Unravelling the Chemolithoautotrophic Metabolism of the Carboxydotrophic Knallgas Bacterium Hydrogenophaga pseudoflava

The sustainable production of chemicals is critical to reducing greenhouse gas emissions and environmental pollution. Gaseous feedstocks from industrial waste streams and waste gasification, including CO, CO 2 and H 2 , represent abundant and underutilized resources for biotechnology. Syngas fermentation has been predominantly studied under anaerobic conditions, whereas aerobic processes may offer advantages for the biosynthesis of energy‐intensive products. One promising organism is the aerobic carboxydotroph Hydrogenophaga pseudoflava DSM 1084, whose chemolithoautotrophic metabolism remains poorly understood. To address this, we performed proteome analysis under different growth conditions and characterized deletion mutants of key enzymes involved in CO and H 2 oxidation. Deletion mutants showed that only CO dehydrogenase I and the membrane‐bound hydrogenase are essential for CO and H 2 oxidation, respectively. The role of the putative second CO dehydrogenase remains to be elucidated, and the soluble hydrogenase may contribute to redox balancing under microaerobic or anaerobic conditions. In addition, the H 2 ‐sensing regulatory system appears to be nonfunctional, and the transhydrogenase PntAB supports redox homeostasis during chemolithoautotrophic growth. Electrons from CO or H 2 are transferred via the reduced quinone pool to NADH through proton‐motive force‐driven reverse electron transfer. Nevertheless, growth on CO remains slower than on CO 2 /H 2 . In contrast to CO 2 /H 2 , an unphysiologically high CO concentration (80%) led to a major proteome rearrangement, with 1879 of 3640 reliably quantified proteins showing altered abundance compared to fructose‐grown cells, including a pronounced stress response. Under such conditions, cells are likely to invest additional energy to maintain redox balance. Together with the less energy‐conserving autotrophic branch of the respiratory chain and cytoplasmic CO oxidation, this results in inefficient CO conversion and reduced growth.