Abstract
Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these man-made compartments can be identified readily by fluorescence-activated sorting at rates >107 GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components.
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References
Retterer, S. T. & Simpson, M. L. Microscale and nanoscale compartments for biotechnology. Curr. Opin. Biotechnol. 23, 522–528 (2012).
Walde, P. Building artificial cells and protocell models: experimental approaches with lipid vesicles. Bioessays 32, 296–303 (2010).
Decher, G. Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science 277, 1232–1237 (1997).
Donath, E., Sukhorukov, G. B., Caruso, F., Davis, S. A. & Mohwald, H. Novel hollow polymer shells by colloid-templated assembly of polyelectrolytes. Angew. Chem. Int. Ed. 37, 2202–2205 (1998).
Stadler, B. et al. Polymer hydrogel capsules: en route toward synthetic cellular systems. Nanoscale 1, 68–73 (2009).
Tong, W. J., Song, X. X. & Gao, C. Y. Layer-by-layer assembly of microcapsules and their biomedical applications. Chem. Soc. Rev. 41, 6103–6124 (2012).
Peyratout, C. S. & Dahne, L. Tailor-made polyelectrolyte microcapsules: from multilayers to smart containers. Angew. Chem. Int. Ed. 43, 3762–3783 (2004).
Mak, W. C., Cheung, K. Y. & Trau, D. Diffusion controlled and temperature stable microcapsule reaction compartments for high-throughput microcapsule-PCR. Adv. Funct. Mater. 18, 2930–2937 (2008).
Price, A. D., Zelikin, A. N., Wark, K. L. & Caruso, F. A biomolecular ‘ship-in-a-bottle’: continuous RNA synthesis within hollow polymer hydrogel assemblies. Adv. Mater. 22, 720–723 (2010).
Baumler, H. & Georgieva, R. Coupled enzyme reactions in multicompartment microparticles. Biomacromolecules 11, 1480–1487 (2010).
Pescador, P., Toca-Herrera, J. L., Donath, E. & Katakis, I. Efficiency of a bienzyme sequential reaction system immobilized on polyelectrolyte multilayer-coated colloids. Langmuir 24, 14108–14114 (2008).
Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nature Chem. Biol. 5, 567–573 (2009).
Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).
Schaerli, Y. & Hollfelder, F. The potential of microfluidic water-in-oil droplets in experimental biology. Mol. Biosyst. 5, 1392–1404 (2009).
Leemhuis, H., Stein, V., Griffiths, A. D. & Hollfelder, F. New genotype–phenotype linkages for directed evolution of functional proteins. Curr. Opin. Struct. Biol. 15, 472–478 (2005).
Lin, H. & Cornish, V. W. Screening and selection methods for large-scale analysis of protein function. Angew. Chem. Int. Ed. 41, 4402–4425 (2002).
Jäckel, C. & Hilvert, D. Biocatalysts by evolution. Curr. Opin. Biotechnol. 21, 753–759 (2010).
Tracewell, C. A. & Arnold, F. H. Directed enzyme evolution: climbing fitness peaks one amino acid at a time. Curr. Opin. Chem. Biol. 13, 3–9 (2009).
Romero, P. A. & Arnold, F. H. Exploring protein fitness landscapes by directed evolution. Nature Rev. Mol. Cell Biol. 10, 866–876 (2009).
Yang, Y., Baker, J. A. & Ward, J. R. Decontamination of chemical warfare agents. Chem. Rev. 92, 1729–1743 (1992).
Morales-Rojas, H. & Moss, R. A. Phosphorolytic reactivity of o-iodosylcarboxylates and related nucleophiles. Chem. Rev. 102, 2497–2521 (2002).
Tawfik, D. S. & Griffiths, A. D. Man-made cell-like compartments for molecular evolution. Nature Biotechnol. 16, 652–656 (1998).
Miller, O. J. et al. Directed evolution by in vitro compartmentalization. Nature Methods 3, 561–570 (2006).
Huebner, A. et al. Quantitative detection of protein expression in single cells using droplet microfluidics. Chem. Commun. 1218–1220 (2007).
Kintses, B. et al. Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chem. Biol. 19, 1001–1009 (2012).
Weaver, J. C., Bliss, J. G., Powell, K. T., Harrison, G. I. & Williams, G. B. Rapid clonal growth measurements at the single-cell level – gel microdroplets and flow-cytometry. Bio-Technology 9, 873–877 (1991).
Dejugnat, C. & Sukhorukov, G. B. PH-responsive properties of hollow polyelectrolyte microcapsules templated on various cores. Langmuir 20, 7265–7269 (2004).
