Absolute Proteome Quantification in the Gas-Fermenting Acetogen .

Microbes that can recycle one-carbon (C) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model acetogen Clostridium autoethanogenum grown autotrophically on three gas mixtures (CO, CO+H, or CO+CO+H). We detect the prioritization of proteome allocation for C fixation and the significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are likely relevant for CO oxidation, H metabolism, and ethanol production. The integration of proteomic and metabolic flux data demonstrated that enzymes catalyze high fluxes with high concentrations and high catalytic rates. We show that flux adjustments were dominantly accompanied by changing enzyme catalytic rates rather than concentrations. Acetogen bacteria are important for maintaining biosustainability as they can recycle gaseous C waste feedstocks (e.g., industrial waste gases and syngas from gasified biomass or municipal solid waste) into fuels and chemicals. Notably, the acetogen is being used as a cell factory in industrial-scale gas fermentation. Here, we perform reliable absolute proteome quantification for the first time in an acetogen. This is important as our work advances both rational metabolic engineering of acetogen cell factories and accurate reconstruction of their phenotypes. Furthermore, this absolute proteomics data set serves as a reference toward a better systems-level understanding of the ancient metabolism of acetogens.