Ultrahigh-Throughput Detection of Enzymatic Alcohol Dehydrogenase Activity in Microfluidic Droplets with a Direct Fluorogenic Assay.


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Authors
Klaus, Miriam 
Zurek, Paul Jannis 
Kaminski, Tomasz S 
Pushpanath, Ahir 
Neufeld, Katharina 
Abstract

The exploration of large DNA libraries of metagenomic or synthetic origin is greatly facilitated by ultrahigh-throughput assays that use monodisperse water-in-oil emulsion droplets as sequestered reaction compartments. Millions of samples can be generated and analysed in microfluidic devices at kHz speeds, requiring only micrograms of reagents. The scope of this powerful platform for the discovery of new sequence space is, however, hampered by the limited availability of assay substrates, restricting the functions and reaction types that can be investigated. Here, we broaden the scope of detectable biochemical transformations in droplet microfluidics by introducing the first fluorogenic assay for alcohol dehydrogenases (ADHs) in this format. We have synthesized substrates that release a pyranine fluorophore (8-hydroxy-1,3,6-pyrenetrisulfonic acid, HPTS) when enzymatic turnover occurs. Pyranine is well retained in droplets for >6 weeks (i. e. 14-times longer than fluorescein), avoiding product leakage and ensuring excellent assay sensitivity. Product concentrations as low as 100 nM were successfully detected, corresponding to less than one turnover per enzyme molecule on average. The potential of our substrate design was demonstrated by efficient recovery of a bona fide ADH with an >800-fold enrichment. The repertoire of droplet screening is enlarged by this sensitive and direct fluorogenic assay to identify dehydrogenases for biocatalytic applications.

Description
Keywords
alcohol dehydrogenase, directed evolution, droplet microfluidics, functional metagenomics, ultrahigh-throughput screening, Alcohol Dehydrogenase, Fluorescent Dyes, High-Throughput Screening Assays, Lab-On-A-Chip Devices, Molecular Structure, Particle Size
Journal Title
Chembiochem
Conference Name
Journal ISSN
1439-4227
1439-7633
Volume Title
Publisher
Wiley
Sponsorship
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (722610)
European Commission Horizon 2020 (H2020) ERC (695664)
Engineering and Physical Sciences Research Council (EP/H046593/1)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (750772)
ERC, H2020 Marie-Curie