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Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)
Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)
Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 ?M in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ?100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.
0027-8424
E7383-E7389
Gielen, Fabrice
c77341af-6e84-468f-a89e-0dcda0a75139
Hours, Raphaelle
678ac001-f89f-4b52-bf13-891e6587eded
Emond, Stephane
91595bb0-ad6f-474f-bcf9-d5b404bfd262
Fischlechner, Martin
b3930129-0775-4c05-81c7-475934df97ee
Schell, Ursula
85db1151-b737-490f-b541-7b69e73fd1a6
Hollfelder, Florian
a9f01280-f05d-4057-b5d7-85eac249e477
Gielen, Fabrice
c77341af-6e84-468f-a89e-0dcda0a75139
Hours, Raphaelle
678ac001-f89f-4b52-bf13-891e6587eded
Emond, Stephane
91595bb0-ad6f-474f-bcf9-d5b404bfd262
Fischlechner, Martin
b3930129-0775-4c05-81c7-475934df97ee
Schell, Ursula
85db1151-b737-490f-b541-7b69e73fd1a6
Hollfelder, Florian
a9f01280-f05d-4057-b5d7-85eac249e477

Gielen, Fabrice, Hours, Raphaelle, Emond, Stephane, Fischlechner, Martin, Schell, Ursula and Hollfelder, Florian (2016) Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS). Proceedings of the National Academy of Sciences, 113 (47), E7383-E7389. (doi:10.1073/pnas.1606927113).

Record type: Article

Abstract

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 ?M in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ?100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.

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Accepted/In Press date: 12 October 2016
e-pub ahead of print date: 7 November 2016
Published date: 22 November 2016
Organisations: Chemistry

Identifiers

Local EPrints ID: 404964
URI: http://eprints.soton.ac.uk/id/eprint/404964
ISSN: 0027-8424
PURE UUID: cb958ee6-3152-4bd2-bd0e-a1be32e6dcc4

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Date deposited: 25 Jan 2017 14:29
Last modified: 06 Oct 2020 22:56

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Contributors

Author: Fabrice Gielen
Author: Raphaelle Hours
Author: Stephane Emond
Author: Martin Fischlechner
Author: Ursula Schell
Author: Florian Hollfelder

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