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Rapid Screening of Diverse Biotransformations for Enzyme Evolution

preprint
submitted on 12.11.2020, 17:59 and posted on 16.11.2020, 06:59 by Emily Kempa, James L. Galman, Fabio Parmeggiani, James Marshall, Julien Malassis, Clement Fontenelle, Jean-Baptiste Vendeville, Bruno Linclau, Simon J. Charnock, Sabine Flitsch, Nicholas Turner, Perdita Barran

The lack of label-free high-throughput screening technologies presents a major bottleneck in the identification of active and selective biocatalysts, with the number of variants often exceeding the capacity of traditional analytical platforms to assess their activity in a practical timescale. Here we show the application of direct infusion mass spectrometry (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ~40 seconds per sample. The heat-map output allows rapid selection of active enzymes within 96-well plates facilitating identification of industrially relevant biocatalysts. This DiBT-MS screening workflow has been applied to the directed evolution of a phenylalanine ammonia lyase (PAL), enhancing its activity towards electron-rich cinnamic acid derivatives which are relevant to lignocellulosic biomass degradation. Additional benefits of the screening platform include the discovery of biocatalysts (kinases, imine reductases) with novel activities and the incorporation of ion mobility technology for the identification of product hits with increased confidence.

Funding

This work would not have been possible without funding made available from the Biotechnology and Biological Sciences Research Council (BBSRC), (BB/M011208/1, BBSRC-FAPESP grant BB/P01738X/1, IBCarb BB/L013762/1, BB/M027791/1, BB/M02903411, BB/M029034/1 BB/M028836/1) and the European Research Council (ERC), (788231-ProgrES-ERC-2017-ADG).

History

Email Address of Submitting Author

perdita.barran@manchester.ac.uk

Institution

The University of Manchester

Country

United Kingdom

ORCID For Submitting Author

0000-0002-7720-586X

Declaration of Conflict of Interest

no conflict of interest

Version Notes

version one

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