13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry.
(13)C-based metabolic flux analysis ((13)CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured (13)C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-(13)C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-(13)C] glucose. Decreasing the [1-(13)C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected (13)C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.
Biochemical Engineering Institute, Saarland University, Campus A1.5, 66123 Saarbrücken, Germany.
Yongbo
Y
Yuan
Tae Hoon
TH
Yang
Elmar
E
Heinzle
D000596:Amino Acids / Q000378:metabolism; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D005285:Fermentation; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D053858:Metabolic Networks and Pathways
D000596:Amino Acids; D002247:Carbon Isotopes; D005947:Glucose