Chemical Biology: From Mechanism to Droplet Microfluidics (and Back)
Nature has evolved the most amazing functional biomolecules and we want to know how they work. Enzymes are the all-purpose catalysts that make the Chemistry of Life run smoothly and efficiently, under the mildest, ‘greenest’ conditions – and protein binders are involved in governing many biological processes.
The Hollfelder group is interested in gaining a fundamental understanding of the principles responsible for molecular recognition processes in chemistry and biology. We probe whether these principles enable us to describe, manipulate and ultimately make functional molecules. Using an eclectic mix of techniques we extend the mechanistic lessons learned to potential applications in biotechnology, synthetic chemistry and medicine.
Research objectives
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How does protein evolution work and where do functional proteins come from?
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Can we explain biological catalysis and molecular recognition in chemical and biophysical terms?
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Where does extreme miniaturisation of experiments (into picoliter droplets made in microfluidic devices) help to answer chemical and biological questions?
Key publications
Gantz, M.; Neun, S.; Medcalf, E. J.; van Vliet, L. D.; Hollfelder, F. Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments. Chem Rev 2023, 123, 5571-5611. 0.1021/2Facs.chemrev.2c00910
Salmen, F.; De Jonghe, J.; Kaminski, T. S.; Alemany, A.; Parada, G. E.; Verity-Legg, J.; Yanagida, A.; Kohler, T. N.; Battich, N.; van den Brekel, F.; Ellermann, A. L.; Arias, A. M.; Nichols, J.; Hemberg, M.; Hollfelder, F. *; van Oudenaarden, A.* (2022). High-throughput total RNA sequencing in single cells using VASA-seq. Nat Biotechnol, 10.1038/s41587-022-01361-8
Gantz, M.; Aleku, G. A.; Hollfelder, F. (2022). Ultrahigh-throughput screening in microfluidic droplets: a faster route to new enzymes. Trends Biochem Sci, 47, 451-452. 10.1016/j.tibs.2021.11.001
Fryer, T.; Rogers, J. D.; Mellor, C.; Kohler, T. N.; Miner, R.; Hollfelder, F. (2022). Gigavalent Display of Proteins on Monodisperse Polyacrylamide Hydrogels as a Versatile Modular Platform for Functional Assays and Protein Engineering. ACS Cent Sci 2022, 10.1021/acscentsci.2c00576.
Lindenburg, L.; Hollfelder, F. (2021) "NAD-display": Ultrahigh-Throughput in Vitro Screening of NAD(H) Dehydrogenases Using Bead Display and Flow Cytometry. Angew Chem Int Ed Engl, 60, 9015-9021. 10.1002/anie.202013486
Knyphausen, P.; Lindenburg, L.; Hollfelder, F. (2021) Error-Free Synthetic DNA by Molecular Dictation. Trends Biotechnol., 39: 861-865.
Zurek, P. J.; Knyphausen, P.; Neufeld, K.; Pushpanath, A.; Hollfelder, F. (2020) UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution. Nat Commun., 11, 6023. 10.1038/s41467-020-19687-9
Tauzin, A. S.; Pereira, M. R.; Van Vliet, L. D.; Colin, P. Y.; Laville, E.; Esque, J.; Laguerre, S.; Henrissat, B.; Terrapon, N.; Lombard, V.; Leclerc, M.; Dore, J.; Hollfelder, F.; Potocki-Veronese, G (2020). Investigating host-microbiome interactions by droplet based microfluidics. Microbiome, 8, 141. 10.1186/s40168-020-00911-z
Skamaki, K.; Emond, S.; Chodorge, M.; Andrews, J.; Rees, D. G.; Cannon, D.; Popovic, B.; Buchanan, A.; Minter, R. R.; Hollfelder, F (2020). In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region. PNAS, 117, 27307-27318. 10.1073/pnas.2002954117
Emond, S.; Petek, M.; Kay, E. J.; Heames, B.; Devenish, S. R. A.; Tokuriki, N.; Hollfelder, F. (2020) Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis. Nat. Commun., 11: 3469. 10.1038/s41467-020-17061-3
van Loo B, Bayer CD, Fischer G, Jonas S, Valkov E, Mohamed MF, Vorobieva A, Dutruel C, Hyvönen M, Hollfelder F (2019). Balancing specificity and promiscuity in enzyme evolution: Multidimensional activity transitions in the alkaline phosphatase superfamily. J. Am. Chem. Soc., 141(1):370-387. doi: 10.1021/jacs.8b10290
Miton CM, Jonas S, Fischer G, Duarte F, Mohamed MF, van Loo B, Kintses B, Kamerlin SCL, Tokuriki N, Hyvönen M, Hollfelder F (2018). Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset. Proc. Natl. Acad. Sci. U.S.A., 115(31):E7293-E7302. doi: 10.1073/pnas.1607817115
Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F (2016). Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Proc. Natl. Acad. Sci. U.S.A., 113(47):E7383-E7389. doi: 10.1073/pnas.1606927113
Colin PY, Kintses B, Gielen F, Miton CM, Fischer G, Mohamed MF, Hyvönen M, Morgavi DP, Janssen DB, Hollfelder F (2015). Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics. Nat. Commun., 6:10008. doi: 10.1038/ncomms10008
Fischlechner M, Schaerli Y, Mohamed MF, Patil S, Abell C, Hollfelder F (2014). Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nat. Chem., 6(9):791-796. doi: 10.1038/nchem.1996
Microfluidic droplets
Ultra-high-throughput screening and sequence space