Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption

Nature Biotechnology volume 32, pages 473–478 (2014)Cite this article

Subjects

Abstract

Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Here we present a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase. We use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 104 cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization. We also enriched L-lactate–producing Escherichia coli clones 5,800× from a population containing one L-lactate producer per 104 D-lactate producers. Our approach has broad applications for single-cell analyses, such as in strain selection for the overproduction of fuels, chemicals and pharmaceuticals.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Microfluidic high-throughput screening platform.
Figure 2: Development of screen for high xylose–consuming strains.
Figure 3: Enrichment of high xylose–consuming strain (H131).
Figure 4: Results from genomic DNA library screen.
Figure 5: Shake flask data from lactate-producing strains and enrichment of L-lactate–producing strain.

Similar content being viewed by others

References

  1. Alper, H., Fischer, C., Nevoigt, E. & Stephanopoulos, G. Tuning genetic control through promoter engineering. Proc. Natl. Acad. Sci. USA 102, 12678–12683 (2005).

    Article  CAS  Google Scholar 

  2. Bailey, J.E. et al. Inverse metabolic engineering: a strategy for directed genetic engineering of useful phenotypes. Biotechnol. Bioeng. 52, 109–121 (1996).

    Article  CAS  Google Scholar 

  3. Martin, V.J.J., Pitera, D.J., Withers, S.T., Newman, J.D. & Keasling, J.D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802 (2003).

    Article  CAS  Google Scholar 

  4. Atsumi, S., Hanai, T. & Liao, J.C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008).

    Article  CAS  Google Scholar 

  5. Alper, H., Moxley, J., Nevoigt, E., Fink, G.R. & Stephanopoulos, G. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314, 1565–1568 (2006).

    Article  CAS  Google Scholar 

  6. Doshi, R., Nguyen, T. & Chang, G. Transporter-mediated biofuel secretion. Proc. Natl. Acad. Sci. USA 110, 7642–7647 (2013).

    Article  CAS  Google Scholar 

  7. Liu, X., Sheng, J. & Curtiss, R. Fatty acid production in genetically modified cyanobacteria. Proc. Natl. Acad. Sci. USA 108, 6899–6904 (2011).

    Article  CAS  Google Scholar 

  8. Wei, N., Xu, H.Q., Kim, S.R. & Jin, Y.S. Deletion of FPS1, encoding Aquaglyceroporin Fps1p, improves xylose fermentation by engineered Saccharomyces cerevisiae. Appl. Environ. Microbiol. 79, 3193–3201 (2013).

    Article  CAS  Google Scholar 

  9. Sonderegger, M., Schumperli, M. & Sauer, U. Selection of quiescent Escherichia coli with high metabolic activity. Metab. Eng. 7, 4–9 (2005).

    Article  CAS  Google Scholar 

  10. Weaver, J.C., Williams, G.B., Klibanov, A. & Demain, A.L. Gel microdroplets - rapid detection and enumeration of individual microorganisms by their metabolic-activity. Bio/Technology 6, 1084–1089 (1988).

    CAS  Google Scholar 

  11. Tawfik, D.S. & Griffiths, A.D. Man-made cell-like compartments for molecular evolution. Nat. Biotechnol. 16, 652–656 (1998).

    Article  CAS  Google Scholar 

  12. Aharoni, A., Amitai, G., Bernath, K., Magdassi, S. & Tawfik, D.S. High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. Chem. Biol. 12, 1281–1289 (2005).

    Article  CAS  Google Scholar 

  13. Miller, O.J. et al. Directed evolution by in vitro compartmentalization. Nat. Methods 3, 561–570 (2006).

    Article  CAS  Google Scholar 

  14. Link, D.R., Anna, S.L., Weitz, D.A. & Stone, H.A. Geometrically mediated breakup of drops in microfluidic devices. Phys. Rev. Lett. 92, 054503 (2004).

    Article  CAS  Google Scholar 

  15. Ahn, K., Agresti, J., Chong, H., Marquez, M. & Weitz, D.A. Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels. Appl. Phys. Lett. 88, 264105 (2006).

    Article  Google Scholar 

  16. Ahn, K. et al. Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices. Appl. Phys. Lett. 88, 024104 (2006).

    Article  Google Scholar 

  17. Huebner, A. et al. Quantitative detection of protein expression in single cells using droplet microfluidics. Chem. Commun. (Camb.) 12, 1218–1220 (2007).

    Article  Google Scholar 

  18. Agresti, J.J. et al. Ultrahigh-throughput screening in drop-based microfluidics for directed evolution. Proc. Natl. Acad. Sci. USA 107, 4004–4009 (2010).

    Article  CAS  Google Scholar 

  19. Zhou, M., Diwu, Z.J., Panchuk-Voloshina, N. & Haugland, R.P. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253, 162–168 (1997).

    Article  CAS  Google Scholar 

  20. Zhu, A., Romero, R. & Petty, H.R. Amplex UltraRed enhances the sensitivity of fluorimetric pyruvate detection. Anal. Biochem. 403, 123–125 (2010).

    Article  CAS  Google Scholar 

  21. Chundawat, S.P.S., Venkatesh, B. & Dale, B.E. Effect of particle size based separation of milled corn stover on AFEX pretreatment and enzymatic digestibility. Biotechnol. Bioeng. 96, 219–231 (2007).

