Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains

Ross Thyer, Raghav Shroff, Dustin R. Klein, Simon D'Oelsnitz, Victoria C. Cotham, Michelle Byrom, Jennifer S. Brodbelt, Andrew D. Ellington

    Research output: Contribution to journalArticlepeer-review

    39 Scopus citations

    Abstract

    Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production. We apply an engineered β-lactamase containing an essential diselenide bond to enforce selenocysteine dependence during continuous evolution of recoded E. coli strains. Evolved strains maintain an expanded genetic code indefinitely. We engineer a fluorescent reporter to quantify selenocysteine incorporation in vivo and show complete decoding of UAG codons as selenocysteine. Replacement of native, labile disulfide bonds in antibody fragments with diselenide bonds vastly improves resistance to reducing conditions. Highly seleno-competent bacterial strains enable industrial-scale selenoprotein expression and unique diselenide architecture, advancing our ability to customize the selenoproteome.

    Original languageEnglish (US)
    Pages (from-to)624-631
    Number of pages8
    JournalNature Biotechnology
    Volume36
    Issue number7
    DOIs
    StatePublished - Aug 1 2018

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering
    • Applied Microbiology and Biotechnology
    • Molecular Medicine
    • Biomedical Engineering

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