Abstract
Enzymes are responsible for many chemical reactions which support life and are used in numerous industries. Laboratory evolution experiments can mimic the Darwinian process of mutation and selection pressure, yielding newly evolved catalysts. Recently developed single-cell microencapsulation techniques enable the screening of ultralarge enzyme-mutant pools, greatly increasing the odds of isolating improved catalysts. The encapsulation process uses droplet microfluidics, a groundbreaking technology for the miniaturization and automation of biochemical assays which has already demonstrated its effectiveness at evolving enzymes and identifying ultrarare catalysts from the environment. This chapter reviews how large-scale single-cell assays provide an efficient route toward the identification of biocatalysts with novel or improved function. Single-cell microencapsulation enables retention of genotype (gene sequence) and phenotype (enzyme function) within a stable microcompartment. In practice, single-gene mutants expressed by single cells with their corresponding enzyme are encapsulated into monodisperse water-in-oil microdroplets where the assay takes place. This enables the screening of ultralarge 108 members libraries in a day with a concomitant reduction of 1000-fold in assay-reagent costs and a 1000-fold increase in throughput compared to conventional tools. This technology has already been widely adopted for the accelerated discovery and evolution of biocatalysts used in many application domains from the decontamination of polluted environments, bioenergy production, to the degradation of plastics and the discovery of antimicrobials.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abate AR, Hung T, Mary P, Agresti JJ, Weitz DA (2010) High-throughput injection with microfluidics using picoinjectors. Proc Natl Acad Sci U S A 107(45):19163–19166. https://doi.org/10.1073/pnas.1006888107
Agresti JJ, Antipov E, Abate AR, Ahn K, Rowat AC, Baret JC, Marquez M, Klibanov AM, Griffiths AD, Weitz DA (2010) Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc Natl Acad Sci U S A 107(9):4004–4009. https://doi.org/10.1073/pnas.0910781107
Beneyton T, Wijaya IPM, Postros P, Najah M, Leblond P, Couvent A, Mayot E, Griffiths AD, Drevelle A (2016) High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics. Sci Rep 6:27223. https://doi.org/10.1038/srep27223
Beneyton T, Thomas S, Griffiths AD, Nicaud JM, Drevelle A, Rossignol T (2017) Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica. Microb Cell Fact 16:18. https://doi.org/10.1186/s12934-017-0629-5
Chen KQ, Arnold FH (1993) Tuning the activity of an enzyme for unsual environments - sequential random mutagenesis of subtilisin-E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A 90(12):5618–5622. https://doi.org/10.1073/pnas.90.12.5618
Cherf GM, Cochran JR (2015) Applications of yeast surface display for protein engineering. In: Liu B (ed) Yeast surface display: methods, protocols, and applications, Methods in molecular biology, vol 1319. Humana Press Inc, Totowa, pp 155–175. https://doi.org/10.1007/978-1-4939-2748-7_8
Colin PY, Kintses B, Gielen F, Miton CM, Fischer G, Mohamed MF, Hyvonen M, Morgavi DP, Janssen DB, Hollfelder F (2015) Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics. Nat Commun 6:10008. https://doi.org/10.1038/ncomms10008
Collins DJ, Neild A, deMello A, Liu AQ, Ai Y (2015) The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. Lab Chip 15(17):3439–3459. https://doi.org/10.1039/c5lc00614g
Courtois F, Olguin LF, Whyte G, Bratton D, Huck WTS, Abell C, Hollfelder F (2008) An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets. ChemBioChem 9(3):439–446. https://doi.org/10.1002/cbic.200700536
Cuesta SM, Rahman SA, Furnham N, Thornton JM (2015) The classification and evolution of enzyme function. Biophys J 109(6):1082–1086. https://doi.org/10.1016/j.bpj.2015.04.020
