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

Abstract

Carbazomycins (1-8) 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 (1 and 2) were isolated from Streptomyces luteosporeus 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 1 and 2 were proposed. The in vivo genetic manipulation of the O-methyltransferase-encoding gene cbzMT proved indispensable for 1 and 2 biosynthesis. Size exclusion chromatography indicated that CbzMT was active as a dimer. In vitro biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4, producing monomethylated 2 and dimethylated 1. Monomethylated carbazomycin B (2) is not easily methylated; however, CbzMT seemingly prefers the dimethylation of the dihydroxyl substrate (12) to 1, 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.

Keywords

One-strain-many-compounds (OSMAC) / Carbazole / Carbazomycins / O-methyltransferase / Iterative methylation

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files or are available upon request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Baixin Lin: Writing - original draft, Investigation, Formal analysis, Data curation. Dashan Zhang: Formal analysis. Junbo Wang: Formal analysis. Yongjian Qiao: Formal analysis. Jinjin Wang: Methodology. Zixin Deng: Methodology. Lingxin Kong: Writing - review & editing, Writing - original draft, Formal analysis. Delin You: Writing - review & editing, Supervision, Project administration, Conceptualization.

Acknowledgments

This work was supported by a grant from the National Key research and development Program of China (2021YFA0909500, 2021YFC2100100), and National Natural Science Foundation of China (32170077, 32170075).

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