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

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 ComX₁₆₈ pheromone is farnesylated by ComQ₁₆₈, producing an inducer of natural competence. While ComQ₁₆₈'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 ComQ₁₆₈ can be used as a synthetic biology tool to farnesylate cyclic antimicrobial peptides. ComQ₁₆₈ 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-ComQ₁₆₈ 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 ComQ₁₆₈ with both ComX and daptomycin, highlighting a C-terminal region potentially important for substrate interaction. These insights offer a promising starting point for engineering ComQ₁₆₈ variants with expanded substrate scopes. In summary, this work demonstrates for the first time that ComQ₁₆₈, 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.