Determination of full C-13 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 pur poses. C-13 NMR spectroscopy is a key technique in the experimental determi nation of isotopomer distributions. However, only subsets of isotopomers ca n be quantitated using this technique due to redundancies in the scalar cou pling patterns and due to invisibility of the C-12 isotope in NMR. Therefor e, we developed and describe in this paper a H-1 NMR spectroscopy method th at allows to determine the complete isotopomer distribution in metabolites having a backbone consisting of up to at least four carbons. The proposed p ulse sequences employ up to three alternately applied frequency-selective i nversion pulses in the C-13 channel. In a first application study, the comp lete isotopomer distribution of aspartate isolated from [1-C-13]ethanol-gro wn Ashbya gossypii was determined. A tentative model of the central metabol ism of this organism was constructed and used for metabolic flux analysis. The aspartate isotopomer NMR data played a key role in the successful deter mination of the flux through the peroxisomal glyoxylate pathway. The new NM R method can be highly instrumental in generating the data upon which isoto pomer labeling experiments for flux analysis, that are becoming increasingl y important, are based. (C) 2000 Elsevier Science B.V. All rights reserved.