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

Kuru, Erkin; Määttälä, Rosa-Maria; Noguera, Karen; Stork, Devon A.; Narasimhan, Kamesh; Rittichier, Jonathan; Wiegand, Daniel; Church, George M.

doi:10.1021/acschembio.0c00055

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

Journal Article · Tue Jun 30 00:00:00 EDT 2020 · ACS Chemical Biology

DOI:https://doi.org/10.1021/acschembio.0c00055· OSTI ID:1637333

^[1]; Määttälä, Rosa-Maria ^[2]; Noguera, Karen ^[3]; Stork, Devon A. ^[3]; Narasimhan, Kamesh ^[3]; Rittichier, Jonathan ^[1];

^[1];

^[1]

Harvard Medical School, Boston, MA (United States); Wyss Inst. for Biologically Inspired Engineering, Boston, MA (United States)
Harvard Medical School, Boston, MA (United States); MCPHS Univ., Boston, MA (United States)
Harvard Medical School, Boston, MA (United States)

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.

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Research Organization:: Harvard Medical School, Boston, MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: FG02-02ER63445

OSTI ID:: 1637333

Journal Information:: ACS Chemical Biology, Vol. 15, Issue 7; ISSN 1554-8929

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 7 works

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

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