Biosynthesis of the redox cofactor mycofactocin comprises oligoglycosylation by MftF in Mycolicibacterium smegmatis

Mycofactocin (MFT) is a redox cofactor involved in alcohol metabolism of mycobacteria including . In recent years, a preliminary biosynthetic model of MFT has been established by studies, while the final structure of MFT remained elusive. Here, we report the discovery of MFT by metabolomics and establish a model of its biosynthesis in . Structure elucidation revealed that MFT is decorated with up to nine β-1,4-linked glucose residues. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the cofactor function of MFT by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results close an important gap of knowledge, will guide future studies into the physiological roles of MFT in bacteria and may inspire its utilization as a biomarker or potential drug target to combat mycobacterial diseases.