MftG is crucial for ethanol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Mycofactocin is a redox cofactor essential for the alcohol metabolism of Mycobacteria. While the biosynthesis of mycofactocin is well established, the genemftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes strictly requiremftbiosynthetic genes and are found in 75% of organisms harboring these genes. Gene deletion experiments inMycolicibacterium smegmatisdemonstrated a growth defect of the ΔmftGmutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.