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 adapted the thermophilic acetogen Thermoanaerobacter kivui to use CO as sole carbon and energy source. Isolate CO-1 exhibited extremely 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 was attributed to the mobilization of a CO-inducible megatransposon originating from the locus responsible for autotrophy in T . kivui . Transcriptomics illuminated the crucial role maintaining redox balance likely plays during carboxydotrophic growth. These novel insights were exploited to rationally engineer T . kivui to grow on CO. Collectively, our work elucidates a primary mechanism responsible for the acquisition of carboxydotrophy in homoacetogens and showcases how transposons can orchestrate evolution.