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A Novel High-Throughput Assay for Monitoring Enzymatic Reductive Amination Reactions

Dr Godwin Aleku, Pharmaceutical Sciences, Kings College London

Abstract: Enzymatic reductive amination allows direct and selective access to primary, secondary and tertiary chiral amines under environment-friendly reaction conditions. Enzyme engineering efforts for this important class of bio-transformations are crucial to allow for the potential industrial applications but currently rely on tedious and time-consuming screening of large libraries and the use of expensive LC/LCMS systems. As an alternative, we have developed the rapid "RedAm detect" assay for quantitative monitoring of reductive aminase activity of cell extracts, which has allowed us to perform the high-throughput screening of 56 site saturation mutant libraries in two reductive aminases. As a result, several functionally important residues in these enzymes were identified. These data were further analysed alongside other characterised RedAms where extensive functional hotspot mapping has been performed, enabling us to pinpoint regions/motifs in RedAms that allow profound improvements/alterations of the functional traits of these significant enzymes. Our work lays an important foundation towards mapping sequence-activity relationships in RedAms at the enzyme family level and paves the way for predictable, faster, and cost-effective engineering of RedAms for applications.

Bio:Dr Aleku obtained a PhD in Chemical Biology in 2017, working with Professor Nicholas Turner FRS at the Department of Chemistry, University of Manchester. He was later a postdoctoral research associate at the Manchester Institute of Biotechnology in the group of Professor David Leys, followed by an EMBO New Venture Fellowship at Roland Riek's Biomolecular NMR lab in ETH Zurich. In early 2020, he moved to the University of Cambridge where he was a Leverhulme/Isaac Newton Trust Early Career Research Fellow with Professor Florian Hollfelder and a Junior Research Fellow at Wolfson College, Cambridge. Dr Aleku joined King's College London in late 2022 to take up a lectureship in Chemical Biology and Drug Discovery within the School of Cancer and Pharmaceutical Sciences. He leads interdisciplinary research focusing on developing clean enzyme-based sustainable pharmaceutical synthesis methods and applying biocatalysis to drug discovery. His foundational work on Reductive Aminases (RedAms) has been recognized by a Royal Society Horizon Prize (Chemistry-Biology interface) in 2021. He has been recently awarded a Royal Society grant (2023) and the EPSRC New Investigator Award (2024).

Engineering novel FRET biosensors for in vivo visualisation of endogenous phytohormone dynamics.

Dr Maxime Josse, Sainsbury Laboratory Cambridge University

The cellular dynamics of plant phytohormone auxin (IAA) act as a central information processing system for plant physiology and development. The auxin research field is highly advanced with elegant functional and mathematical models of how auxin levels impact development, but these are largely based on either destructive, lower-resolution direct measurements (e.g. GC-MS) or indirect transcriptional/degron-based measurements in vivo. In order to track auxin dynamics directly, in real-time and with subcellular resolution in a minimally invasive manner, direct genetically encoded biosensors are needed. We and others have taken on the goal of engineering direct biosensors for auxin based on FRET (Förster Resonance Energy Transfer). A milestone was reached with AuxSen, which is the first auxin FRET biosensor to be deployed in plants. Unfortunately, AuxSen has a moderate affinity for auxin in the low µM range that will undoubtedly miss important auxin dynamics expected to occur in the low nM range. Via repurposing prokaryotic auxin binding protein, we have engineered a range of novel IAA Genetically-encoded Optical biosensors (IAAGO1/2/3) for auxin. Our initial biosensor protein IAAGO1 had IAA affinity KdIAA ~1.5 µM with high signal-to-noise ratio, from which we used directed evolution and an iterative structure-guided design-build-test process of biosensor engineering to develop the most recent versions (IAAGO2 and IAAGO3) covering an array of affinity (KdIAA 40-1200 nM) and responding to auxin with high spatio-temporal resolution in vitro, in E. coli, yeast, and in planta. Live imaging of IAAGOs in plant model Arabidopsis already unveils the detection of novel subcellular auxin dynamics in cells and growing organs.