Genetic engineering of carbon monoxide dehydrogenases produces distinct autotrophic phenotypes in Clostridium autoethanogenum

Carbon monoxide dehydrogenase (CODH) enzymes couple carbon fixation to energy conservation in acetogens and serve as potential regulatory modules for tuning autotrophic metabolism. Intriguingly, the model-acetogen Clostridium autoethanogenum lost its unique truncation in the bifunctional CODH (acsA), essential for autotrophy, during autotrophic adaptive laboratory evolution while obtaining superior phenotypes.

Additionally, protein expression of the monofunctional CODH cooS1 is high and conditionally-regulated in C. autoethanogenum. Here, we genetically engineered CODHs in C. autoethanogenum by replacing the stop codon in acsA with leucine (strain Leu_SNP) or serine (Ser_SNP), and deleting cooS1 (ΔcooS1). Phenotyping in autotrophic batch and chemostat cultures revealed altered growth profiles and significant redistribution of carbon and redox flows in SNP strains, whereas ΔcooS1 showed moderate and condition-dependent effects.