Design and Evolution of Artificial Enzymes with Non-Canonical Organocatalytic Residues

Traditional methods of producing essential chemicals such as medicines, pesticides and fuels are inefficient, expensive and create a huge burden on the environment. In order to maintain our current standard of living and to make essential products available to the global population, we urgently need to develop clean, efficient and sustainable manufacturing technologies to replace traditional chemical processes. An exciting technology that is already being widely adopted by major chemical companies is called Biocatalysis, whereby microorganisms (e.g. bacteria and yeast) are used to produce large amounts of enzymes (nature's catalysts) which are subsequently used in environmentally friendly processes to efficiently convert starting materials into desired products and high-value intermediates. Importantly, scientists are now able to quickly modify and optimize the natural function and properties of an enzyme to make it suitable for its desired application through a process called directed evolution, which mimics Darwinian evolution on a laboratory timescale. In principal, through directed evolution it should ultimately be possible to replace those chemical processes for which there is a natural counterpart with greener, more efficient biocatalytic alternatives. Unfortunately, to produce our essential chemicals we rely heavily on a series of non-natural reactions, and enzymes capable of performing these transformations simply do not exist. This means that for a typical multi-step sequence required to produce an essential chemical, existing technology may only allow us to replace one or two steps with a clean enzymatic process, with the remaining steps still reliant on hazardous chemical reactions. My research aims to overcome these significant limitations by creating enzymes which are able to efficiently catalyze synthetically valuable, non-natural reactions.

Nature's enzymes are made up from various combinations of only twenty standard amino acid building blocks which are generally not suitable to promote non-natural reactions. To achieve the ambitious goal of creating artificial enzymes, we will supply microorganisms with the necessary tools to produce biocatalysts which contain new functional, catalytic amino acids with unique properties. These residues are carefully designed so that they can be produced cheaply, cleanly and efficiently and have the necessary functionality to perform not one, but many important non-natural reactions which are currently carried out using hazardous chemical reagents. The primitive and promiscuous enzymes initially produced are expected to display low activity compared with natural enzymes, since they have not been subjected to millions of years of natural evolutionary processes to optimize their function. However, directed evolution offers an ideal method to rapidly optimize the activity of these biocatalysts to produce specialized, robust enzymes suitable for use in manufacturing processes. These enzymes can be used as standalone catalysts to make high-value intermediates or in multi-step biocatalytic pathways to produce new and existing medicines, pesticides and fuels. Since these essential products will be produced cheaply in an environmentally friendly manner, they will be widely accessible for use by the global population.

This project involves the development of robust enzymes with novel activities, which promote key synthetic transformations not observed in nature's biosynthetic repertoire. Such designer biocatalysts are required to expand the reaction scope offered by the existing biocatalytic toolbox, which remains limited when compared with the diversity of chemical methodology available.
Our approach involves the evolution of pyrrolysyl-tRNA/pyrrolysyltRNA synthetase pairs which allow non-canonical amino acids containing key organocatalytic motifs to be incorporated into proteins with high fidelity. Expanding the repertoire of genetically encodable amino acids to include novel organocatalytic residues promises to combine the impressive reaction scope of small molecule organocatalysts with the unrivalled rate enhancements provided by enzymes. The genetic incorporation of N-methyl histidine, which has comparable mode of reactivity to that of the widely employed organocatalyst 4-dimethyaminopyridine, into protein scaffolds will provide a tuneable and versatile platform for the site-selective modification of biologically structures, providing rapid access to pharmacologically relevant analogues. The incorporation of amino acids containing secondary amine functionality into selected protein scaffolds will generate a series of first generation biocatalysts capable of promoting diverse transformations via enamine and iminium ion intermediates, including nitroalkane additions, cyclopropanations, Friedel-Crafts reactions and Diels-Alder cycloadditions. Optimization of these biocatalysts for desired transformations will be achieved via directed evolution using an expanded genetic code.
Successful implementation of the proposed research will inspire the design of artificial enzymes for a huge diversity of synthetically valuable transformations, facilitating the development of sustainable biocatalytic manufacturing processes for the production of high-value chemicals.

This statement seeks to address the questions of who will benefit and how will they benefit from the proposed research.
1. Consumers and the wider community. The technology developed during this project will ultimately allow essential chemicals such as pharmaceuticals, agrochemicals and fuels to be manufactured at a lower cost with reduced environmental impact. This will make these essential chemicals accessible to a greater proportion of the global population, significantly enhancing the quality of life of the individuals involved. The wider community will also benefit from scientific engagement with the PI and PDRA through outreach activities.
2. Pharmaceutical, agrochemical, biotechnological and related industries. The chemical industry is under increasing legislative pressure to reduce its carbon footprint. The technology, and specifically the catalysts, developed during this project will allow the development of sustainable and cost-effective biocatalytic routes to high-value products. This technology will also allow the identification and development of new and improved products that were not accessible using existing methodology.
3. Academic communities. Successful implementation of proposed research will result in high-impact publications in internationally renowned journals and thus generate significant interest from experimentalists and theorists across the chemical and biological sciences. Computational biologists/chemists will have access to an expanded set of catalytic residues as they seek to design enzymes with novel activities. Synthetic chemists will benefit from access to robust and efficient biocatalysts which promote valuable transformations that may not be accessible using traditional chemical methodology. Finally, this project seeks to create tuneable supramolecular catalysts by merging the fields of biocatalysis and organocatalysis, which is expected to generate considerable interest within the respective academic communities.
4. UoM, MIB and School of Chemistry. The host institutes/departments will benefit from new cross disciplinary collaborations within the UoM and from increased engagement with industrial partners and world leading academic researchers/institutes (e.g. Prof. Donald Hilvert, ETH). These host institutes will also benefit from the commercial exploitation of IP generated throughout the duration of this fellowship.
5. PI. Successful implementation of the research programme will allow the PI to build an international reputation as a world leading researcher in his field. Effective dissemination of project outputs to the scientific community will be achieved through publications in high quality, international peer-reviewed journals and through presentations at conferences and academic/industrial institutes. This fellowship will also give the PI the opportunity to generate new collaborations and build upon existing ones. These collaborations will form the basis for future joint grant applications.
6. PDRA, PhD and MChem students. Under the mentoring of the PI, PDRAs, PhD students and MChem students will be trained in both theoretical and practical aspects of synthetic chemistry, biocatalysis, molecular biology and protein engineering. These skills that are highly relevant for a future career in the pharmaceutical, biotechnology, bioenergy and related industries. The individuals involved will also benefit from interactions with academic and industrial partners.