A megatransposon drives the adaptation of Thermoanaerobacter kivui to carbon monoxide

Acetogens are promising industrial biocatalysts for upgrading syngas, a gas mixture containing CO, H 2 and CO 2 into fuels and chemicals. However, CO severely inhibits growth of many acetogens, often requiring extensive adaptation to enable efficient CO conversion (carboxydotrophy). Here, we adapt the thermophilic acetogen Thermoanaerobacter kivui to use CO as sole carbon and energy source. Isolate CO-1 exhibits rapid growth on CO and syngas (co-utilizing CO, H 2 and CO 2 ) in batch and continuous cultures (µ max ~ 0.25 h −1 ). The carboxydotrophic phenotype is attributed to the mobilization of a CO-dependent megatransposon originating from the locus responsible for autotrophy in T. kivui . Transcriptomics reveal the crucial role the redox balance plays during carboxydotrophic growth. These insights are exploited to rationally engineer T. kivui to grow on CO. Collectively, our work elucidates a primary mechanism responsible for the acquisition of carboxydotrophy in acetogens and showcases how transposons can orchestrate evolution.