Posttranslationally Acting Arginases Provide a Ribosomal Route to Non‐proteinogenic Ornithine Residues in Diverse Peptide Sequences

Abstract

Ornithine is a component of many bioactive nonribosomal peptides but is challenging to incorporate into ribosomal products. We recently identified OspR, a cyanobacterial arginase-like enzyme that installs ornithines in the antiviral ribosomally synthesised and posttranslationally modified peptide (RiPP) landornamide A. Here we report that OspR belongs to a larger family of peptide arginases from diverse organisms and RiPP types. In E. coli, seven selected enzymes converted arginine into ornithine with little preference for the leader type. A broad range of peptide sequences was modified, including polyarginine repeats. We also generated analogues of ornithine-containing nonribosomal peptides using RiPP technology. Five pseudo-nonribosomal products with ornithines at the correct positions were obtained, including a brevicidine analogue containing ornithine and a d-amino acid installed by the peptide epimerase OspD. These results suggest new opportunities for peptide bioengineering. * = d-amino acid. C) The osp gene cluster from Kamptonema sp. PCC 6506 encoding the arginase-like enzyme OspR.

Engineering lanthipeptides by introducing a large variety of RiPP modifications to obtain new-to-nature bioactive peptides

“…In addition, studies by Piel’s group have shown that OspR was able to modify three Arg residues to the non-canonical ornithine in another linear brevicidine mimic. In addition, the epimerase OspD could introduce a D-amino acid in this mimic, although the position of the D-amino acid did not match the D-amino acid positions in natural brevicidine (Mordhorst et al 2020 ). These results indicate that there are a variety of RiPP tools available that can potentially be combined to introduce NRP structural features into ribosomal peptides (Fig.…”

“…Further modification work mainly focuses on the incorporation of D-amino acids, the non-canonical amino acid ornithine, and the mimicking of a fatty acid chain by enzymatic methods. Piel’s group has demonstrated the possibility of introducing D-amino acids and ornithines onto similar linear peptide sequences (Mordhorst et al 2020 ).…”

“…To overcome the challenges of NRPS re-engineering to generate improved NRP variants, a strategy has been proposed to use lanthipeptides as starting points to synthesize peptides with similar NRP structural features by employing RiPP biosynthetic pathways. (Van Der Velden et al 2017 , Mordhorst et al 2020 , 2023 , Zhao et al 2020 , Ruijne and Kuipers 2021 ). As shown in Fig.…”