O-methyltransferase CbzMT catalyzes iterative 3,4-dimethylations for carbazomycin biosynthesis.

Carbazomycins (-) are a subgroup of carbazole derivatives that contain oxygen at the C3 and C4 positions and an unusual asymmetric substitution pattern. Several of these compounds exhibit antifungal and antioxidant activities. To date, no systematic biosynthetic studies have been conducted on carbazomycins. In this study, carbazomycins A and B ( and ) were isolated from NRRL 2401 using a one-strain-many-compound (OSMAC)-guided natural product mining screen. A biosynthetic gene cluster (BGC) was identified, and possible biosynthetic pathways for and were proposed. The genetic manipulation of the O-methyltransferase-encoding gene proved indispensable for and biosynthesis. Size exclusion chromatography indicated that CbzMT was active as a dimer. biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4, producing monomethylated and dimethylated . Monomethylated carbazomycin B () is not easily methylated; however, CbzMT seemingly prefers the dimethylation of the dihydroxyl substrate () to , even with a low conversion efficiency. These findings not only improve the understanding of carbazomycin biosynthesis but also expand the inventory of OMT-catalyzing iterative methylations on different acceptor sites, paving the way for engineering biocatalysts to synthesize new active carbazomycin derivatives.