Ultrahigh-throughput screening enables efficient single-round oxidase remodelling

Aaron Debon, Moritz Pott, Richard Obexer, Anthony P. Green, Lukas Friedrich, Andrew D. Griffiths, Donald Hilvert*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)

15 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)740-747
Number of pages8
JournalNature Catalysis
Volume2
Issue number9
Early online date13 Sep 2019
DOIs
Publication statusPublished - Sep 2019

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    Debon, A., Pott, M., Obexer, R., Green, A. P., Friedrich, L., Griffiths, A. D., & Hilvert, D. (2019). Ultrahigh-throughput screening enables efficient single-round oxidase remodelling. Nature Catalysis, 2(9), 740-747. https://doi.org/10.1038/s41929-019-0340-5