Adaptive laboratory evolution of Clostridium autoethanogenum to metabolize CO2 and H2 enhances growth rates in chemostat and unravels proteome and metabolome alterations

Gas fermentation of CO₂ and H₂ 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 CO₂-to-ethanol processes. As we have previously characterized its CO₂/H₂ chemostat growth, here we use adaptive laboratory evolution (ALE) with the aim of improving growth with CO₂/H₂. Seven ALE lineages were generated, all with improved specific growth rates. ALE conducted in the presence of 2% CO along with CO₂/H₂ generated Evolved lineage D, which showed the highest ethanol titres amongst all the ALE lineages during the fermentation of CO₂/H₂. 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 CO₂/H₂ metabolism via these multi-omics data and link these results to mutations, suggesting novel targets for metabolic engineering in this bacterium.