Microbes that can recycle one-carbon (C1) 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 CO2 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 on three gas mixtures. We detect prioritisation of proteome allocation for C1 fixation and 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 important. Integration of proteomic and metabolic flux data demonstrated that enzymes catalyse high fluxes with high concentrations and high in vivo catalytic rates. We show that flux throughput was dominantly controlled through enzyme catalytic rates rather than concentrations. Our work serves as a reference dataset and advances systems-level understanding and engineering of acetogens.

For absolute proteome quantification in Clostridium autoethanogenum using stable-isotope labelled (SIL)-protein spike-in standards, we synthesised 20 heavy-labelled lysine and arginine SIL-proteins of key C. autoethanogenum using a cell-free wheat germ extract platform. This PRM dataset is for the quantification of the synthesised SIL-protein spike-in standard stocks.

Firstly, 20 μL of UA was added to 4 μL of the SIL-protein standard stock used to determine the stock concentration, and the mix was vortexed. Then, 1 μL of 0.2 M DTT (Promega) was added, followed by vortexing and incubation for 1 h at 37 °C to reduce disulphide bonds. Sulfhydryl groups were alkylated with 2 µL of 0.5 M iodoacetamide (IAA; Sigma-Aldrich), vigorous vortexing, and incubation for 30 min at room temperature in the dark. Next, 75 µL of 25 mM ammonium bicarbonate was added to dilute UA down to 2 M concentration. Subsequently, 2 pmol (2 µL of stock) of the non-labelled AQUA® peptide HLEAAKGYSFTTTAEKAAELHK (Sigma-Aldrich) containing the quantification tag sequence GYSFTTTAEK was added to enable quantification of SIL-protein stock concentrations using MS analysis based on the ratio of heavy-to-light GYSFTTTAEK signals (see below). Protein digestion was performed for 16 h at 37 °C with 0.1 µg of Trypsin/Lys-C mix (1 µL of stock; Promega) and stopped by lowering pH to 3 by the addition of 5 µL of 10% (v/v) trifluoroacetic acid (TFA). Samples were desalted using C18 ZipTips (Merck Millipore) as follows: the column was wetted using 0.1% (v/v) formic acid (FA) in 100% acetonitrile (ACN), equilibrated with 0.1% FA in 70% (v/v) ACN, and washed with 0.1% FA before loading the sample and washing again with 0.1% FA. Peptides were eluted from the ZipTips with 0.1% FA in 70% ACN. Finally, samples were dried using a vacuum-centrifuge (Eppendorf) at 30 °C until dryness followed by reconstitution in 12 µL of 0.1% FA in 5% ACN for subsequent MS analysis.