期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：47
页码：E7383-E7389
DOI：10.1073/pnas.1606927113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceDirected enzyme evolution is a powerful approach for discovering new catalysts with applications in green chemistry and elsewhere. However, "hits" in sequence space are rare: If too few members of a library are examined, the chances of success of a campaign are limited. Ultrahigh-throughput screening in emulsion droplets has dramatically increased the odds but requires a fluorescent reaction product that triggers selection of hits. We now introduce an absorbance-based microfluidic droplet sorter that broadens the scope of assays to those producing UV/Vis-active chromophores and demonstrate its usefulness by evolving a dehydrogenase based on the screening of half a million library members. Making ultrahigh-throughput screening possible for previously inaccessible reactions enables much wider use of microfluidic droplet sorters for laboratory evolution. 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 {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.
