Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 113, Issue 47, Pages E7383-E7389Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1606927113
Keywords
protein engineering; directed evolution; microfluidics; ultrahigh-throughput; emulsion droplets
Categories
Funding
- Engineering and Physical Sciences Research Council
- Biological and Biotechnological Research Council (BBSRC)
- Johnson Matthey
- postdoctoral Marie-Curie fellowships
- BBSRC [BB/L002469/1, BB/K013629/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/L002469/1, BB/K013629/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [1406246] Funding Source: researchfish
Ask authors/readers for more resources
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 mu 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 similar to 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; k(cat) increased > 2.7-fold), soluble protein expression levels (up 60%), and thermostability (T-m, 12 degrees 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available