New technique dramatically reduces data acquisition time and amplifies metabolite identification.
More rapid and improved estimates of metabolic fluxes in cells are possible thanks to a new technique that combines 13C-metabolic flux analysis (13C-MFA) with non-uniform sampling nuclear magnetic resonance (NMR) spectroscopy data.
Key insights into metabolic networks provided by the new technique could be used for synthetic biology-based efforts to modify living systems for production of metabolites or other products of interest, such as biofuels or fine chemicals.
The use of 13C-MFA can provide key insights into the metabolic networks of microbial cells that are used for producting biofuels or valuable chemicals. This technique can be combined with either NMR spectrometry or mass spectrometry to infer metabolic fluxes within cells based on the characteristic rearrangement of 13C tracers through metabolic pathways. However, position-specific 13C-labeling of metabolites has been particularly difficult to obtain using conventional NMR or mass spectrometry techniques, hindering accurate estimations of metabolic fluxes. To overcome this problem, researchers from the Department of Energy’s Environmental Molecular Sciences Laboratory (EMSL; a national scientific user facility), Washington State University, Duke University Medical Center, and Miami University developed a new technique that combines 13C-MFA with non-uniform sampling (NUS), which dramatically reduces the time required to collect high-resolution NMR data. NUS techniques acquire only a subset of NMR data points and use sophisticated reconstruction methods that ultimately allow extraction of complete sets of chemical shift information. Using EMSL’s 600 MHz and 800 MHz NMR spectrometers, the research team demonstrated that their approach provides detailed information about position-specific labeling patterns that can be incorporated into metabolic flux models. By enabling more accurate estimations of metabolic fluxes in complex biological systems, the new technique could shed light on environmental nutrient cycling and enhance synthetic biology-based engineering efforts to modify living systems for production of metabolites or other products of interest, such as biofuels or fine chemicals.
BER PM Contact
Paul Bayer, SC-23.1, 301-903-5324
Patrick Reardon, EMSL, 509-371-7673, Patrick.Reardon@pnnl.gov
This work was supported by the U.S. Department of Energy’s Office of Science, Office of Biological and Environmental Research, including support of EMSL, an Office of Science user facility; a William Wiley distinguished postdoctoral fellowship from EMSL; and an EMSL intramural research project entitled “Development of an Integrated EMSL Mass Spectrometry and Nuclear Magnetic Resonance Metabolic Flux Analysis Capability in Support of Systems Biology: Test Application for Biofuels Production.”
Reardon, P. N., C. L. Marean-Reardon, M. A. Bukovec, B. E. Coggins, and N. G. Isern. 2016. “3D TOCSY-HSQC NMR for Metabolic Flux Analysis Using Non-Uniform Sampling,” Analytical Chemistry 88(5), 2825-31. DOI: 10.1021/acs.analchem.5b04535. (Reference link) .
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