<i>Streptomyces albus</i>: A New Cell Factory for Non-Canonical Amino Acids Incorporation into Ribosomally Synthesized Natural Products

The incorporation of noncanonical amino acids (ncAAs) with different side chains into a peptide is a promising technique for changing the functional properties of that peptide. Of particular interest is the incorporation of ncAAs into peptide-derived natural products to optimize their biophysical properties for medical and industrial applications. Here, we present the first instance of ncAA incorporation into the natural product cinnamycin in streptomycetes using the orthogonal pyrrolysyl-tRNA synthetase/tRNA<sup>Pyl</sup> pair from <i>Methanosarcina barkeri</i>. This approach allows site-specific incorporation of ncAAs <i>via</i> the read-through of a stop codon by the suppressor tRNA<sup>Pyl</sup>, which can carry different pyrrolysine analogues. Five new deoxycinnamycin derivatives were obtained with three distinct pyrrolysine analogues incorporated into diverse positions of the antibiotic. The combination of partial hydrolysis and MS/MS fragmentation analysis was used to verify the exact position of the incorporation events. The introduction of ncAAs into different positions of the peptide had opposite effects on the peptide’s biological activity.