Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Methylobacterium extorquens AM1 is a facultative methylotroph, which is a potential candidate to be used in commercial processes to convert simple one-carbon compounds to a variety of multicarbon chemicals and products. To better understand C-1 metabolism in M. extorquens AMI at the systems level, metabolite profiling tools were developed and applied in this bacterium. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC x GC-TOFMS) was used to obtain metabolite profiles of M. extorquens AMI (primarily organic acids) and to identify the metabolite differences between cells grown on methanol (C-1 substrate) and succinate (multicarbon substrate). In this study, a list of compounds that included amino acids and major intermediates of central C-1 and multicarbon metabolism were studied as target metabolites. For these, calibration curves were obtained for absolute quantification by spiking different amounts of standard mixtures to cell cultures. Parallel factor analysis (PARAFAC) was used for accurate peak quantification. Unknown chemical differences between cells grown on methanol and succinate were identified by applying Fisher ratio analysis at a selective mass channel (m/z 147). Thirty-six compounds were discovered to be statistically differentially expressed between C-1 and multicarbon metabolism. Among these, 13 were identified by matching to library mass spectra, and the rest were novel compounds that were not included in libraries. These differentially expressed compounds have provided clues to new pathways that are specifically linked to C-1 metabolism.