Quantitative Isotope-Dilution High-Resolution-Mass-Spectrometry Analysis of Multiple Intracellular Metabolites in <i>Clostridium autoethanogenum</i> with Uniformly ¹³C‑Labeled Standards Derived from Spirulina

We have investigated the applicability of commercially available lyophilized spirulina (<i>Arthrospira platensis</i>), a microorganism uniformly labeled with ¹³C, as a readily accessible source of multiple ¹³C-labeled metabolites suitable as internal standards for the quantitative determination of intracellular bacterial metabolites. Metabolites of interest were analyzed by hydrophilic-interaction liquid chromatography coupled with high-resolution mass spectrometry. Multiple internal standards obtained from uniformly (U)-¹³C-labeled extracts from spirulina were used to enable isotope-dilution mass spectrometry (IDMS) in the identification and quantification of intracellular metabolites. Extraction of the intracellular metabolites of <i>Clostridium autoethanogenum</i> using 2:1:1 chloroform/methanol/water was found to be the optimal method in comparison with freeze–thaw, homogenization, and sonication methods. The limits of quantification were ≤1 μM with excellent linearity for all of the calibration curves (<i>R</i>² ≥ 0.99) for 74 metabolites. The precision and accuracy were found to be within relative standard deviations (RSDs) of 15% for 49 of the metabolites and within RSDs of 20% for all of the metabolites. The method was applied to study the effects of feeding different levels of carbon monoxide (as a carbon source) on the central metabolism and Wood–Ljungdahl pathway of <i>C. autoethanogenum</i> grown in continuous culture over 35 days. Using LC-IDMS with U-¹³C spirulina allowed the successful quantification of 52 metabolites in the samples, including amino acids, carboxylic acids, sugar phosphates, purines, and pyrimidines. The method provided absolute quantitative data on intracellular metabolites that was suitable for computational modeling to understand and optimize the <i>C. autoethanogenum</i> metabolic pathways active in gas fermentation

Additional file 1: of Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium

Discrepancies occurring between the current and Brown et al. finished genome sequence of C. autoethanogenum. This table shows all of the discrepancies that occur when our finished genome sequence (CLAU) is mapped against the Brown et al. finished genome sequence (BRO). Mutation column describes the mutation occurring in the CLAU genome compared to the BRO genome. Gene / region gives the gene name where the discrepancy occurs, ← / ← or similar denotes that the discrepancy occurred in a non-coding region between the named genes. Homopolymer length indicates the number of the same base occurring consecutively at the site of the discrepancy. Amino acid length gives the annotated protein length of the gene in which the discrepancy occurs, *indicates protein codes for multiple stop codons and ^indicates that no stop codon was found in the annotation. The sequence identity is relative to the CLAU C. autoethanogenum genome sequence when protein BLAST searched on the NCBI database. CLAU, C. autoethanogenum finished genome sequence in present study; CLJU, C. ljungdahlii DSM 13528 finished genome sequence (GCA_000143685.1); BRO, Brown et al. C. autoethanogenum finished genome sequence (GCA_000484505.1); CAUT, Bruno-Barcena et al. C. autoethanogenum draft genome sequence (GCA_000427255.1); NF, not found. (DOCX 73 kb