AbstractThe human pathogen Mycobacterium tuberculosis depends on host fatty acids as a carbon source. However, fatty acid β-oxidation is mediated by redundant enzymes, which hampers the development of antitubercular drugs targeting this pathway. Here, we show that rv0338c, which we refer to as etfD, encodes a membrane oxidoreductase essential for β-oxidation in M. tuberculosis. An etfD deletion mutant is incapable of growing on fatty acids or cholesterol, with long-chain fatty acids being bactericidal, and fails to grow and survive in mice. Analysis of the mutant’s metabolome reveals a block in β-oxidation at the step catalyzed by acyl-CoA dehydrogenases (ACADs), which in other organisms are functionally dependent on an electron transfer flavoprotein (ETF) and its cognate oxidoreductase. We use immunoprecipitation to show that M. tuberculosis EtfD interacts with FixA (EtfB), a protein that is homologous to the human ETF subunit β and is encoded in an operon with fixB, encoding a homologue of human ETF subunit α. We thus refer to FixA and FixB as EtfB and EtfA, respectively. Our results indicate that EtfBA and EtfD (which is not homologous to human EtfD) function as the ETF and oxidoreductase for β-oxidation in M. tuberculosis and support this pathway as a potential target for tuberculosis drug development.