Imine Reductases: Biochemistry, Engineering and Application

The synthesis of biologically active pharmaceutical and agrochemical compounds requires that chemical intermediates are synthesised in a very specific way, giving only one of two possible isomeric forms, as the unwanted form may be inactive or even toxic. Some of the most important chemical intermediates in pharmaceuticals synthesis are amines, nitrogen containing compounds that form part of well-known drugs such as beta-blockers and painkillers such as ibuprofen. It is a significant challenge to synthesise amines in single isomer form, and increasingly both academics and industrialists are turning to enzymes - nature's biological catalysts - as an efficient way of making these compounds. Not only do enzymes naturally possess the required selectivity to make single isomers, they are also attractive from the perspective of sustainable, green chemistry, as they work at ambient temperature and pressure, do not require toxic chemical reagents to work, and generate lower hazard waste than some non-biological catalysts. Many kinds of enzyme have been used to make single-isomer amines, but a new class of enzymes 'imine reductases (IREDs)' has recently been unearthed, which offers new and improved ways to synthesise these important compounds. One of the major advantages relates to reaction yield; while many of the competing enzymatic routes result in only 50% maximal theoretical yield, IREDs can deliver 100% yield of the single isomer form required. In this project we propose to take our early work on IREDs and to develop this with a view to offering valuable new enzyme catalysts for industry. We will first engineer commonly used industrial bacteria to make large amounts of the IRED enzymes, and then we will thoroughly test their ability to catalyse the industrial reactions of interest. We will use X-ray crystallography to determine the molecular structure of these enzymes, and this will allow us to make conclusions about how they work at a molecular level. If we learn more about how the enzymes work, we can then use protein engineering techniques to make the enzymes work better on the reactions of interest, but also expand their applicability so that they work on new reactions that are not catalysed by the natural enzymes. We can also use the structural information to improve the way that the enzymes will perform under industrial process conditions. In the end, a comprehensive review of this class of IREDs will be performed, resulting in important new information on a largely unexplored group of enzymes, and valuable catalysts for the production of important industrial synthetic intermediates.

Efficient methods for the asymmetric synthesis of chiral amines are important for the pharmaceutical industry as amines constitute prestige targets in pharmaceutical chemistry. Increasingly, industry is turning to biocatalytic routes for the production of chiral amines as enzymes not only offer superior selectivity in the production of single enantiomer products, but comply with many of the precepts of green and sustainable chemistry. Many different kinds of enzymes have been applied to the enantioselective synthesis of amines, including hydrolases, amine oxidases and transaminases, but these can suffer from limited yields, poor stability and reaction equilibrium issues. One under-explored area of biocatalysis for the production of chiral amines is the use of putative 'imine reductases' (IREDs). The transformation of prochiral imines to chiral amines using IREDs would offer quantitative asymmetric route to amine products of high optical purity. In this project, we will build on preliminary investigations to conduct a comprehensive study into the biochemistry and application of NAD(P)H-dependent IREDs. Target genes will be cloned, expressed and the IREDs applied to the synthesis of a wide range of amine products in both single-enzyme reactions and as part of cascades with other amine-transforming enzymes such as transaminases and amine oxidases. The structures of interesting IREDs will be pursued using X-ray crystallography, and structural information used to guide mutational investigations into mechanism and specificity in these enzymes. Structures will also be used to guide targeted combinatorial mutagenesis experiments that look to alter substrate range and improve activity of IREDs. In vitro evolution techniques based on random mutagenesis, in combination with relevant screens, will also be used to improve catalytic characteristics and process suitability.

The development of selective, efficient and green methods of chemicals synthesis has wide-ranging consequences that go far beyond the academic sphere. The pharmaceutical chemicals industry is one of the major beneficiaries of research into these improved synthetic methods, which delivers technological and economically beneficial solutions to the problems of synthesising chiral intermediates in an efficient and environmentally benign manner. The investigation and development of such techniques is increasingly important as the proportion of IB processes within industrial chemistry is thought to increase to 20% by 2015 (Source: Horizon 2020 EU Framework Programme). The PI and CoPI collaborate extensively with representatives of major UK and other international phamaceutical companies (GSK, Dr Reddy's, Merck, Codexis et al.). Some of these activities are enabled through the Centre of Excellence for Biocatalysis, Biotransformations and Biomanufacture (CoEBio3) of which the Co-PI is director. The contacts within CoEBio3 will allow communication of results, when appropriate, through face-to-face meetings and presentations to both individual companies and meetings of the combined industrial affiliates of CoEBio3. Industrial representatives are often laboratory scientists in biotransformation laboratories who will be able to quickly transfer information on new techniques and developments to their own groups within companies, within a 2-4 y timescale, dependent on the publication of results. Given the importance of the pharmaceutical sector to the UK, improved processes for the chemical industry will make a clear contribution to economic competitiveness.
Outside the academic and industrial parties that most directly gain advantage from new technology advances within the project, relevant UK government stakeholders also derive benefit in general from successful developments in Industrial Biotechnology, as these help to publicise the benefits of investing in IB and also inform the development of future policy. Organisations such as the BiS-funded Bioscience Knowledge Transfer Network are well placed to use these case studies to champion IB in the presence of companies who had not previously considered using this technology. The general benefits of IB such as that used in the project can be communicated through Bioscience KTN activites such as visits, newsletters, webinars and workshops.
Last, the wider beneficiaries of the work will be the general public. As consumers of pharmaceutical products, the public benefits from the more efficient production of these compounds, and also from the development of new pharmaceuticals that can be accessed using new technology. More generally and in the longer term, the incorporation of industrial biotechnology techniques into pharmaceutical manufacture allows the public to benefit from the improved lifestyles and environment associated with a more efficient, sustainable and environmentally benign chemicals industry. The impact of these developments on the public can be communicated at local and international level using press releases, articles in popular science journals, and at public engagement events such as the York Festival of Ideas and the Manchester Science Festival.