Beyond Transaminases: Evolution of native Amine Dehydrogenases for Chiral Primary Amine Synthesis.

Background:

Chiral amines are important functional groups that feature in up to 50% of small molecule drugs. The chemistry use to make them is often non-selective, hazardous or toxic and so there is significant pressure to develop green and sustainable methods of chiral amine synthesis. It is now recognised that enzyme technology or ‘biocatalysis’ can play a full role in the industrial synthesis of chiral amines, as these catalysts offer advantages of selectivity and sustainability can be applied on industrial scale.  One of the major enzymatic reactions of choice for the production of chiral primary amines is the ‘reductive amination’ of a ketone directly to the amine. In some industrial examples, the enzyme used is a transaminase (TA), which uses an ammonia donor, such as alanine or isopropylamine, and a cofactor called PLP, to accomplish the transformation. However, despite significant advances, TA technology suffers from problems including unfavourable reaction equilibria and also the need for the ammonia donor. In collaboration with colleagues at Genoscope in France, we have recently reported the discovery of ‘Amine Dehydrogenase’ enzymes (AmDHs) that also catalyze the formation of chiral primary amines from ketones, but using only ammonia as the source of the amine nitrogen [Grogan, Vergne (2019) Nature Catal., 2, 324]. We have performed preliminary studies on AmDHs and determined that they have great potential as biocatalysts for the synthesis of primary amines. We have also identified many interesting primary amine products as pharmaceutical precursors, which we propose may be accessible using AmDH catalysis, if the enzymes could be evolved for alternative specificity. In this project we will be the first to apply directed evolution methods to the alteration or improvement of AmDH activity for process applications.

Objectives:

To apply directed evolution to AmDHs for the creation of process-suitable biocatalysts for chiral primary amine synthesis.

Experimental Approach:

[1] Synthesise ketone precursors and product standards for  pharmaceutical amine targets; [2] Screen natural AmDH library for activity towards model ketones using HPLC; [3] Apply random mutagenesis methods to selected AmDHs and use high-throughput (HT) colorimetric screening to select mutants; [4] Structural characterisation of evolved AmDHs and rational mutagenesis for further improvement; [5] Application of evolved AmDHs to preparative scale aminations and characterisation of products.

Novelty:

Following the discovery of natural AmDHs, there have been no examples of their engineering using directed evolution for improved characteristics.  The potential is great, as evolved catalysts would present a superior alternative to industrial TA catalysts for primary chiral amine synthesis.

Training:

The student will be supervised in the Unsworth lab and in the Grogan group in the YSBL and will be trained in synthetic organic chemistry and relevant analytsis (NMR, IR, MS etc.), gene cloning, construct engineering, heterologous expression, biotransformations, gene mutation and structural enzymology using X-ray crystallography. The project will also provide training in assays using HPLC, GC and GCMS and plate-based UV spectrophotometry for HT screening. The student will also benefit from programmes of postgraduate study and skills training offered through the Department of Chemistry, and frequent written and oral presentations. Regular contact with collaborators Genoscope through Zoom meetings will be maintained. Genoscope has generously offered to host the student at some point during the PhD and York will also host a Genoscope PhD student for a return visit. These interactions will ensure access to additional training, expertise and guidance throughout the project.

In Collaboration with Dr Carine Vergne-Vaxelaire, Genoscope, France.

All Chemistry research students have access to our innovative Doctoral Training in Chemistry (iDTC): cohort-based training to support the development of scientific, transferable and employability skills: https://www.york.ac.uk/chemistry/postgraduate/training/idtc/ 

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For more information about the project, click on the supervisor's name above to email the supervisor. For more information about the application process or funding, please click on email institution

This PhD will formally start on 1 October 2023. Induction activities may start a few days earlier.

To apply for this project, submit an online PhD in Chemistry application: https://www.york.ac.uk/study/postgraduate/courses/apply?course=DRPCHESCHE3