Daniel R (2005) The metagenomics of soil. Nat Rev Microbiol 3(6):470–478. https://doi.org/10.1038/nrmicro1160
Dorr M, Fibinger MPC, Last D, Schmidt S, Santos-Aberturas J, Bottcher D, Hummel A, Vickers C, Voss M, Bornscheuer UT (2016) Fully automatized high-throughput enzyme library screening using a robotic platform. Biotechnol Bioeng 113(7):1421–1432. https://doi.org/10.1002/bit.25925
Fallah-Araghi A, Baret JC, Ryckelynck M, Griffiths AD (2012) A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution. Lab Chip 12(5):882–891. https://doi.org/10.1039/c2lc21035e
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. https://doi.org/10.1038/nchem.1996
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 113(47):E7383–E7389. https://doi.org/10.1073/pnas.1606927113
Gielen F, Butz M, Rees EJ, Erdelyi M, Moschetti T, Hyvonen M, Edel JB, Kaminski CF, Hollfelder F (2017) Quantitative affinity determination by fluorescence anisotropy measurements of individual nanoliter droplets. Anal Chem 89(2):1092–1101. https://doi.org/10.1021/acs.analchem.6b02528
Gielen F, Colin P-Y, Mair P, Hollfelder F (2018) Ultrahigh-throughput screening of single-cell lysates for directed evolution and functional metagenomics. Methods Mol Biol (Clifton, NJ) 1685:297–309. https://doi.org/10.1007/978-1-4939-7366-8_18
Hasan S, Geissler D, Wink K, Hagen A, Heiland JJ, Belder D (2019) Fluorescence lifetime-activated droplet sorting in microfluidic chip systems. Lab Chip 19(3):403–409. https://doi.org/10.1039/c8lc01278d
Holland-Moritz DA, Wismer M, Mann B, Farasat I, Guetschow E, Sun S, Moore J, Kennedy R, Mangion I, Welch CJ, Devine P (2019) Mass activated droplet sorting (MADS) enables high throughput screening of enzymatic reactions at nanoliter scale. Angew Chem Int Ed Engl. https://doi.org/10.1002/anie.201913203
Holland-Moritz DA, Wismer MK, Mann BF, Farasat I, Devine P, Guetschow ED, Mangion I, Welch CJ, Moore JC, Sun SW, Kennedy RT (2020) Mass activated droplet sorting (MADS) enables high-throughput screening of enzymatic reactions at nanoliter scale. Angew Chem-Int Edit:59, 4470. https://doi.org/10.1002/anie.201913203
Huebner A, Olguin LF, Bratton D, Whyte G, Huck WTS, de Mello AJ, Edel JB, Abell C, Hollfelder F (2008) Development of quantitative cell-based enzyme assays in microdroplets. Anal Chem 80(10):3890–3896. https://doi.org/10.1021/ac800338z
Hung S-T, Mukherjee S, Jimenez R (2020) Enrichment of rare events using a multi-parameter high throughput microfluidic droplet sorter. Lab Chip 20:834. https://doi.org/10.1039/c9lc00790c
Jiang LG, Zeng Y, Zhou HB, Qu JAY, Yao SH (2012) Visualizing millisecond chaotic mixing dynamics in microdroplets: a direct comparison of experiment and simulation. Biomicrofluidics 6(1):12. https://doi.org/10.1063/1.3673254
Karamitros CS, Morvan M, Vigne A, Lim J, Gruner PJ, Beneyton T, Vrignon J, Konrad M, Baret JC (2020) Bacterial expression systems for enzymatic activity in droplet-based microfluidics. Anal Chem. https://doi.org/10.1021/acs.analchem.9b04969
Kemna EW, Schoeman RM, Wolbers F, Vermes I, Weitz DA, Van Den Berg A (2012) High-yield cell ordering and deterministic cell-in-droplet encapsulation using Dean flow in a curved microchannel. Lab Chip 12(16):2881–2887
Khersonsky O, Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. In: Kornberg RD, Raetz CRH, Rothman JE, Thorner JW (eds) Annual review of biochemistry, Annual review of biochemistry, vol 79. Annual Reviews, Palo Alto, pp 471–505. https://doi.org/10.1146/annurev-biochem-030409-143718
Kintses B, Hein C, Mohamed MF, Fischlechner M, Courtois F, Leine C, Hollfelder F (2012) Picoliter cell lysate assays in microfluidic droplet compartments for directed enzyme evolution. Chem Biol 19(8):1001–1009. https://doi.org/10.1016/j.chembiol.2012.06.009
