Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS)
Proceedings of the National Academy of Sciences of the United States
National Academy of Sciences
MetadataShow full item record
Gielen, F., Hours, R., Emond, S., Fischlechner, M., Schell, U., & Hollfelder, F. (2016). Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Proceedings of the National Academy of Sciences of the United States, 113 (47), E7383-E7389. https://doi.org/10.1073/pnas.1606927113
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 μM 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.
protein engineering, directed evolution, microfluidics, ultrahigh-throughput, emulsion droplets
This research was funded by the Engineering and Physical Sciences Research Council (studentship to RH and an Impact Acceleration Account Partnership Development Award), the Biological and Biotechnological Research Council (BBSRC) and Johnson Matthey. SE and MF were supported by postdoctoral Marie-Curie fellowships.
External DOI: https://doi.org/10.1073/pnas.1606927113
This record's URL: https://www.repository.cam.ac.uk/handle/1810/261651