August 2024
·
66 Reads
·
2 Citations
Peptide-based antibiotics (PBAs), including antimicrobial peptides (AMPs) and their synthetic mimics, have received significant interest due to their diverse and unique bioactivities. The integration of high-throughput sequencing and bioinformatics tools has dramatically enhanced the discovery of enzymes, allowing researchers to identify specific genes and metabolic pathways responsible for producing novel PBAs more precisely. Cell-free systems (CFSs) that allow precise control over transcription and translation in vitro are being adapted, which accelerate the identification, characterization, selection, and production of novel PBAs. Furthermore, these platforms offer an ideal solution for overcoming the limitations of small-molecule antibiotics, which often lack efficacy against a broad spectrum of pathogens and contribute to the development of antibiotic resistance. In this review, we highlight recent examples of how CFSs streamline these processes while expanding our ability to access new antimicrobial agents that are effective against antibiotic-resistant infections.
![Design of γ-keto and hydrazino esters and ribosome-mediated synthesis of pyridazinone bonds
A Four γ-keto (orange) and B two hydrazino (green) esters were synthesized with an activated leaving group (CME, DNB, and ABT). DNB or ABT were used for the substrates that do not contain an aromatic moiety and the ABT-activated substances were only synthesized when the DNB substrates were found to be water-insoluble (Supplementary Information; 1-CME, 2-CME, 3-DNB, 3-ABT, 4-DNB; 5-CME, 6-DNB, 6-ABT). The substrates were charged to tRNA by the appropriate Fx and introduced to an in vitro translation reaction containing wild-type ribosomes. C In vitro translation reactions were carried out with pairs of γ-keto ester substrates (A) and hydrazino ester substrates (B). Ribosome-catalyzed synthesis of eight different pyridazinone rings was observed. The relative percent yield of the target oligomer of all species was determined by the peak area corresponding to the theoretical mass/the sum of areas of the whole peaks shown in the mass spectrum, as shown in matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectra (Supplementary Information). Percent yield is based on n = 3 reactions. D, E MALDI-TOF mass spectra of oligomers polymerized by the ribosome in vitro with a pyridazinone bond formed between 1 and 5, and 1 and 6, respectively. The calculated masses of the products in D are [M + H]⁺ = 1362, [M + Na]⁺ = 1384 and in E are [M + H]⁺ = 1286, [M + Na]⁺ = 1308. See SI for MALDI-TOF mass spectra of the other pyridazinone bonds represented in C. The non-target products at masses 1058 and 1080 (#) and 1305 and 1327 (*) are a reporter strep-tag alone (TrpSerHisProGlnPheGluLys) and the peptide containing a misincorporated Ser60,61 at the Thr (ACC) codon (1(Ser)TrpSerHisProGlnPheGluLys, see Supplementary Fig. 2 for details). Spectra in D and E are representative of n = 3 independent experiments.](https://www.researchgate.net/publication/364679508/figure/fig2/AS:11431281091938676@1666667499084/Design-of-g-keto-and-hydrazino-esters-and-ribosome-mediated-synthesis-of-pyridazinone_Q320.jpg)
![Ribosomal synthesis of alternating copolymers with a pyridazinone backbone
A We designed an additional amino acid, γKPheA (7), bearing a ketone on its γ-carbon of the sidechain, for sequential polymerization of pyridazinones bonds on a biopolymer chain. Compounds 7 and 6 were charged to tRNAPro1E2(GGU) and tRNAGluE2(GAU) by flexizyme, respectively, and added to an in vitro transcription and translation reaction. The genetic template was designed to consecutively incorporate the monomers in an alternating fashion (ABAB- or ABABAB-type). The resulting peptides-pyridazinone hybrids were purified via the streptavidin tag (WSHPQFEK) and characterized by MALDI-TOF mass spectrometry. B MALDI mass spectrum of the StrepII-7676 peptide (relative peak area: 14.8%) and its molecular structure, calculated mass: [M + H]⁺ = 1791; [M + Na]⁺ = 1813 (C) MALDI mass spectrum of the StrepII-767676 (relative peak area: 16.9%) peptide and its molecular structure, calculated mass: [M + H]⁺ = 2034; [M + Na]⁺ = 2056. Spectra are representative of n = 3 independent experiments.](https://www.researchgate.net/publication/364679508/figure/fig4/AS:11431281091929601@1666667499167/Ribosomal-synthesis-of-alternating-copolymers-with-a-pyridazinone-backbone-A-We-designed_Q320.jpg)
![Ribosomal synthesis of N-terminal functionalized peptides with backbone-extended monomers
a All backbone-extended amino acids (3–15) charged to tRNAfMet(CAU) by Fx were incorporated into the N-terminus of a peptide by ribosome-mediated polymerization in the PURExpressTM system. The peptides were purified via the Streptavidin tag (WSHPQFEK) and characterized by MALDI mass spectrometry. The observed mass of each peptide corresponds to the theoretical mass, which is b [M + H]⁺ = 1345; [M + Na]⁺ = 1367, c [M + H]⁺ = 1359; [M + Na]⁺ = 1381, d [M + H]⁺ = 1373; [M + Na]⁺ = 1395, e [M + H]⁺ = 1369; [M + Na]⁺ = 1391, f [M + Na]⁺ = 1351, g [M + H]⁺ = 1379; [M + Na]⁺ = 1401, h [M + H]⁺ = 1371; [M + Na]⁺ = 1393, i [M + H]⁺ = 1372; [M + Na]⁺ = 1394, j [M + H]⁺ = 1343; [M + Na]⁺ = 1365, k [M + Na]⁺ = 1365, l [M + H]⁺ = 1357; [M + Na]⁺ = 1379, m [M + H]⁺ = 1371; [M + Na]⁺ = 1393, n [M + H]⁺ = 1371; [M + Na]⁺ = 1393. The peaks denoted with an asterisk are a truncated peptide not bearing the target substrate at the N-terminus ([M + H]⁺ = 1246; [M + Na]⁺ = 1268). Data are representative of three independent experiments.](https://www.researchgate.net/publication/343916034/figure/fig4/AS:959175274283009@1605696614386/Ribosomal-synthesis-of-N-terminal-functionalized-peptides-with-backbone-extended_Q320.jpg)