Koster S, Angile FE, Duan H, Agresti JJ, Wintner A, Schmitz C, Rowat AC, Merten CA, Pisignano D, Griffiths AD, Weitz DA (2008) Drop-based microfluidic devices for encapsulation of single cells. Lab Chip 8(7):1110–1115. https://doi.org/10.1039/b802941e
Lagus TP, Edd JF (2013) High-throughput co-encapsulation of self-ordered cell trains: cell pair interactions in microdroplets. RSC Adv 3(43):20512–20522. https://doi.org/10.1039/c3ra43624a
Larsen AC, Dunn MR, Hatch A, Sau SP, Youngbull C, Chaput JC (2016) A general strategy for expanding polymerase function by droplet microfluidics. Nat Commun 7(9). https://doi.org/10.1038/ncomms11235
Liu X, Painter RE, Enesa K, Holmes D, Whyte G, Garlisi CG, Monsma FJ, Rehak M, Craig FF, Smith CA (2016) High-throughput screening of antibiotic-resistant bacteria in picodroplets. Lab Chip 16(9):1636–1643. https://doi.org/10.1039/c6lc00180g
MacConnell AB, Price AK, Paegel BM (2017) An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening. ACS Comb Sci 19:181–192. https://doi.org/10.1021/acscombsci.6b00192
Macosko EZ, et al. (2015) Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161:1202–1214. https://doi.org/10.1016/j.cell.2015.05.002
Mahler L, Tovar M, Weber T, Brandes S, Rudolph MM, Ehgartner J, Mayr T, Figge MT, Roth M, Zang E (2015) Enhanced and homogeneous oxygen availability during incubation of microfluidic droplets. RSC Adv 5(123):101871–101878. https://doi.org/10.1039/c5ra20118g
Mair P, Gielen F, Hollfelder F (2017) Exploring sequence space in search of functional enzymes using microfluidic droplets. Curr Opin Chem Biol 37:137–144. https://doi.org/10.1016/j.cbpa.2017.02.018
Mazutis L, Gilbert J, Ung WL, Weitz DA, Griffiths AD, Heyman JA (2013) Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc 8(5):870–891. https://doi.org/10.1038/nprot.2013.046
Miller OJ, Bernath K, Agresti JJ, Amitai G, Kelly BT, Mastrobattista E, Taly V, Magdassi S, Tawfik DS, Griffiths AD (2006) Directed evolution by in vitro compartmentalization. Nat Methods 3(7):561–570. https://doi.org/10.1038/nmeth897
Najah M, Calbrix R, Mahendra-Wijaya IP, Beneyton T, Griffiths AD, Drevelle A (2014) Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms. Chem Biol 21(12):1722–1732. https://doi.org/10.1016/j.chembiol.2014.10.020
Neun S, Kaminski TS, Hollfelder F (2019) Single-cell activity screening in microfluidic droplets. Methods Enzymol 628:95–112. https://doi.org/10.1016/bs.mie.2019.07.009
Neylon C (2004) Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res 32(4):1448–1459. https://doi.org/10.1093/nar/gkh315
Obexer R, Godina A, Garrabou X, Mittl PRE, Baker D, Griffiths AD, Hilvert D (2017) Emergence of a catalytic tetrad during evolution of a highly active artificial aldolase. Nat Chem 9(1):50–56. https://doi.org/10.1038/nchem.2596
Olsen MJ, Stephens D, Griffiths D, Daugherty P, Georgiou G, Iverson BL (2000) Function-based isolation of novel enzymes from a large library. Nat Biotechnol 18(10):1071–1074. https://doi.org/10.1038/80267
Ostafe R, Prodanovic R, Ung WL, Weitz DA, Fischer R (2014) A high-throughput cellulase screening system based on droplet microfluidics. Biomicrofluidics 8(4):4. https://doi.org/10.1063/1.4886771
Pandya C, Farelli JD, Dunaway-Mariano D, Allen KN (2014) Enzyme promiscuity: engine of evolutionary innovation. J Biol Chem 289(44):30229–30236. https://doi.org/10.1074/jbc.R114.572990
Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10(12):866–876. https://doi.org/10.1038/nrm2805
Romero PA, Tran TM, Abate AR (2015) Dissecting enzyme function with microfluidic-based deep mutational scanning. Proc Natl Acad Sci U S A 112(23):7159–7164. https://doi.org/10.1073/pnas.1422285112
Schmitz JE, Daniel A, Collin M, Schuch R, Fischetti VA (2008) Rapid DNA library construction for functional genomic and metagenomic screening. Appl Environ Microbiol 74(5):1649–1652. https://doi.org/10.1128/aem.01864-07
