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.

We used anchor proteins with absolutely quantified concentrations using stable-isotope labelled (SIL)-protein spike-in standards (from the Anchors dataset) to estimate proteome-wide label-free concentrations in Clostridium autoethanogenum through establishing a linear correlation between protein concentrations and their measured DIA MS intensities. The optimal label-free quantification model was determined using the aLFQ package in R (Rosenberger et al. 2014; DOI: 10.1093/bioinformatics/btu200). 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 and spiked into C. autoethanogenum culture samples that were sampled 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: 15 µg of protein for all 12 culture samples (biological quadruplicates from CO, syngas, and high-H2 CO) plus ‘sample spike-in standard mix’ (SIL-protein quantities matching estimated intracellular anchor protein quantities) for performing absolute proteome quantification in C. autoethanogenum 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.