    Article  CAS  Google Scholar 

  22. Hahn-Hagerdal, B., Karhumaa, K., Jeppsson, M. & Gorwa-Grauslund, M.F. in Biofuels (ed. L. Olsson) 147–177 (Springer, Berlin, 2007).

  23. Kuyper, M. et al. Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res. 5, 925–934 (2005).

    Article  CAS  Google Scholar 

  24. van Maris, A.J.A. et al. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie van Leeuwenhoek 90, 391–418 (2006).

    Article  Google Scholar 

  25. Jeffries, T.W. Engineering yeasts for xylose metabolism. Curr. Opin. Biotechnol. 17, 320–326 (2006).

    Article  CAS  Google Scholar 

  26. Wagschal, K., Franqui-Espiet, D., Lee, C.C., Robertson, G.H. & Wong, D.W.S. Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates. Appl. Environ. Microbiol. 71, 5318–5323 (2005).

    Article  CAS  Google Scholar 

  27. Smith, D.R. et al. Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res. 18, 1638–1642 (2008).

    Article  CAS  Google Scholar 

  28. Ohnishi, J. et al. A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant. Appl. Microbiol. Biotechnol. 58, 217–223 (2002).

    Article  CAS  Google Scholar 

  29. Zhou, H., Cheng, J.-S., Wang, B.L., Fink, G.R. & Stephanopoulos, G. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metab. Eng. 14, 611–622 (2012).

    Article  CAS  Google Scholar 

  30. Zhang, J.Z. Evolution by gene duplication: an update. Trends Ecol. Evol. 18, 292–298 (2003).

    Article  Google Scholar 

  31. Grabar, T.B., Zhou, S., Shanmugam, K.T., Yomano, L.P. & Ingram, L.O. Methylglyoxal bypass identified as source of chiral contamination in L(+) and D(−)-lactate fermentations by recombinant Escherichia coli. Biotechnol. Lett. 28, 1527–1535 (2006).

    Article  CAS  Google Scholar 

  32. Baret, J.-C., Beck, Y., Billas-Massobrio, I., Moras, D. & Griffiths, A.D. Quantitative cell-based reporter gene assays using droplet-based microfluidics. Chem. Biol. 17, 528–536 (2010).

    Article  CAS  Google Scholar 

  33. Yamashita, M. et al. Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472. J. Biosci. Bioeng. 89, 350–360 (2000).

    Article  CAS  Google Scholar 

  34. Sun, H., Treco, D., Schultes, N.P. & Szostak, J.W. Double-strand breaks at an initiation site for meiotic gene conversion. Nature 338, 87–90 (1989).

    Article  CAS  Google Scholar 

  35. Siegel, A.C., Bruzewicz, D.A., Weibel, D.B. & Whitesides, G.M. Microsolidics: fabrication of three-dimensional metallic microstructures in poly(dimethylsiloxane). Adv. Mater. 19, 727–733 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge support by Department of Energy Grant DE-FC36-07G017058, Royal Dutch Shell, and the Singapore-MIT Alliance. The authors would like to thank A. Abate for writing the LabView detection/sorting code, K. Humphry for her initial help on the project, Raindance Technologies for providing fluorinated surfactants and oils. We also thank the Ingram laboratory for providing strains TG108 and TG113.

Author information

Author notes

  1. Benjamin L Wang and Adel Ghaderi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Benjamin L Wang, Adel Ghaderi, Hang Zhou & Gregory Stephanopoulos

  2. Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA

    Jeremy Agresti & David A Weitz

  3. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    Gerald R Fink

Authors
  1. Benjamin L Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adel Ghaderi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeremy Agresti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David A Weitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gerald R Fink
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gregory Stephanopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.L.W., A.G. and G.S. planned the experiments, interpreted results and wrote the manuscript. B.L.W. developed the integrated microfluidic screening system and performed the xylose consumption experiments. A.G. performed the lactate production experiments. H.Z. created the xylose-consuming strains. H.Z. and B.L.W. constructed the yeast genomic DNA library and performed analysis on the high xylose–consuming strain. G.R.F. provided guidance in designing and constructing the yeast genomic DNA library. J.A. built the microfluidic detection and sorting stand. D.A.W. and J.A. provided technical advice regarding the microfluidic droplet system.

Corresponding author

Correspondence to Gregory Stephanopoulos.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figues 1–15, Supplementary Table 1 and Supplementary Notes 1–8 (PDF 1430 kb)

Supplementary Video 1

Droplet formation of yeast or E. coli cells in growth medium (MOV 24156 kb)

Supplementary Video 2

Alternating sequence of cell-containing droplets and assay droplets (MOV 27739 kb)

Supplementary Video 3

Droplet coalescence of cell-containing droplets and assay droplets (MOV 15440 kb)

Supplementary Video 4

Sorting of “desired” droplets into the upper channel (MOV 21111 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, B., Ghaderi, A., Zhou, H. et al. Microfluidic high-throughput culturing of single cells for selection based on extracellular metabolite production or consumption. Nat Biotechnol 32, 473–478 (2014). https://doi.org/10.1038/nbt.2857

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2857

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research