We report a tunable chemical genetics approach for enhancing genetic code expansion in different wild-type bacterial strains that employs apidaecin-like, anti-microbial 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 non-essential RF1 activity and promoted multi-site non-standard 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, DH10B strains and A. tumefaciens with the >
20-fold improvement observed in probiotic E. coli Nissle 1917. In-cell expression of these peptides promoted multi-site 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 ~2 fold boost to nsAA incorporation independent of the peptide. Lastly, in-cell expression of an apidaecin-like peptide library allowed the discovery of new peptide variants with reduced toxicity that still improved multi-site 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.