Determination of full 13C isotopomer distributions for metabolic flux analysis using heteronuclear spin echo difference NMR spectroscopy

C-13-isotopomer labeling experiments play an increasingly important role in the analysis of intracellular metabolic fluxes for genetic engineering purposes. C-13 NMR spectroscopy is a key technique in the experimental determination of isotopomer distributions. However, only subsets of isotopomers can be quantitated using this technique due to redundancies in the scalar coupling patterns and due to invisibility of the C-12 isotope in NMR. Therefore, we developed and describe in this paper a H-1 NMR spectroscopy method that allows to determine the complete isotopomer distribution in metabolites having a backbone consisting of up to at least four carbons. The proposed pulse sequences employ up to three alternately applied frequency-selective inversion pulses in the C-13 channel. In a first application study, the complete isotopomer distribution of aspartate isolated from [1-C-13]ethanol-grown Ashbya gossypii was determined. A tentative model of the central metabolism of this organism was constructed and used for metabolic flux analysis. The aspartate isotopomer NMR data played a key role in the successful determination of the flux through the peroxisomal glyoxylate pathway. The new NMR method can be highly instrumental in generating the data upon which isotopomer labeling experiments for flux analysis, that are becoming increasingly important, are based. (C) 2000 Elsevier Science B.V. All rights reserved.