Abstract
Borosins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylated backbones. Although the first mature compound of borosin was reported in 1997, the biosynthetic pathway was elucidated 20 years later. Until this work, borosins have been able to be categorized into 11 types based on the features of their protein structure and core peptides. Type III borosins were reported only in fungi initially. In order to explore the sources and potential of type III borosins, a precise genome mining work of type III borosins was conducted in bacteria and KchMA's self-methylation activity was validated by biochemical experiment. Furthermore, a commercial protease and AI-assisted rational design was employed to engineer KchMA for the capacity to produce various N-methylated peptides. Our work demonstrates that type III borosins are abundant not only in eukaryotes but also in bacteria and have immense potential as a tool for synthetic biology.