45. Low Labeling ¹³C Metabolic Flux Analysis of Saccharomyces cerevisiae 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 ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated in this paper. We measured the ¹³C enrichment of proteinogenic amino acids in hydrolyzates using GC–C–IRMS from a series of parallel batch cultivations of Saccharomyces cerevisiae, which was cultured by mixtures of natural glucose and [1−¹³C] glucose, containing 0, 0.5, 1, and 2 % [1−¹³C] glucose, respectively. By decreasing the [1−¹³C] glucose content, kinetic isotope effects played an increasing role but could be corrected. The ¹³C metabolic algorithm and matrix algorithms were optimized in this study. The central metabolism of vivo fluxes were determined by the calculation method optimization. The obtained flux distribution was similar to published results, which obtained from GC–MS method using conventional high labeling (99 %). The GC–C–IRMS-based method involves low labeling (0.5 %) degree of expensive tracer substrate, and suits well for larger laboratory and industrial pilot-scale fermentations.