(288f) Investigation of Metabolic Pathways Via Isotopomer-Assisted Metabolite Analysis

High throughput mass spectrometry based metabolomics technologies are rapidly developed. They are able to measure both concentrations of a large amount of metabolites and their isotopomer labelings with high precision and a low per- experiment cost. 13C-assisted metabolite analysis methods can be particularly useful to trace metabolic pathways and allow branching and circular pathway fluxes to be determined, because the labeling pattern of metabolites is sensitive to the amount of flux through them. The isotopomer analysis also provides a rich dataset for validation of the genome predicted pathways and reveals the novel enzyme activities in microorganisms. Proteinogenic amino acids are often used for isotopomer analysis because they acquire the labeling pattern of their metabolic precursors. Recently, we have applied 13C-assisted metabolite analysis for studying Thermoanaerobacter sp. X514 and Dehalococcoides ethenogenes 195, which are important for bio-ethanol production and TCE bioremediation respectively. Specifically, we grew cells with 13C labeled carbon substrates and measured the resulting isotopic label patterns of cellular metabolites using GC-MS. Supported by the genome annotation, qPCR and the enzymatic activity experiments, the label information in key amino acids has clarified ambiguities in the genome annotation and identified two novel metabolic pathways: 1) Thermoanaerobacter sp. X514 and Dehalococcoides ethenogenes 195 may contain an undocumented Re-type citrate synthase which is different from that recently reported in Clostridium kluyveri and Desulfovibrio vulgaris Hildenborough. 2) Isoleucine in both species is derived from acetyl-CoA and pyruvate via the citramalate pathway rather than synthesized from threonine via threonine ammonia-lyase. Further searches from the Joint Genome Institute database (http://www.jgi.doe.gov) indicate that a few other anaerobic bacteria may contain both Re-type citrate synthase and citramalate synthase (CimA), both of which are phylogenetically related to the isopropylmalate synthase/homocitrate synthase family. The carbon flux analysis results also indicate that Thermoanaerobacter sp. X514 doesn't contain an active oxidative pentose phosphate pathway, and Dehalococcoides ethenogenes 195 can fix CO2 for biomass synthesis (>15% of total carbon in biomass) via pyruvate synthase (DET0724-0727) and pyruvate carboxylase (DET0119-0120). This study has demonstrated that in vivo 13C-assisted metabolite analysis is a useful tool for studying fundamental metabolic pathways, and provides complementary and direct information for genomic analysis of cellular metabolism.