Acetogenesis to ethanologenesis: facilitating NADH oxidation via reductive acetate uptake

Acetate is not always an end product in acetogen metabolism.The reaction mechanism of butyrate production with acetate uptake was identified.Greater NADH oxidation facilitates the catabolic (carbon monoxide oxidation) reaction rate.The metabolic shift from acetogenesis to ethanologenesis occurs via complete acetate uptake.The shift to a single volatile product enables the yield and selectivity of target products to be enhanced. (Homo)acetogens, including Clostridium spp., represent an enigma in metabolic flexibility and diversity. Eubacterium callanderi KIST612 is an acetogen that produces n-butyrate with carbon monoxide (CO) as the carbon and energy source; however, the production route is unknown. Here, we report that its distinctive butyrate formation links to reductive acetate uptake, suggesting that acetate (the end-product) is reuptake, leading to a physiological advantage through NADH oxidation. Thus, we introduced an ethanol production pathway from acetyl-CoA as a competitive pathway for butyrate production. Consequently, the metabolic pathway in our mutants switched from acetogenesis to ‘ethanologenesis’, eliminating butyrate production and the uptake of previously produced acetate. The metabolic shifts occurred toward greater NADH oxidation, facilitating CO oxidation and productivity, which is a survival mechanism at the thermodynamic edge. This metabolic shift to a single product holds potential to revolutionize product separation strategies in synthetic gas (syngas)-based biorefineries. [Display omitted] Acetate is not always an end product in acetogenesis. This study identified butyrate production with acetate uptake, a finding that led to the development of a strain for ethanol production with complete acetate uptake. The shift from acetogenesis to ethanologenesis could reduce operating costs in downstream processed for commercial synthetic gas (syngas) fermentation. In acetogens, acetate production is crucial for energy conservation under autotrophic conditions. Theoretical ATP yield has been a key factor when engineering strains for butyrate, ethanol, and other products via carbon monoxide (CO) fermentation. In the present study, we proposed a strategy to overcome low ATP yields by enhancing metabolic reaction rates linked to NADH oxidation and energy conservation. We identified the butyrate production pathway and demonstrated the approach by constructing an ethanol-producing strain. Ethanol production with complete acetate uptake were vali