Tokuriki, N. et al. Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme. Nature Commun. 3, 1257 (2012).
Dicosimo, R., McAuliffe, J., Poulose, A. J. & Bohlmann, G. Industrial use of immobilized enzymes. Chem. Soc. Rev. 42, 6437–6474 (2013).
Theberge, A. B. et al. Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology. Angew. Chem. Int. Ed. 49, 5846–5868 (2010).
Ahn, K. et al. Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices. Appl. Phys. Lett. 88, 024104 (2006).
Baret, J. C. et al. Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab Chip 9, 1850–1858 (2009).
Agresti, J. J. et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc. Natl Acad. Sci. USA 107, 4004–4009 (2010).
Hawley, T. S. & Hawley, R. G. Flow Cytometry Protocols (Humana Press, 2004).
Fernandez-Alvaro, E. et al. A combination of in vivo selection and cell sorting for the identification of enantioselective biocatalysts. Angew. Chem. Int. Ed. 50, 8584–8587 (2011).
Yang, G. & Withers, S. G. Ultrahigh-throughput FACS-based screening for directed enzyme evolution. Chembiochem 10, 2704–2715 (2009).
Becker, S. et al. Single-cell high-throughput screening to identify enantioselective hydrolytic enzymes. Angew. Chem. Int. Ed. 47, 5085–5088 (2008).
Skirtach, A. G., Yashchenok, A. M. & Mohwald, H. Encapsulation, release and applications of LbL polyelectrolyte multilayer capsules. Chem. Commun. 47, 12736–12746 (2011).
Moya, S., Sukhorukov, G. B., Auch, M., Donath, E. & Mohwald, H. Microencapsulation of organic solvents in polyelectrolyte multilayer micrometer-sized shells. J. Colloid Interface Sci. 216, 297–302 (1999).
Schoffelen, S. & van Hest, J. C. Chemical approaches for the construction of multi-enzyme reaction systems. Curr. Opin. Struct. Biol. 23, 613–621 (2013).
Liu, Y. et al. Biomimetic enzyme nanocomplexes and their use as antidotes and preventive measures for alcohol intoxication. Nature Nanotech. 8, 187–192 (2013).
Oroz-Guinea, I. & Garcia-Junceda, E. Enzyme catalysed tandem reactions. Curr. Opin. Chem. Biol. 17, 236–249 (2013).
Garcia-Junceda, E. Multi-Step Enzyme Catalysis: Biotransformations and Chemoenzymatic Synthesis (VCH, 2008).
Ricca, E., Brucher, B. & Schrittwieser, J. H. Multi-enzymatic cascade reactions: overview and perspectives. Adv. Synth. Catal. 353, 2239–2262 (2011).
Zhang, Y-H. P. Simpler is better: high-yield and potential low-cost biofuels production through cell-free synthetic pathway biotransformation (SyPaB). ACS Catal. 1, 998–1009 (2011).
Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).
Pirie, C. M., De Mey, M., Jones Prather, K. L. & Ajikumar, P. K. Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis. ACS Chem. Biol. 8, 662–672 (2013).
Zhao, H. Synthetic Biology (Academic Press, 2013).
Devenish, S. R., Kaltenbach, M., Fischlechner, M. & Hollfelder, F. in Protein Nanotechnology: Protocols, Instrumentation and Applications Vol. 996 (ed. Gerrard, J.) 269–286 (2013).
Acknowledgements
This research was funded by the Engineering and Physical Sciences Research Council, by European Union Marie-Curie fellowships (to M.F. and M.F.M.) and by fellowships from the Schering Foundation, the Cambridge Overseas Trust and Trinity Hall (to Y.S.). F.H. is a European Research Council Starting Investigator. We thank N. Miller for help with FACS and several colleagues for helpful discussions and comments on the manuscript. We thank RainDance Technologies (Lexington, USA) for a sample of the EA-surfactant.
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M.F. developed the concept and designed, conducted and analysed the experiments. Y.S. developed the concept and designed, conducted and analysed preliminary experiments. M.F., Y.S., M.F.M. and F.H. wrote the manuscript. M.M. and S.P. synthesized enzyme substrates. F.H. and C.A. directed the research.
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Fischlechner, M., Schaerli, Y., Mohamed, M. et al. Evolution of enzyme catalysts caged in biomimetic gel-shell beads. Nature Chem 6, 791–796 (2014). https://doi.org/10.1038/nchem.1996
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DOI: https://doi.org/10.1038/nchem.1996
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