by small molecule organocatalysts which are able to promote a plethora of valuable transformations not observed in Nature. Our approach merges the complementary disciplines of organocatalysis and biocatalysis, combining the mechanistic and functional versatility of small molecule systems with the enormous rate accelerations and reaction selectivities achievable within evolvable protein scaffolds.The project is firmly embedded at the interface of chemistry, biology and biophysics, a key driver for the BBSRC in the 'Exploiting new ways of working' agenda. It draws on key bioscience skills, including protein engineering, directed evolution and enzyme characterization, and specifically exploits a multi-disciplinary approach to the creation of enzymes with novel function, which is a major objective in the Industrial Biotechnology sector.