Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of C-13 enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the C-13 enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-C-13]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the C-13 enrichment. The approach to correct for these effects in metabolic flux studies using delta C-13 measurement by GC-C-IRMS uses two parallel experiments applying substrate with natural abundance and C-13-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of 13C in proteinogenic amino acids over the whole range of applied concentrations of [1-C-13]glucose. The current findings indicate a great potential of GC-C-IRMS for labeling quantification in C-13 metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors. (c) 2008 Elsevier Inc. All rights reserved.