Metagenomic Imine Reductases for Biocatalytic Reductive Aminations

Abstract

Enzymes are protein biomolecules that enhance the overall rate of reaction. The quest for novel biocatalysts which originate from diverse sequence spaces with enhanced scopes and properties is a never-ending task, which has been fundamental to the success of biocatalysis. One rich source of novel biocatalysts is the use of metagenomics, whereby the microorganism requires no pre-culturing to extract the DNA. Offering a platform where enzymes with unique properties can be identified. Many enzyme families have yet to be explored for the identification of novel biocatalysts through metagenomics, including imine reductases (IREDs). Imine reductases are a nicotinamide co-factor dependent family of oxidoreductases. Synthetically, these enzymes are capable forming a range of chiral amines through reductive amination. Described in this project is the identification through a metagenomics approach, the largest panel (384 enzymes) of IREDs reported to date which are the most diverse with respect to phylogenetics. To facilitate screening, these enzymes are arrayed into microtiter plates to allow for rapid characterisation. Complementing this was the development of tetrazolium-based colorimetric screen allowing for high-throughput characterisation. These enzymes were also characterised with respect to expression and phylogenetics. To challenge these enzymes to see if they can outcompete previous IREDs for reductive amination in the preparation of high-value compounds, 3 specific substrate scopes were pursued. With excellent selectivity and activity, dimethylamine derivatives, α-methylbenzylamine derivatives and N-substituted β-amino ester derivatives through a DKR mechanism were obtained, where examples were shown on preparative scale. In a similar manner, an architecture of N-substituted α-amino esters, also performed on preparative scale. The ability to screen large libraries of IRED variants has also been demonstrated through the development of formazan-based solid phase screen. A suitable candidate was identified from the metagenomic collection meeting certain criterion. Where the enzymes were subsequently characterised with respect to both amine and carbonyl partners for reductive amination. Furthermore, engineering was also undertaken to infer thermostability, where the IRED variant displayed a significantly higher thermostability compared to the parent, rendering this compatible for the solid-phase screen.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor) & Nicholas Turner (Supervisor)