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Use FACS Sorting in Metabolic Engineering of Escherichia coli for Increased Peptide Production

  • Qiong ChengEmail author
  • Kristin Ruebling-Jass
  • Jianzhong Zhang
  • Qi Chen
  • Kevin M. Croker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 834)

Abstract

Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced as recombinant forms by microbial fermentation. Targeted metabolic engineering of the production strains has usually been the approach taken to increase protein production, and this approach requires sufficient knowledge about cell metabolism and regulation. Random screening is an alternative approach that could circumvent the knowledge requirement, but is hampered by lack of suitable high-throughput screening methods. We developed a novel fluorescence-activated cell sorting (FACS) method to screen for cells with increased peptide production. Using a model peptide rich in certain amino acids, we showed that increased fluorescence clones sorted from a plasmid expression library contained genes encoding rate-limiting enzymes for amino acid synthesis. These expression clones showed increased peptide production. This demonstrated that FACS could be used as a very powerful tool for metabolic engineering. It can be generally applied to other products or processes if the desired phenotype could be correlated with a fluorescence or light scattering parameter on the FACS.

Key words

Fluorescence-activated cell sorting Lumio labeling Peptide production Inclusion body Amino acid synthesis Metabolic engineering 

References

  1. 1.
    Herzenberg LA, Parks D, Sahaf B et al (2002) The history and future of the fluorescence activated cell sorter and flow cytometry: A view from Stanford. Clinical Chemistry 48:1819–27.PubMedGoogle Scholar
  2. 2.
    Tracy BP, Gaida SM, Papoutsakis ET (2010) Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes. Curr Opinion in Biotechnol 21:85–99.CrossRefGoogle Scholar
  3. 3.
    Mattanovich D, Borth N (2006) Applications of cell sorting in biotechnology. Review. Microb Cell Fact 5:12–22.CrossRefGoogle Scholar
  4. 4.
    Nebe-von-Caron G, Stephens PJ, Hewitt CJ et al (2000) Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Methods 42:97–114.PubMedCrossRefGoogle Scholar
  5. 5.
    Johnson IS (1983) Human insulin from recombinant DNA technology. Science 219:632–7.PubMedCrossRefGoogle Scholar
  6. 6.
    Brassard DL, Grace MJ, Bordens RW (2002) Interferon-alpha as an immunotherapeutic protein. J Leukoc Biol 71:565–81.PubMedGoogle Scholar
  7. 7.
    Stübgen JP (2009) Recombinant interferon-beta therapy and neuromuscular disorders. J Neuroimmunol 212:132–41.PubMedCrossRefGoogle Scholar
  8. 8.
    Ng T, Marx G, Littlewood T, Macdougall I (2003) Recombinant erythropoietin in clinical practice. Postgrad Med J 79:367–76.PubMedCrossRefGoogle Scholar
  9. 9.
    Maurer KH (2004) Detergent proteases. Curr Opin Biotechnol 15:330–4.PubMedCrossRefGoogle Scholar
  10. 10.
    Misawa S, Kumagai I (1999) Refolding of therapeutic proteins in Escherichia coli as inclusion bodies. Biopolymers 51:297–307.PubMedCrossRefGoogle Scholar
  11. 11.
    Fischer B, Sumner I, Goodenough P (1993) Isolation, renaturation, and formation of disulfide bonds of enkaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnol Bioeng 41:3–13.PubMedCrossRefGoogle Scholar
  12. 12.
    Griffin BA, Adams SR, Jones J, Tsien RY (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269–72.PubMedCrossRefGoogle Scholar
  13. 13.
    Adams SR, Campbell RE et al (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications. J AM Chem Soc 124:6063–76.PubMedCrossRefGoogle Scholar
  14. 14.
    Berry A (1996) Improving production of aromatic compounds in Escherichia coli by metabolic engineering. Trends Biotechnol 14: 250–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Hayzer DJ, Leisinger T (1980) The gene-enzyme relationships of proline biosynthesis in Escherichia coli. J Gen Microbiol 118: 287–93.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Qiong Cheng
    • 1
    Email author
  • Kristin Ruebling-Jass
    • 1
  • Jianzhong Zhang
    • 1
  • Qi Chen
    • 1
  • Kevin M. Croker
    • 1
  1. 1.DuPont Central Research and DevelopmentWilmingtonUSA

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