Bio-MAANICC - Biocatalytic Manufacturing of beta-Amino Acids: Nucleophilic addition to an Imine for C-C bond formation

Many of the conventional methods used for creating the chemical components of goods used in daily life are expensive and result in a large amount of wasted material that requires extra care to be safely discarded. Yet despite pursuing efforts toward a minimal environmental effect, there is typically still an unavoidable negative impact, and the burden of these necessary steps and less efficient processes are passed on to consumers in the form of higher prices and/or restricted availability of a wide range of products from medicines, pesticides, plastics and fuels. Alternative manufacturing processes that are efficient and most importantly sustainable must be developed and implemented wherever possible to reduce the environmental burden of these traditional methods and move toward environmentally friendly routes.
One area of research that has been gaining attention and praise in recent years for providing alternative production methods is the field of biocatalysis. The defining characteristic of biocatalysis is the use of microorganisms to produce enzymes (natural, functioning protein catalysts) that are then used to perform desirable reactions as a replacement for conventional chemical production methods. Because these enzymes, or biocatalysts, come from living organisms, the reactions they perform are generally very specific and the processes in which they are used are normally very mild and 'green,' often enabling production methods that give reduced waste and a much lower environmental impact. However, the fact that these biocatalysts are often so specific for one reaction can be problematic when they are intended to be used to make valuable materials that are not found in Nature. To circumvent this problem, scientists have developed many methods to modify enzymes to improve characteristics for use in these non-natural reactions. This practice, called enzyme engineering, gives researchers the opportunity to customize the biocatalyst to fit the process in which it will be used, and has become widely implemented within chemical and pharmaceutical companies as a means to use natural and renewable catalysts to produce non-natural materials using sustainable, cost-effective production methods.
Due to the success of biocatalysts in large scale manufacturing, industries are continuously looking for ways to install biocatalytic processes, as well as for enzymes that catalyse desirable new reactions not available among known biocatalysts. These additions can lead to the development of new and better products and innovative methods to replace current chemical processes with an equivalent biocatalytic route. Working to contribute to this desired expansion, the research proposed here aims to develop a series of engineered enzymes that catalyse a synthetically valuable reaction not currently offered by known biocatalysts. The utility of these biocatalysts will be demonstrated through the production of high-value chemicals found in pharmaceuticals and natural products. The goal is to provide a productive and environmentally friendly route for manufacturing a variety of chemical building blocks which will facilitate research toward determining and advancing their potential in medical applications. In order to accomplish this, we will establish a new biocatalytic strategy to make these compounds through the use of a novel biocatalyst, and apply enzyme engineering methods to quickly optimize its activity. The development of biological cost-effective and clean production methods will also be a hugely beneficial resource for future production of medicines and materials containing these valuable intermediates, helping ensure that these products can be globally available and environmentally sustainable.

This project aims to develop a series of biocatalysts that perform the synthetically valuable reaction of carbon-carbon bond formation through nucleophilic addition to an imine as an enzymatic Mannich reaction. Enzymes that can catalyse carbon-carbon bond forming reactions are highly desirable for synthetic applications, but the reaction catalysed via addition to an imine is notably absent from the current toolbox of applied biocatalysts as this is rare as a naturally occurring enzymatic reaction. Therefore, establishing a preliminary panel of enzymes catalysing this unique transformation with broad substrate specificity will facilitate new routes to chemical space previously not accessible through biocatalysis.
This research programme focuses on enzyme engineering to alter substrate acceptance for both the carbon nucleophile and the imine electrophile in the carbon-carbon bond forming reaction, followed by extension to a multi-enzyme biocatalytic cascade for production of precursor compounds. Biocatalyst optimization for this transformation will be accomplished through multiple strategies of directed evolution using high-throughput assays to screen for improved activity toward non-natural substrates. Generating a family of these engineered enzymes with varied specificity for the identity of the nucleophile, the electrophile or both substrates will enable a vast expansion of the synthetic potential of these biocatalysts. We will display this utility through the production of a diverse panel of chiral synthons used as pharmaceutical and agrochemical intermediates and in natural products. Successful demonstration of this new biocatalyst in biotechnological applications will close a major gap in the types of chemistry available through biocatalysis, and hopefully stimulate further research into its use in methods of production for other valuable chemicals, at the same time as encouraging a continued search for biocatalysts that catalyse novel enzymatic reactions.

The project proposed here will uniquely contribute to expanding the potential of industrial biotechnology within the UK through providing a novel, exploitable technology, and therefore broaden the scope of applications accessible to this growing industry of increasing global importance. The biocatalysts to be developed have prospective applications in producing high value chemicals that can be used to advance research in multiple separate research disciplines, such as organocatalyst and pharmaceutical development, as well as other medical functions, and consequently can have an impact on a wider community.
Academic communities - Successfully accomplishing the research objectives proposed within this Fellowship will lead to multiple publications of achievements in high-impact journals, which will generate significant interest among researchers in chemical and biological disciplines. Biologists and biochemists will have access to novel biocatalysts which catalyse a rare enzymatic reaction, but have been characterized and developed for broader applications in synthesis. Creating the technology base for production of a wider chemical diversity in greater availability by these engineered enzymes will greatly benefit synthetic and medicinal chemists through providing a pool of advanced intermediates that will facilitate research into creating the next generation of medicines and chiral organocatalysts with improved properties.
Chemical, pharmaceutical, agrochemical and biotechnological industries - The biocatalysts developed through this research programme will enable streamlined production of a class of compounds that had previously been predominantly impractical to manufacture at large scale. This new method of production and the corresponding increase in product availability will, in turn, benefit broader industrial production of a number of chemicals used in the synthesis of pharmaceuticals, chiral ligands, agrochemicals and other fine chemicals. A transfer in manufacturing to include this biocatalytic step or these biocatalytically derived intermediates is likely to lead to a more sustainable production process.
General public and wider community - The wider public community will primarily see the impact of this research in the form of new and improved chemical goods (medicines, agrochemicals, etc). Based on the industrial and production benefits listed above, consumers may also see these products available at reduced prices as a result of any savings in manufacturing costs being reflected in final market prices. The combined improved availability, reduced environmental impact of their production and potentially lower price point will enable these goods to be accessible to a larger portion of the global population.
University of Manchester, Manchester Institute of Biotechnology, Department of Chemistry - The institute and department hosting this research will benefit from the cutting edge biocatalytic resources developed during this project, and successful completion of project objectives will add to the well-established international acclaim of the host organization and institute. Furthermore, these host institutes will benefit from new and expanded collaborations with academic researchers and industrial partners, as well as through commercial exploitation of IP generated through this research fellowship.
Research fellow - Successfully accomplishing the project aims set out in this proposal will allow the research fellow to disseminate these notable research outputs through high-impact publications in prestigious international peer-reviewed journals, earning him a reputation as an up and coming researcher in the field of biocatalysis. Additionally, these achievements will give the fellow the opportunity to forge new collaborations and establish a valuable foundation for a path toward becoming an independent researcher and applying for future funding.