Sciambi A, Abate AR (2015) Accurate microfluidic sorting of droplets at 30 kHz. Lab Chip 15(1):47–51. https://doi.org/10.1039/c4lc01194e
Sjostrom SL, Bai YP, Huang MT, Liu ZH, Nielsen J, Joensson HN, Svahn HA (2014) High-throughput screening for industrial enzyme production hosts by droplet microfluidics. Lab Chip 14(4):806–813. https://doi.org/10.1039/c3lc51202a
Su X, Schmitz G, Zhang M, Mackie RI, Cann IK (2012) Heterologous gene expression in filamentous fungi. Adv Appl Microbiol 81:1–61. https://doi.org/10.1016/B978-0-12-394382-8.00001-0
Tawfik DS, Griffiths AD (1998) Man-made cell-like compartments for molecular evolution. Nat Biotechnol 16(7):652–656. https://doi.org/10.1038/nbt0798-652
Tovar M, Hengoju S, Weber T, Mahler L, Choudhary M, Becker T, Roth M (2019) One sensor for multiple colors: fluorescence analysis of microdroplets in microbiological screenings by frequency-division multiplexing. Anal Chem 91(4):3055–3061. https://doi.org/10.1021/acs.analchem.8b05451
Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U (2011) A flow cytometry-based screening system for directed evolution of proteases. J Biomol Screen 16(3):285–294. https://doi.org/10.1177/1087057110396361
Uchiyama T, Miyazaki K (2009) Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr Opin Biotechnol 20(6):616–622. https://doi.org/10.1016/j.copbio.2009.09.010
van Loo B, Heberlein M, Mair P, Zinchenko A, Schuurmann J, Eenink BDG, Holstein JM, Dilkaute C, Jose J, Hollfelder F, Bornberg-Bauer E (2019) High-throughput, lysis-free screening for sulfatase activity using escherichia coli autodisplay in microdroplets. ACS Synth Biol 8(12):2690–2700. https://doi.org/10.1021/acssynbio.9b00274
van Vliet LD, Colin PY, Hollfelder F (2015) Bioinspired genotype-phenotype linkages: mimicking cellular compartmentalization for the engineering of functional proteins. Interface Focus 5(4). https://doi.org/10.1098/rsfs.2015.0035
Wang BL, Ghaderi A, Zhou H, Agresti J, Weitz DA, Fink GR, Stephanopoulos G (2014) Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption. Nat Biotechnol 32(5):473–U194. https://doi.org/10.1038/nbt.2857
Wang X, Ren L, Su Y, Ji Y, Liu Y, Li C, Li X, Zhang Y, Wang W, Hu Q, Han D, Xu J, Ma B (2017a) Raman-activated droplet sorting (RADS) for label-free high-throughput screening of microalgal single-cells. Anal Chem 89(22):12569–12577. https://doi.org/10.1021/acs.analchem.7b03884
Wang XX, Ren LH, Su YT, Ji YT, Liu YP, Li CY, Li XR, Zhang Y, Wang W, Hu Q, Han DX, Xu J, Ma B (2017b) Raman-activated droplet sorting (RADS) for label-free high-throughput screening of microalgal single-cells. Anal Chem 89(22):12569–12577. https://doi.org/10.1021/acs.analchem.7b03884
Westmann CA, Alves LD, Silva-Rocha R, Guazzaroni ME (2018) Mining novel constitutive promoter elements in soil metagenomic libraries in escherichia coli. Front Microbiol 9:15. https://doi.org/10.3389/fmicb.2018.01344
Yang KK, Wu Z, Arnold FH (2019) Machine-learning-guided directed evolution for protein engineering. Nat Methods 16(8):687–694. https://doi.org/10.1038/s41592-019-0496-6
Zinchenko A, Devenish SRA, Kintses B, Colin PY, Fischlechner M, Hollfelder F (2014) One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution. Anal Chem 86(5):2526–2533. https://doi.org/10.1021/ac403585p
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Nature Singapore Pte Ltd, part of Springer Nature
About this entry
Cite this entry
Gielen, F. (2022). Single-Cell Microencapsulation for Evolution and Discovery of Biocatalysts. In: Santra, T.S., Tseng, FG. (eds) Handbook of Single-Cell Technologies. Springer, Singapore. https://doi.org/10.1007/978-981-10-8953-4_47
Download citation
DOI: https://doi.org/10.1007/978-981-10-8953-4_47
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-10-8952-7
Online ISBN: 978-981-10-8953-4
eBook Packages: EngineeringReference Module Computer Science and Engineering