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Rapid screening of diverse biotransformations for enzyme evolution

Rapid screening of diverse biotransformations for enzyme evolution
Rapid screening of diverse biotransformations for enzyme evolution
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 time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s 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) as a case study, enhancing its activity toward 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.
mass spectrometry, desorption electrospray ionization, high-throughput screening, Biotransformation, enzyme evolution, biocatalysis
2691-3704
508–516
Kempa, Emily E.
2ac727d4-d3c6-4392-8ebf-0c61bce70fa1
Galman, James L.
d91afad8-b0b7-4027-95a8-6bd5586ef1f7
Parmeggiani, Fabio
61347984-e2cd-404f-b9ab-3521ef5c5a14
Marshall, James R.
a6681329-aa05-4381-b8a9-8a435aa51bb1
Malassis, Julien GJ
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Fontenelle, Clement Q
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Vendeville, Jean B
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Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Charnock, Simon J.
817ac902-d84c-4deb-ba93-cc8fa0e0024d
Flitsch, Sabine L.
74eec2dd-f329-4f0f-999d-73edbcf34e96
Turner, Nicholas J
da1a6276-4d43-4767-923b-11889e780231
Barran, Perdita E.
108a9d6f-ea25-4864-be0b-ef843ee59179
Kempa, Emily E.
2ac727d4-d3c6-4392-8ebf-0c61bce70fa1
Galman, James L.
d91afad8-b0b7-4027-95a8-6bd5586ef1f7
Parmeggiani, Fabio
61347984-e2cd-404f-b9ab-3521ef5c5a14
Marshall, James R.
a6681329-aa05-4381-b8a9-8a435aa51bb1
Malassis, Julien GJ
5cd4224d-e4db-4835-863c-56b709511ccd
Fontenelle, Clement Q
c746c5ea-96bc-46c2-849d-47215ab58c03
Vendeville, Jean B
9f2c6155-cf23-4733-9402-33633fb5bb47
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Charnock, Simon J.
817ac902-d84c-4deb-ba93-cc8fa0e0024d
Flitsch, Sabine L.
74eec2dd-f329-4f0f-999d-73edbcf34e96
Turner, Nicholas J
da1a6276-4d43-4767-923b-11889e780231
Barran, Perdita E.
108a9d6f-ea25-4864-be0b-ef843ee59179

Kempa, Emily E., Galman, James L., Parmeggiani, Fabio, Marshall, James R., Malassis, Julien GJ, Fontenelle, Clement Q, Vendeville, Jean B, Linclau, Bruno, Charnock, Simon J., Flitsch, Sabine L., Turner, Nicholas J and Barran, Perdita E. (2021) Rapid screening of diverse biotransformations for enzyme evolution. JACS AU, 1 (4), 508–516. (doi:10.1021/jacsau.1c00027).

Record type: Article

Abstract

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 time scale. Here, we show the application of direct infusion of biotransformations to the mass spectrometer (DiBT-MS) screening to a variety of enzymes, in different formats, achieving sample throughputs equivalent to ∼40 s 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) as a case study, enhancing its activity toward 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.

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Accepted/In Press date: 8 April 2021
Published date: 26 April 2021
Keywords: mass spectrometry, desorption electrospray ionization, high-throughput screening, Biotransformation, enzyme evolution, biocatalysis

Identifiers

Local EPrints ID: 450575
URI: http://eprints.soton.ac.uk/id/eprint/450575
ISSN: 2691-3704
PURE UUID: 75b15513-071f-47ad-be68-1082787551f8
ORCID for Clement Q Fontenelle: ORCID iD orcid.org/0000-0002-1630-3407
ORCID for Bruno Linclau: ORCID iD orcid.org/0000-0001-8762-0170

Catalogue record

Date deposited: 04 Aug 2021 16:33
Last modified: 05 Aug 2021 01:36

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Contributors

Author: Emily E. Kempa
Author: James L. Galman
Author: Fabio Parmeggiani
Author: James R. Marshall
Author: Julien GJ Malassis
Author: Clement Q Fontenelle ORCID iD
Author: Jean B Vendeville
Author: Bruno Linclau ORCID iD
Author: Simon J. Charnock
Author: Sabine L. Flitsch
Author: Nicholas J Turner
Author: Perdita E. Barran

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