Release Factor Inhibiting Antimicrobial Peptides Improve Nonstandard Amino Acid Incorporation in Wild-type Bacterial Cells

Abstract

We 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 concentrationdependent 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 inframe 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 apidaecinlike 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.

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination in bacteria

“…Indeed, the Api sample SC scores at the RF2-specific UGA codons were somewhat lower than those at the RF1-specific UAG codons, but the difference was fairly modest and generally followed the trend observed in the untreated control ( Figure 3—figure supplement 2 ). This result shows that Api efficiently acts upon both class 1 RFs, thereby contradicting a recent suggestion that Api preferentially traps RF1 over RF2 ( Kuru et al, 2020 ). In search for other features that could be associated with a more pronounced Api-induced ribosome stalling at the ends of specific genes, we analyzed the C-terminal sequences of the proteins encoded in ORFs with high SC-scores ( Figure 3B ).…”

“…Additionally, unlike miscoding antibiotics, Api does not reduce the general accuracy of translation and thus, could be exploited for medical applications where a premature stop codon readthrough is desirable ( Huang et al, 2019 ; Keeling et al, 2014 ). Api-mediated stop codon suppression can be also instrumental in synthetic biology, for example, for enhancing the incorporation of non-canonical amino acids ( Florin et al, 2017 ; Kuru et al, 2020 ).…”

Genome-wide effects of the antimicrobial peptide apidaecin on translation termination

“…Indeed, the Api sample SC scores at the RF2-specific UGA codons were somewhat lower than those at the RF1-specific UAG codons, but the difference was fairly mild and generally followed the trend observed in the untreated control ( Figure 3-figure supplement 2). This result shows that Api efficiently acts upon both class 1 RFs, thereby contradicting a recent suggestion that Api preferentially traps RF1 over RF2 (Kuru et al, 2020). In search for other features that could be associated with a more pronounced Api-induced ribosome stalling at the ends of specific genes, we analyzed the Cterminal sequences of the proteins encoded in ORFs with high SC-scores ( Figure 3B).…”

“…Additionally, unlike miscoding antibiotics, Api does not reduce the general accuracy of translation and thus, could be exploited for medical applications where premature stop codon readthrough is desirable (Huang et al, 2019;Keeling et al, 2014). Api-mediated stop codon suppression can be also instrumental in synthetic biology, e.g., for enhancing incorporation of non-canonical amino acids (Florin et al, 2017;Kuru et al, 2020).…”

Identification of novel translated small ORFs in Escherichia coli using complementary ribosome profiling approaches

“…Apidaecin uptake requires the sbmA gene, which encodes an inner-membrane transport protein (32); sbmA is missing in some Gram-negative and all Gram-positive bacterial species. If apidaecin uptake is low, an alternative approach would be to encode and express apidaecin, a small protein, inside the bacteria (33,34). Similarly, Onc112, which functions similarly to retapamulin, is a small protein that could be encoded and expressed within cells (35).…”