Reverse‐Engineered Gas‐Fermenting Acetogen Strains Recover Enhanced Phenotypes From Autotrophic Adaptive Laboratory Evolution

Gas-fermenting acetogens, such as Clostridium autoethanogenum, have emerged as promising biocatalysts capable of converting CO and CO₂-containing gases into fuels and chemicals relevant for a circular economy. However, the functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in C. autoethanogenum. In this study, we deleted one of the targets-CLAU_0471 (proposed amino acid permease)-with high mutation occurrence in ALE isolates and extensively characterised the autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterised two previously reverse-engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two-component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g., low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3-butanediol production, while RE1 performed similarly to the best-performing previously characterised ALE isolate LAbrini. The three reverse-engineered strains showed similarities in proteome expression, and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype-phenotype relationships for a better understanding of the metabolism of an industrially relevant acetogen.