Microfluidic droplets as tools for high-throughput biology: Enzyme evolution, recruitment and discovery based on catalytic promiscuity
September 24-28, 2017
‘Promiscuous’ enzymes possess additional activities in addition to their native ones, challenging the textbook adage “one enzyme – one activity”. The observation of strong promiscuous activities in the alkaline phosphatase (AP) superfamily - where one active site can catalyse up to six chemically distinct hydrolytic reactions with promiscuous second order rate accelerations between 10e9 and 10e17 - suggests that even broadly promiscuous catalysis can be rather efficient. To efficiently explore the interconversion of promiscuous enzyme, we use picoliter water-in–oil emulsion droplets produced in microfluidic devices as high-throughput screening reactors. We present new workflows that allow screening of >10e6 clones and permit successful selections from single protein and metagenomic libraries, where lower throughput approaches have failed.
• Ultrahigh-throughput Discovery of Promiscuous Enzymes by Picodroplet Functional Metagenomics. Colin, P.-Y.; Kintses, B.; Gielen, F.; Miton, C. M.; Mohamed, M. F.; Fischer, G.; Hyvonen, M.; Morgavi, D. P.; Janssen, D. B.; Hollfelder, F., Nature Communications 2015, 6:10008. doi: 10.1038/ncomms10008.
• Enzyme engineering in biomimetic compartments. Colin, P. Y, Zinchenko, A & Hollfelder, F. Curr Opin Struct Biol. 2015, 33:42-51(doi: 10.1016/j.sbi.2015.06.001)
• Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Fischlechner, M.; Schaerli, Y.; Mohamed, M. F.; Patil, S.; Abell, C.; Hollfelder, F., Nat Chem 2014, 6 (9), 791-6
• Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F. Proc Natl Acad Sci U S A. 2016 Nov 22;113(47):E7383-E7389.
Florian Hollfelder, "Microfluidic droplets as tools for high-throughput biology: Enzyme evolution, recruitment and discovery based on catalytic promiscuity" in "Enzyme Engineering XXIV", Pierre Monsan, Toulouse White Biotechnology, France Magali Remaud-Simeon, LISBP-INSA, University of Toulouse, France Eds, ECI Symposium Series, (2017). https://dc.engconfintl.org/enzyme_xxiv/149