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. Developed with 2% CO supplementation of CO₂/H₂, Evolved lineage D has the highest ethanol/acetate of ALE lineages when fermenting 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. Complete multi-omics analyses at steady-state revealed that although Evolved D has widespread proteome changes, intracellular metabolites prevent phenotype shifts. Yet, we observe numerous insights to CO₂/H₂ metabolism via these multi-omics results and link these to mutations, suggesting novel targets for metabolic engineering.