BackgroundOxygen tolerant complex metal-dependent formate dehydrogenases hold potential for biotechnological applications.Principle FindingsIn this work, we report the functional expression of the complex, molybdenum-dependent soluble formate dehydrogenase encoded by thefdsGBACDoperon fromCupriavidus necator(CnFDH) inEscherichia coli.Expression of the operon from plasmids or from a copy integrated in the chromosome enabled growth of an energy-auxotrophic selection strain on formate as sole energy source under aerobic conditions. Growth could be accelerated in turbidostat, leading to a drop of the generation time of 1 hour. While no mutation was found in the operon of evolved isolates, genome sequencing revealed non-synonymous point mutations in the genefocAcoding for a bidirectional formate transporter carried in all isolates sequenced. Reverting the mutations led to a drop in the growth rate demonstrating thefocAmutation as principle target of continuous culture adaptation.SignificanceA member of the oxygen-tolerant subclass of complex FDH showed stable formate oxidation activity when expressed in the heterologous hostE. coli, a model organism of biotechnology. The integration of the operon in the chromosome offers the possibility of structure/function studies and activity enhancements throughin vivomutagenesis, which can also be applied to CO2reduction in appropriate selection hosts.