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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.
Highlights
Ultrahigh-throughput screening of enzymes with droplet microfluidics has established itself as a valuable tool for interrogation of libraries with > 107 members
The compounds 4, 7 and 10 differ from one another with respect to the steric demands around the hydroxy group of the alcohol dehydrogenases (ADHs) recognition site, so that a range of molecular recognition features is covered by the collection: as an example of a primary alcohol substrate H-HTPS 4 was generated; Me-HTPS 7 and Phe-HTPS 10 were produced as examples for secondary alcohols containing either a small methyl (7) or a sterically more demanding phenyl group (10), respectively
The establishment of the first single-step fluorogenic assay for ADHs amenable to ultrahigh throughput in droplets will provide ready access to highly desirable biocatalysts for the dehydrogenation of alcohols and the reverse reaction, ketoreduction
Summary
Compartments to pL volumes, droplet microfluidics drastically increases throughput while reducing reagent consumption by 6–8 orders of magnitude.[1] These advances improve the success rate of screening campaigns by enabling the testing of larger sample numbers.[2] Droplet screening has been used for the directed evolution of existing biocatalysts[3] and for the functional identification of novel biocatalysts from metagenomic DNA libraries.[4] The latter holds enormous potential, because the identification of new catalysts enables the functional annotation of previously unknown sequence space,[5] thereby complementing the continuously increasing amount of available – yet often incompletely or incorrectly annotated – sequence data.[6]. In order to make droplet microfluidics a universal discovery tool, several challenges remain: (i) Expanding the scope of reactions that can be assayed. The currently available droplet assays do not cover the many different chemistries encountered in natural repertoires. The UV/Vis-active reaction product has to remain contained in the droplet in which it was generated to quantitatively label the library member for selection.
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