Efficient in vivo synthesis of lasso peptide pseudomycoidin proceeds in the absence of both the leader and the leader peptidase

from the ribosomally synthesized core part by a macrolactam synthetase, in the absence of the leader protease. The minimalism of the pseudomycoidin synthesis system makes it a powerful model to generate pseudomycoidin-based lasso-peptide libraries and to study the poorly understood process of lasso formation. We detected two additional pseudomycoidin modifications: phosphorylation of a terminal residue that was previously observed in another lasso peptide, followed by glycosylation, which was not observed heretofore. We speculate that these bulky C-terminal modifications may help maintain the threaded lasso topology of the compound synthesized by the macrolactam synthetase.