Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO 2 and H 2 enhances growth rates in chemostat and unravels proteome and metabolome alterations.

Gas fermentation of CO 2 and H 2 is an attractive means to sustainably produce fuels and chemicals. Clostridium autoethanogenum is a model organism for industrial CO to ethanol and presents an opportunity for CO 2 -to-ethanol processes. As we have previously characterized its CO 2 /H 2 chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO 2 /H 2 . Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO 2 /H 2 generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO 2 /H 2 . Chemostat comparison against the parental strain shows no change in acetate or ethanol production, while Evolved D could achieve a higher maximum dilution rate. Multi-omics analyses at steady state revealed that Evolved D has widespread proteome and intracellular metabolome changes. However, the uptake and production rates and titres remain unaltered until investigating their maximum dilution rate. Yet, we provide numerous insights into CO 2 /H 2 metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.