Identification of a chlorophenol O-methyltransferase from a mycobacterial isolate involved in the formation of odorous chloroanisoles in chlorinated water

Odorous chloroanisoles, particularly 2,4,6-trichloroanisole (2,4,6-TCA), are frequently detected in aquatic environments. Although several fungal species are known to catalyze the transformation of chlorophenols into chloroanisoles, thereby causing undesirable taste and odor alterations in wine, bacterial contributions to this transformation may also be significant in water and wastewater treatment processes. In this study, the whole genome of Mycolicibacterium sp. CB14, a bacterial isolate with a high capacity for 2,4,6-TCA production, was analyzed. The strain exhibited high resistance to chlorination and showed its highest growth rate at 30 °C. Among the 58 putative methyltransferases identified through whole-genome analysis, three enzymes were selected based on BLASTp analysis against a known fungal chlorophenol O -methyltransferase. One of these three enzymes was shown to produce 2,4,6-TCA when its gene was heterologously expressed in Escherichia coli BL21(DE3). To our knowledge, this is the first study to report the successful heterologous expression of a mycobacterial TCA-producing enzyme. The prevalence of the identified bacterial chlorophenol O -methyltransferase was confirmed, at least among mycobacterial species, through comparisons between the identified amino acid sequence and registered sequences from various species. This study provides a basis for developing qPCR assays to assess the production potential of odorous chloroanisoles and for the trace-level quantification of these compounds in aquatic environments.