Engineering Biocatalysts for the Next Generation of omega-Transaminase Processes

Chiral amines are prevalent in natural products, which often display potent biological activity. Such chiral amine motifs are also frequently found in pharmaceutical drug compounds and chemical building blocks meaning that the development of environmentally benign and sustainable routes to produce these important motifs is extremely desirable. Nature synthesizes these complex and valuable molecules through the action of highly specialized enzymes. These natural catalysts enable an extremely efficient biosynthesis from simple starting materials, installing functional groups with exceptional levels of selectivity.

Chemical catalysts are frequently designed to mimic the action of enzymes and are often capable of achieving impressive selectivity. However, unlike enzymes, processes involving these catalysts usually involve high temperatures, sub-optimal pH, organic solvent and complex purification methods.

Enzymes called omega-transaminases (TAs) catalyze the conversion of commercially available or easily accessible starting materials to high-value amines. These biocatalysts require an additional donor molecule to provide the amine functional group. This donor is ultimately converted to a by-product and the desired amine product is formed. However, the reaction is freely reversible and unless this by-product is removed from the reaction, low yields of the desired amine will be isolated, as the enzyme will more readily catalyse the reverse reaction to regenerate starting materials. A number of elegant approaches have been reported which remove this ketone by-product and allow access to appreciable quantities of the chiral amine. These strategies include the addition of expensive enzymes or the use of extremely large quantities of the amine donor in combination with the technically challenging removal of ketone by-products. One such approach, which relies on an extensively modified TA, is currently used for the industrial synthesis of the antidiabetic drug compound, sitagliptin. However, the approach is far from efficient and the development of this heavily modified TA biocatalyst was enormously challenging, highlighting an immediate need for more sustainable strategies for performing these biotransformations and for developing suitable enzyme catalysts.

This research will build upon recent work reported in our laboratory that describes arguably the most efficient approach to date for performing biotransformations involving TAs. The success of the approach is due to spontaneous precipitation of the by-product, which cannot regenerate starting materials. This polymer is also highly colored and has allowed the development of an effective high-throughput screening strategy that enables the rapid identification of active enzymes.

Our focus now is to optimize the process further and make it more suitable for industrial application. Specifically, low cost amine donor molecules will be used that are spontaneously removed from the reaction in a similar way to our previously reported method. We will also apply a simple high-throughput screening strategy to assist in the genetic engineering of natural enzymes in order to increase the scope of the reactions that they can catalyze and make them suitable for industrial scale synthesis. The enzymes developed in this study will enable cost-effective, sustainable and environmentally neutral methods for the small/medium and industrial scale production of one of the most important compound classes.

The aim of this project is to develop biocatalysts which enable both high-throughput screening and represent suitable catalysts for the industrial scale synthesis of optically pure chiral amines. The overall goal of this research is to make the biocatalytic conversion of ketone to amine the preferred method for small/medium and industrial scale synthesis of chiral amines.

Initial work will focus on Identifying low cost, bulk amine donors which efficiently displace reaction equilibria using a novel high-throughput screen (HTS). Strategies for simplifying product isolation using low-cost reagents will also be investigated. Having identified suitable amine donors, a large library of omega-TA biocatalysts will be made available by our project partner, Prof. Uwe Bornscheuer. HTS techniques will be used to identify variants that display dual activity towards previously reported ortho-xylylenediamine and towards low cost bulk amines. Combination of direct evolution and in silico approaches will be employed to enhance the activity biocatalytic properties of these enzymes. Finally, the substrate scope of selected catalysts will be expanded to encompass bulky ketones using a 'substrate-walking' approach coupled to a HTS.

The biocatalytic processes developed in this project will provide a highly efficient, low cost and environmentally benign method for the small/medium and industrial scale synthesis of chiral amines using omega-transaminase (TA) biocatalysts. The wide-reaching potential of the research will ensure that it positively influences the academic and industrial communities as well as the wider community.

Academic communities (synthetic chemists/medicinal chemists/biochemists/computational scientists) will benefit from:
1) New and improved routes for the synthesis of chiral amines, which are important motifs in pharmaceutical drugs, bioactive natural products and are common synthetic building blocks
2) Access to biocatalysts with suitable properties for application in organic/medicinal chemistry
3) Structural information on omega-transaminase (TA) enzymes and residues responsible for enhanced selectivity and substrate scope
4) High-throughput screening techniques to assist with the development of new biocatalysts

The industrial communities (pharmaceutical and fine chemical companies) will benefit from:
1) Biocatalysts with suitable properties for application from discovery right through to large-scale processes
2) Significant reduction in the cost of processes involving omega-TAs
3) Highly 'green' and efficient approaches for the production of pharmaceuticals and chemical building blocks
4) Reduction in carbon footprint by:
- using stoichiometric equivalents of bulk amine donors leading to reduction in waste
- replacement of transition metal catalyzed or multi-step processes with sustainable biocatalysts
- simplified purification methods

The wider community will benefit from:
1) Lower-cost of existing pharmaceuticals
2) Discovery of new pharmaceuticals
3) Participation and education from the numerous outreach activities organized including events focused at encouraging more young women towards a career in science
4) Enhancing the profile of UK research facilities, including Manchester Metropolitan University

Timescales
There will be immediate benefits to both the academic and industry sectors as low-cost, highly efficient and sustainable approaches for the production of chiral amines are developed. This work will be disseminated from the beginning of the project (July 2015, Biotrans conference) and at numerous occasions throughout the two year period to maximize awareness. As the cost of producing pharmaceuticals decreases, the wider community and economy will benefit from lower cost medicines. It is expected that this research and future research activities will see biocatalytic routes for the production of pharmaceuticals becoming the preferred industrial method. This will lead to extremely positive environmental benefits as 'green' processes replace traditional metal-catalyzed processes.

Training
This multi-disciplinary project will provide excellent training for the PDRA employed. He/she will be trained in aspects of chemistry, biocatalysis, microbiology and molecular biology and benefit from the expertise of the PI, Prof. Turner, Prof. Bornscheuer and industrial experts from Merck and Almac. As the ability to engage in multidisciplinary research is becoming essential in both academic and industrial environments, this project will give the PDRA invaluable experience for future applications. The PI will gain important training in managing an ambitious project, which involves high-profile academic and industrial partners and collaborators and will enable her to build closer links with these institutes. It is expected that the excellent research will generate enormous interest from academic and industrial researchers and will lead to future collaborative funding and support.