Modifying Methylene‐Tetrahydrofolate Reductase to Disrupt Electron Bifurcation in Clostridium autoethanogenum

Methylene‐tetrahydrofolate reductase (MTHFR) is an important enzyme for acetogenic carbon fixation, but the redox mechanism driving this reaction is not clearly understood. Previous enzymology work and energetic accounting on species such as Clostridium autoethanogenum has led to confounding results when placed in the context of in vivo experiments. In this work, we create multiple C. autoethanogenum strains harboring alternative MTHFR enzyme complexes as well as genome‐scale metabolic models to better understand how these organisms conserve energy on gas substrates. The inclusion of a Type‐III MTHFR unexpectedly allows for higher growth than expected and suggests the possibility of an additional redox balancing cycle employed during autotrophic growth.