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Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells
journal contribution
posted on 2020-06-30, 18:35 authored by Erkin Kuru, Rosa-Maria Määttälä, Karen Noguera, Devon A. Stork, Kamesh Narasimhan, Jonathan Rittichier, Daniel Wiegand, George M. ChurchWe report a tunable
chemical genetics approach for enhancing genetic
code expansion in different wild-type bacterial strains that employ
apidaecin-like, antimicrobial peptides observed to temporarily sequester
and thereby inhibit Release Factor 1 (RF1). In a concentration-dependent
matter, these peptides granted a conditional lambda phage resistance
to a recoded Escherichia coli strain with nonessential
RF1 activity and promoted multisite nonstandard amino acid (nsAA)
incorporation at in-frame amber stop codons in vivo and in vitro. When exogenously added, the peptides
stimulated specific nsAA incorporation in a variety of sensitive,
wild-type (RF1+) strains, including Agrobacterium tumefaciens, a species in which nsAA incorporation has not been previously reported.
Improvement in nsAA incorporation was typically 2–15-fold in E. coli BL21, MG1655, and DH10B strains and A. tumefaciens with the >20-fold improvement observed in probiotic E.
coli Nissle 1917. In-cell expression of these peptides promoted
multisite
nsAA incorporation in transcripts with up to 6 amber codons, with
a >35-fold increase in BL21 showing moderate toxicity. Leveraging
this RF1 sensitivity allowed multiplexed partial recoding of MG1655
and DH10B that rapidly resulted in resistant strains that showed an
additional approximately twofold boost to nsAA incorporation independent
of the peptide. Finally, in-cell expression of an apidaecin-like peptide
library allowed the discovery of new peptide variants with reduced
toxicity that still improved multisite nsAA incorporation >25-fold.
In parallel to genetic reprogramming efforts, these new approaches
can facilitate genetic code expansion technologies in a variety of
wild-type bacterial strains.
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BLcoli Nissle 1917. In-cell expressionDH 10B strainsmultisite nsAA incorporationRelease Factor 1lambda phage resistanceapidaecin-like peptide libraryMGtunable chemical genetics approachrelease Factor Inhibiting Antimicro...wild-typerecoded Escherichia coli strainRF 1 sensitivitynsAA incorporationcode expansion technologiesRF 1 activity
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