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Directed evolution of ketoreductases in gel-shell beads

Periodic Reporting for period 1 - KREDs in GSBs (Directed evolution of ketoreductases in gel-shell beads)

Reporting period: 2015-06-01 to 2017-05-31

Optically pure secondary alcohols are valuable intermediates required for the introduction of chiral centres in drugs and agricultural chemicals. These compounds may be prepared through the reduction of the corresponding ketone. The use of enzymatically catalysed reduction of ketones, using ketoreductases (KREDs), has recently started playing an increasingly important role in the industrial-scale production of these compounds. Biocatalysts avoid the use of expensive stoichiometric amounts of chiral reducing agents and permit environmentally friendly, mild processes with less solvents and waste and a lower energy input. To meet the demands of a commercial viability, industry typically formulates strict fitness functions to which the enzyme must adhere. Typically, this would include a desired space-time yield with a minimum enantiomeric excess1. Although limited advances in protein engineering and directed evolution in have helped to improve biocatalysts, a remaining challenge is the matching of selection conditions to the reaction conditions of final industrial application. This project will have a high Scientific Impact on Biotechnology as it introduces a fundamentally new methodology for the development of biocatalysts. Also, comparatively little research has been done in the past on flow processes and directed evolution, a shortcoming this project seeks to address. The outcome of this project will be a high value procedure for converting promising but immature enzymatic processes to fully fledged industrial ones. This project will make a significant contribution to strengthening the European Green Chemistry sector, making this project timely, as the European Chemistry Sector faces the challenges of increased regulation and competition from the USA and BRIC nations. The process improvements stemming from this proposal in the synthesis of pharmaceutical compounds could significantly reduce their cost, impacting the lives of millions of European citizens.
The technical goal of this project was to establish a new way of carrying out “directed evolution” of enzymes that catalyse important reactions that may be of use to industries such as Pharma and in agricultural chemicals. As the name suggests, directed evolution of enzymes involves applying selective pressure on a population of enzyme variants in an effort to select the variant with the most favourable properties amongst them. Such properties may include better activity, better specificity and better stability of the enzyme. Important factors in the success of a directed evolution campaign are i) the quality and size of the DNA library encoding the variants, ii) the quality of the assay with which improved properties are to be detected and iii) the numbers of variants that can be screened given limited resources (time and costs). In this project, we have worked on a system that addresses all three of these factors for the directed evolution of the enzymes we were interested in, dehydrogenases. We are currently preparing a manuscript describing our work that will be submitted to a scientific journal, helping to communicate the insights we gained to the broader research community.
The new assay we have developed will, we believe, support further efforts to improve dehydrogenase enzymes such as KREDs that are of importance to diverse industries including the Pharmaceutical one. Using our technique, it may be possible for researchers in industries to discover better enzymes that will ultimately lead to reduction in costs and of environmental impact of production of for example key intermediates in the production of medicines. Furthermore, during the course of this project, we have gained new insights into the construction of DNA libraries. These insights will be of use to the wider community of researchers carrying out efforts in protein engineering, again helping to bring about improvements in sustainability and cost reductions in all the diverse areas where proteins are required (medicine, agriculture, bioremediation etc).