Designer hybrid chemo-enzymatic catalysts for cofactor recycling

The development of new and efficient catalysts for the synthesis of complex molecules is an ongoing need in the chemical industry, with both bio- and chemo-catalysts being applied at scale. This research is concerned with the design of a novel, hybrid chemo-enzymatic catalyst, which combines the best features of both chemical and enzymatic catalysis, by introducing metal catalysts into natural enzymes. Enzymes have evolved in nature and can be modified by scientists to perform many useful transformations in a very efficient way. However, the modifications are never too far from the natural world, in terms of functionality (i.e, mutation of amino acids to other amino acids). Recently, specific chemical modification of proteins has been developed as an exciting new tool to introduce chemo-catalytic functionalities within a biomolecule, thus expanding the functionality space. The objective of this project is to implement such a modification procedure into an existing enzyme, to change its reactivity and improve a key reaction needed for industrial exploitation of enzymes. The novelty of this design is the use of an enzyme specifically evolved for substrate binding and transition state stabilisation. The longer term vision is to apply hybrid catalysts to completely unnatural catalytic reactions. The use of enzymes that stabilise hydride transfer transition states in conjunction with suitable chemical catalysts could be envisaged for expanding the enzyme functionality to the reduction of more challenging substrates, rarely occurring in nature (e.g. nitriles, amides). Therefore, this research addresses a timely and fascinating question for enzyme study and application: can we design enzymes containing ANY desired catalytic functionality?
The hybrid catalysts prepared here will be used for the synthesis of molecules containing alcohol functions. Chiral alcohols are important constituents of pharmaceuticals and agrochemicals. Therefore, catalysts that facilitate their preparation are important tools for these industries. The use of enzymes as catalysts is very efficient and sustainable, due to their high activities and selectivities, benign reaction conditions and their renewable origin. However, many of the enzymes that can make chiral alcohols enantioselectively have one major shortcoming: they employ the expensive and sensitive nicotinamide adenine dinucleotide phosphate, NADPH cofactor (£1085 per g), which is an essential component of the reaction mixture. The high cost precludes its use as a disposable reagent and continuous regeneration of NADPH in the reaction vessel (in situ) is necessary for economically acceptable transformations using isolated enzymes. Existing regeneration systems are based on either enzymes or chemical catalysts, but they have limited applicability, because they generate by-products, have a low stability or activity, or are inactivated by the presence of the enzyme. Therefore, we will engineer a chemical cofactor regeneration system that will be protected from deactivation, by incorporating it into a protein. We will use the same protein to incorporate the regeneration catalyst and to perform the alcohol synthesis, and in this way we will avoid complex mixtures containing two enzymes. The system will be of relevance to the industries involved in the synthesis of active pharmaceutical ingredients (pharma, custom manufacturing organisations etc), which could potentially apply it as a simple technology for recycling NADPH.

Industrial impact. The proposal addresses two needs of today's chemical industry in the UK: the need for innovative technologies to stimulate sustainable chemicals manufacturing and the need for skilled people with creative ideas and the right skills to implement these technologies. Chemical industries contribute to approx. £600 bn of sales and £195 bn gross value in the UK economy, and the impact of the pharma and API custom manufacturing industries in this sector is considerable. The development of novel technology to improve the effectiveness and to lower the environmental impact of chemicals synthesis will increase UK's competitiveness and will improve society's perception of the chemicals industry. An efficient and robust cofactor regeneration system that can reduce NADP+ using formate will be of considerable interest to the above industries, because it will have the potential to improve processes for manufacturing chiral alcohols. The artificial formate dehydrogenase designed here has the potential to overcome the limitations of native formate dehydrogenase (e.g. low stability, use of two enzymes in the reaction mixture), whilst maintaining its advantages (gaseous co-product, irreversible reaction and ease of work-up). The availability of efficient methods for cofactor regeneration will result in increased uptake of biocatalysis to manufacture molecules containing enantiomerically pure alcohol functions, ultimately resulting in reduced costs and increased process safety. Although this proof of concept will employ rare and costly second and third row transition metals, there is future scope to employ cheaper and more readily available metals, e.g. Mn, Fe etc.
The project will also train a postdoctoral scientist with a highly valued combination of skills for the chemical industries, by integrating experimental design and chemicals synthesis with protein engineering, expression and purification, as well as a range of state-of-the-art analytical techniques. The PDRA will also benefit from working in the Bioprocess, Environmental and Chemical Technologies research group, including access to a wide range of projects, strong industrial collaborations, an appreciation of process economics and environmental impact issues. The PDRA will develop the ability to work in an interdisciplinary environment and to communicate scientific results, both at UoN internal seminars and during international conferences and national meetings.

Impact on the public. The longer term impact on the general public will be to reduce costs of pharmaceutical manufacturing, enabling new, effective clinical therapies to be brought to market. In addition, the consumers of the pharmaceutical products will benefit from more efficient ways of producing biologically active chemicals, which will accelerate the development of new molecules with improved properties. Access to more efficient syntheses of biologically active chemicals will also benefit the agrochemicals sector and will accelerate the development of agrochemicals with lower environmental impact. Another long term effect of the research will be to reduce the environmental impact of the chemicals industry, by providing new biotechnological tools to maximise energy efficiency and minimise waste.