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Ultrahigh-throughput screening enables efficient single-round oxidase remodelling

Abstract

Biocatalysis provides a potentially sustainable means of chemical manufacturing. However, the tailoring of enzymes to industrial processes is often laborious and time consuming, which limits the broad implementation of this approach. High-throughput screening methods can expedite the search for suitable catalysts, but are often constrained by the need for labelled substrates. The generalization of such techniques would therefore significantly expand their impact. Here we have established a versatile ultrahigh-throughput microfluidic assay that enables isolation of functional oxidases from libraries that contain up to 107 members. The increased throughput over prevalent methods led to complete active-site remodelling of cyclohexylamine oxidase in one round of directed evolution. A 960-fold increase in catalytic efficiency afforded an enzyme with wild-type levels of activity for a non-natural substrate, allowing biocatalytic synthesis of a sterically demanding pharmaceutical intermediate with complete stereocontrol. The coupled enzyme assay is label free and can be easily adapted to re-engineer any oxidase.

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Fig. 1: Detection strategy and FADS of CHAO.
Fig. 2: Characterization of the engineered PT.1 variant and wild-type CHAO.

Data availability

The data that support the plots and other findings of this study are available from the corresponding author upon reasonable request.

Code availability

No custom code was used to generate data presented in this study.

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Acknowledgements

This work was generously supported by the Swiss National Science Foundation and the ETH Zurich.

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A.P.G. and R.O. initiated the project; A.D. and M.P. performed the experiments and analysed the data; L.F. performed the molecular modelling; A.D., A.D.G. and D.H. co-wrote the paper.

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Correspondence to Donald Hilvert.

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Supplementary Figs. 1–7, Supplementary Tables 1–6 and Supplementary references.

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Debon, A., Pott, M., Obexer, R. et al. Ultrahigh-throughput screening enables efficient single-round oxidase remodelling. Nat Catal 2, 740–747 (2019). https://doi.org/10.1038/s41929-019-0340-5

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