Ethanol production in syngas-fermenting Clostridium ljungdahlii is controlled by thermodynamics rather than by enzyme expression

Acetogenic bacteria are attracting interest as biocatalysts in the biotechnology industry, since they are able to ferment carbon monoxide (CO)-rich gases. Wild-type strains produce mainly acetate and ethanol, but genetic modifications have already broadened the product portfolio. To enhance the production of intrinsic or heterologous biochemicals, knowledge of the microbial physiology is necessary. This physiology includes two different phases: acidogenesis (growth/acetate production) and solventogenesis (starvation/ethanol production). We operated two sequential, continuous bioreactors with a pure culture of Clostridium ljungdahlii to achieve steady-state conditions in an acetate-and an ethanol-producing stage to spatially separate acidogenesis and solventogenesis. Here, nearly 2000 proteins and their differential abundance between acidogenesis and solventogenesis were identified. In addition, we measured important metabolites. The results showed that nutrient-limited conditions triggered a transition to solventogenesis without altering the differential abundance of enzymes in the central energy metabolism. Our proteomics results revealed that the enzymes for ethanol production (AOR/ADH) were consistently abundant, even during acidogenesis. Based on this work, we developed an overflow model with thermodynamic rather than genetic regulation. The model identifies acetic acid and reduced cofactors as saturation reactants. When the intracellular concentration of undissociated acetic acid reaches a thermodynamic threshold, C. ljungdahlii will be able to shunt surplus reducing equivalents toward ethanol immediately. This is important during retarded growth, when reducing equivalents can no longer be shunted toward biomass production, while the supply of CO-rich gas is still high. Nutrient availability and pH can be manipulated to achieve the desirable level of solventogenesis during bioprocessing.