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Microfluidic Methods for Molecular Biology pp 189–204Cite as

Single-Cell Phenotypic Screening in Inverse Metabolic Engineering

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Abstract

Contrary to classical approaches centering on debottlenecking flux-limiting steps in a metabolic pathway, inverse metabolic engineering (IME) aims at identifying and modulating all gene factors that contribute to an optimal phenotype. Within IME, mutant libraries are generated and screened in order to select mutants with the desired phenotype. The screening process is traditionally performed using microtiter well plates, a laborious and expensive process of limited throughput. Here, we review emerging screening methods that address these throughput and cost-effectiveness shortcomings, but also operate at the single-cell level. We discuss the importance of single-cell analyses in IME and detail two specific single-cell screening approaches: the first is fluorescence-activated cell sorting for phenotypic discrimination based on cytosolic or cell-membrane-bound products. The second is droplet microfluidics for screening of cells capable of overproducing secreted products or overconsuming substrates, properties that require confinement to isolate mutants with specific secretory phenotypes.

Keywords

  • Metabolic engineering
  • Single-cell analysis
  • Microfluidics
  • Flow cytometry

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Authors and Affiliations

  1. Department of Physics, University of Idaho, Moscow, ID, 83844, USA

    A. E. Vasdekis

  2. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    G. Stephanopoulos

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  1. A. E. Vasdekis
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  2. G. Stephanopoulos
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Correspondence to A. E. Vasdekis .

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Editors and Affiliations

  1. Virginia Tech, Blacksburg, Virginia, USA

    Chang Lu

  2. Dpt of Biomedical Engg. & Mechanics, Virginia Tech, Blacksburg, Virginia, USA

    Scott S. Verbridge

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Vasdekis, A.E., Stephanopoulos, G. (2016). Single-Cell Phenotypic Screening in Inverse Metabolic Engineering. In: Lu, C., Verbridge, S. (eds) Microfluidic Methods for Molecular Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-30019-1_9

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