AbstractBacillus subtilis subsp. natto secretes the ComX_natto pheromone as a quorum‐sensing pheromone to produce poly‐γ‐glutamate for biofilm formation. The amino‐acid sequence of the pheromone is Lys‐Trp‐Pro‐Pro‐Ile‐Glu, and the tryptophan residue is post‐translationally modified with a farnesyl group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C‐terminal end of the precursor peptide ComX_natto. Here, we report the functional analysis of ComQ_natto, which catalyzes a unique farnesyl‐transfer reaction. ComQ_natto recognizes not only full‐length ComX_natto but also N‐ and/or C‐terminal truncated ComX_natto analogues and even a single tryptophan for modification with a farnesyl group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQ_natto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.