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 used 19 synthetic heavy SIL variants of key C. autoethanogenum proteins as spike-in standards for quantification of intracellular concentrations of their non-SIL counterparts using a DIA MS approach. Also, we performed a dilution series experiment for the spike-in SIL-proteins to ensure accurate absolute quantification. We refer to these 19 intracellular proteins as anchor proteins that were further used to estimate proteome-wide absolute protein concentrations in C. autoethanogenum (see Label-free dataset). Raw DIA MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD025732.

The 19 spike-in SIL-protein standards that could be used for absolute proteome quantification in C. autoethanogenum were mixed in two lots: 1) ‘sample spike-in standard mix’: SIL-protein quantities matching estimated intracellular anchor protein quantities; 2) ‘dilution series standard mix’: SIL-protein quantities doubling the estimated intracellular anchor protein quantities for the dilution series sample with the highest SIL-protein concentrations. To ensure accurate absolute quantification of anchor protein concentrations, a dilution series experiment was performed to determine the linear dynamic quantification range and LLOQ for each of the 19 spike-in SIL-proteins. Dilution series samples were prepared by making nine 2-fold dilutions of the ‘dilution series spike-in standard mix’ (i.e., 10 samples total for dilution series with a 512-fold concentration span) in a constant C. autoethanogenum cell lysate background (0.07 µg/µL; 10 µg/tube) serving as a blocking agent to avoid loss of purified SIL-proteins (to container and pipette tip walls) and as a background proteome for accurate MS quantification of the linear range and LLOQ for anchor proteins. C. autoethanogenum cultures were sampled for proteomics by immediate pelleting of 2 mL of culture using centrifugation (25,000 × g for 1 min at 4 °C) and stored at -80 °C until analysis. Details of protein extraction, protein quantification, sample preparation, high pH reverse-phase fractionation, and protein digestion are described previously (Valgepea et al. 2018; DOI:10.1186/s13068-018-1052-9). The following starting material was used: 1) 15 µg of protein for all 12 culture samples (biological quadruplicates from CO, syngas, and high-H2 CO) plus ‘sample spike-in standard mix’ for performing absolute proteome quantification in C. autoethanogenum; 2) ten dilution series samples with 10-15 µg of total protein (C. autoethanogenum cell lysate background plus ‘dilution series spike-in standard mix’) for performing the dilution series experiment for the 19 spike-in SIL-proteins. Total peptide concentration in each sample was determined using the PierceTM Quantitative Fluorometric Peptide Assay (Thermo Fisher Scientific) to ensure that the same total peptide amount across samples could be injected for DIA MS analysis.