Development of biocatalytic ene reductase toolkits for stereospecific reductions through evolution of flavoprotein oxidoreductases.

The traditional methods for developing redox biocatalysts have delivered a number of viable manufacturing processes, but development is slow, complex and notoriously unreliable. Therefore, new approaches are needed. We propose to bring together a number of advances in biocatalyst development and general chemicals manufacturing to provide the necessary innovation. The first key advance is that it is no longer necessary to isolate a new biocatalyst for every new reaction target. Directed evolution makes it possible to improve existing biocatalysts, by extending their substrate range, focussing their selectivity and improving their stability to meet the demands of the manufacturing process. This means that stable, simple and well-behaved enzymes can be used as the starting point to develop extremely large portfolios of new biotransformations. Directed evolution can, therefore, be used to develop enzymes for previously inaccessible biotransformations and to replace existing, unexploitable enzymes with much more robust enzymes. Research until now has focussed on extending substrate ranges for specific reaction classes and on tightening selectivity. To deliver true platform biocatalysts, research is also needed to explore the extension of reaction classes, to provide stable, multi-purpose biocatalysis starting from a single enzyme. Such research would offer a realistic prospect to use isolated redox enzymes in a much wider range of manufacturing processes. We have established a platform robotics technology for directed evolution of Old Yellow Enzyme oxidoreductases and we have already made notable progress in broadening the specificities and reactions chemistries of this class of enzyme. The student will build on this success by generating single and multiple site-specific saturation libraries, informed from structural analysis of the target enzymes (PETNR and TOYE) to expand the chemistries available. Key targets include imine and nitro group reduction, which opens up new synthetic capabilities for this class of enzyme, as well as expanding the specificity range of existing chemistries catalysed by these enzymes (Toogood et al AdvSynCat, 350, 2789-2803 (2008); Fryszkowska et al Adv. Syn. Catal, 351, 2976-2990 (2009). The second key development is that redox biotransformations can be operated independently of coenzymes by using light-activated catalysis. Overall, the role of a coenzyme is simply to feed electrons into the enzyme active site. To bypass coenzyme requirements, we aim to deliver light-driven and tunable biocatalysts that can be integrated into next generation microreactors for the synthesis of high value compounds in a flexible and highly controllable format. Redox enzymes can be photo-activated in the presence of specific light-harvesting dyes which when excited at specific wavelengths efficiently and repeatedly inject electron(s) into the protein cofactors in the presence of a cheap, sacrificial electron donor. We have synthesized a range of light-activating and electron donating groups, each tuned to be activated by different wavelengths. These groups will be attached to the target enzyme catalysts through thiol linkage to generate a cascading series of reactions controlled by light of specific wavelengths where the direction and efficiency of the reaction pathway can be controlled. Such wavelength dependent control will transform the use of redox oxidoreductases in industrial biocatalysis by avoiding use of expensive coenzymes and enabling multiple enzymes to operate in cascade reactions through wavelength dependent control of each biocatalyst. Combined, the project will deliver innovative and affordable biocatalysts for the stereospecific reduction of a wide range of alkene substrates using either conventional coenzyme-driven or light-driven approaches. The approach is transformative and will enable development of generic and robust toolkits for industrial biocatalysis.