Where do we find new enzymes? – Rules and tools
September 15-19, 2019
Enzymes for a variety of useful applications are required, but currently not known. Functional metagenomics and directed evolution promise access to such new catalysts, but the chances of finding them is low. Therefore high-throughput technologies are crucial to fight the odds. We have implemented workflows that allow screening of >10e6 clones and permit successful selections in picoliter water-in–oil emulsion droplets produced in microfluidic devices. While potentially faster, the vastness of sequence space (and the scarcity of ‘solutions’ in it) require strategies and rules for the identification and interconversion of enzymes. In this context ‘promiscuous’ enzymes have prominent roles: these catalysts possess additional activities in addition to their native ones, challenging the textbook adage “one enzyme – one activity”, yet facilitating ‘on-the-spot’ utility for newly encountered challenges and also making future evolutionary transitions easier. Their molecular and mechanistic understanding may help us thus to chart routes towards new catalysts. Recent results of directed evolution, mechanistic investigation by kinetic and structural analysis and technology development are discussed in this context.
• van Loo, B.; Bayer, C. D.; Fischer, G.; Jonas, S.; Valkov, E.; Mohamed, M. F.; Vorobieva, A.; Dutruel, C.; Hyvonen, M.; Hollfelder, F., Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily. J Am Chem Soc 2019, 141 (1), 370-387.
• van Loo, B.; Berry, R.; Boonyuen, U.; Mohamed, M. F.; Golicnik, M.; Hengge, A. C.; Hollfelder, F., Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase. Biochemistry 2019, 58 (10), 1363-1378.
• Miton, C. M.; Jonas, S.; Fischer, G.; Duarte, F.; Mohamed, M. F.; van Loo, B.; Kintses, B.; Kamerlin, S. C. L.; Tokuriki, N.; Hyvonen, M.; Hollfelder, F., Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset. Proc Natl Acad Sci U S A 2018, 115 (31), E7293-E7302.
• 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;113(47):E7383-E7389.
• 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., Ultrahigh-throughput Discovery of Promiscuous Enzymes by Picodroplet Functional Metagenomics. Nature Communications 2015, 6:10008. doi: 10.1038/ncomms10008.
• Enzyme engineering in biomimetic compartments. Curr Opin Struct Biol. 2015, 33:42-51(doi: 10.1016/j.sbi.2015.06.001)
• Fischlechner, M.; Schaerli, Y.; Mohamed, M. F.; Patil, S.; Abell, C.; Hollfelder, F., Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat Chem 2014, 6 (9), 791-6
Florian Hollfelder, "Where do we find new enzymes? – Rules and tools" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/139