Quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum
- 388 Downloads
Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.
To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.
Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.
Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.
Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.
KeywordsTetrahydrofolates Acetogens Wood–Ljungdahl pathway Gas fermentation Metabolome
This study was funded by a Grant from the Australian Research Council, partly funded by LanzaTech (ARC LP140100213). Elements of this research utilised equipment and support provided by the QLD node of Metabolomics Australia, an initiative of the Australian Government being conducted as part of the NCRIS National Research Infrastructure for Australia.
Compliance with ethical standards
Conflict of interest
RSPL, KV, MPH, LKN, EM declare that they have no conflict of interest. LanzaTech has interest in commercial gas fermentation with C. autoethanogenum. RT, SDS, MK are employees of LanzaTech.
Research involving human and animals participants
All authors comply with Springer’s ethical policies. This article does not contain any studies with human participants or animals performed by any of the authors.
- Aiso, K., Nozaki, T., Shimoda, M., & Kokue, E. (1999). Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection. Analytical Biochemistry, 272(2), 143–148. https://doi.org/10.1006/abio.1999.4174.CrossRefPubMedGoogle Scholar
- Fuchs, G. (2011). Alternative pathways of carbon dioxide fixation: Insights into the early evolution of life?. Annual Review of Microbiology. https://doi.org/10.1146/annurev-micro-090110-102801.PubMedGoogle Scholar
- Garratt, L. C., Ortori, C. A., Tucker, G. A., Sablitzky, F., Bennett, M. J., & Barrett, D. A. (2005). Comprehensive metabolic profiling of mono- and polyglutamated folates and their precursors in plant and animal tissue using liquid chromatography/negative ion electrospray ionisation tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 19(17), 2390–2398. https://doi.org/10.1002/rcm.2074.CrossRefPubMedGoogle Scholar
- Huang, L., Zhang, J., Hayakawa, T., & Tsuge, H. (2001). Assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring 5-methyltetrahydrofolate and tetrahydrofolate using high-performance liquid chromatography with fluorescence detection. Analytical Biochemistry, 299(2), 253–259. https://doi.org/10.1006/abio.2001.5421.CrossRefPubMedGoogle Scholar
- Marcellin, E., Behrendorff, J. B., Nagaraju, S., DeTissera, S., Segovia, S., Palfreyman, R., et al. (2016). Low carbon fuels and commodity chemicals from waste gases—Systematic approach to understand energy metabolism in a model acetogen. Green Chemistry, 18, 3020–3028. https://doi.org/10.1039/C5GC02708J.CrossRefGoogle Scholar
- Valgepea, K., de Souza Pinto Lemgruber, R., Meaghan, K., Palfreyman, R. W., Abdalla, T., Heijstra, B. D., et al. (2017). Maintenance of ATP homeostasis triggers metabolic shifts in gas-fermenting acetogens. Cell Systems, 4, 505–515. https://doi.org/10.1016/j.cels.2017.04.008.CrossRefPubMedGoogle Scholar