Finding faster and simpler ways to screen protein sequence space to enable the identification of new biocatalysts for asymmetric synthesis remains both a challenge and a rate-limiting step in enzyme discovery. Biocatalytic strategies for the synthesis of chiral amines are increasingly attractive and include enzymatic asymmetric reductive amination, which offers an efficient route to many of these high-value compounds. Here we report the discovery of over 300 new imine reductases and the production of a large (384 enzymes) and sequence-diverse panel of imine reductases available for screening. We also report the development of a facile high-throughput screen to interrogate their activity. Through this approach we identified imine reductase biocatalysts capable of accepting structurally demanding ketones and amines, which include the preparative synthesis of N-substituted β-amino ester derivatives via a dynamic kinetic resolution process, with excellent yields and stereochemical purities.
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Data supporting the results and conclusions are available within this paper and the Supplementary Information. Both the IREDy-to-go screen and a duplication of the screening plate without the colorimetric screening components containing 0.5 mg of crude lysate of each IRED in each well are freely available through Prozomix Ltd.
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We thank the Industrial Biotechnology Innovation Centre (IBioIC) and Biotechnology and Biological Sciences Research Council (BBSRC) for awarding the CASE studentship to J.R.M. from Prozomix Ltd. P.Y. was supported by a CSC scholarship and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (Grant no. 2016166). T.W.T. was supported by a BBSRC CASE studentship awarded by Pfizer. S.L.M. was supported by a BBSRC CASE studentship from Johnson Matthey. R.B.P. and R.S.H. were supported by the European Research Council (ERC Grant no. 742987). J.M.-S. was funded by grant BB/M006832/1 from the UK Biotechnology and Biological Sciences Research Council. S.J.C., D.J.C., J.D.F., R.A.M.D. and K.M.G. acknowledge the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 685474 for supporting MetaFluidics. N.J.T. is grateful to the ERC for the award of an Advanced Grant (Grant no. 742987). We thank D. Heyes of the Manchester Institute of Biotechnology (MIB) for assistance in gathering the circular dichroism data. We thank Y. Qi of Prozomix Ltd for screening of the Prozomix diaphorases. Prozomix and J.R.M., P.Y., S.L.M., T.W.T., R.B.P., J.M.-S., R.S.H. and N.J.T. also thank other staff of Prozomix for their support.
The authors declare no competing interests.
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Supplementary Sections 1 (1.1–1.4), 2, 3 (3.1, 3.2.1–3.2.7, 3.3.1–3.3.4, 3.4.1 and 3.4.2), 4 (4.1–4.4, 4.4.1-4.4.5, 4.5 and 4.6), 5 (5.1, 5.2, 5.2.1–5.2.4, 5.3–5.5), 6, 7 (7.1–.7.5), 8 and 9.
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Marshall, J.R., Yao, P., Montgomery, S.L. et al. Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination. Nat. Chem. 13, 140–148 (2021). https://doi.org/10.1038/s41557-020-00606-w