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

Abstract The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of 13 C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the 13 C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1- 13 C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the 13 C enrichment. The approach to correct for these effects in metabolic flux studies using δ 13 C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and 13 C-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 13 C in proteinogenic amino acids over the whole range of applied concentrations of [1- 13 C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in 13 C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.