The tryptophan prenyltransferase ComQ from Bacillus subtilis 168 can prenylate daptomycin at Trp1

Achoimre

Prenylation is a common natural modification that enhances peptide bioactivity. Identifying prenyltransferases with broad substrate specificity is vital for enabling the modification of diverse compounds. In Bacillus subtilis 168, the tryptophan at Position 3 from the C terminus of the ComX 168 pheromone is farnesylated by ComQ 168 , producing an inducer of natural competence. While ComQ 168 's native substrate, ComX, is a linear peptide, many bioactive cyclic peptides like daptomycin and polymyxin also contain long lipid tails, similar to farnesylated ComX. This study investigates whether ComQ 168 can be used as a synthetic biology tool to farnesylate cyclic antimicrobial peptides. ComQ 168 was cloned from B. subtilis 168, expressed in E. coli , and purified in soluble fusion form. Its ability to farnesylate ComX, which was generated by a ribosomally synthesized and post‐translationally modified peptide (RiPPs) approach, confirmed its activity and proper folding. Remarkably, TF‐ComQ 168 also catalyzed farnesylation of daptomycin in vitro , most likely at the N‐terminal tryptophan residue. To explore substrate recognition, AlphaFold modeling was used to predict the binding pocket of ComQ 168 with both ComX and daptomycin, highlighting a C‐terminal region potentially important for substrate interaction. These insights offer a promising starting point for engineering ComQ 168 variants with expanded substrate scopes. In summary, this work demonstrates for the first time that ComQ 168 , previously known to modify linear peptides, can also farnesylate cyclic peptides—establishing its potential as a versatile tool in the biosynthetic engineering of bioactive compounds.