13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry |
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| Authors: | Yongbo Yuan Tae Hoon Yang Elmar Heinzle |
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| Affiliation: | 1. USC — Universidade Sagrado Coração, Bauru, São Paulo, 17011-160, Brazil;2. UNESP — Univ Estadual Paulista, Campus de Botucatu, Instituto de Biociências, Departamento de Parasitologia, Botucatu, São Paulo, 18618-970, Brazil;3. UNESP — Univ Estadual Paulista, Campus de Botucatu, Instituto de Biociências, Departamento de Física e Biofísica, Botucatu, São Paulo, 18618-970, Brazil;4. UFRA — Universidade Federal Rural da Amazônia, Laboratório de Pesquisa Carlos Azevedo, Belém, Pará 66077-901, Brazil;1. College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China;2. College of Plant Protection, Agricultural University of Hebei, Baoding 071001, China |
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| Abstract: | 13C-based metabolic flux analysis (13CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured 13C 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 [1-13C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-13C] glucose. Decreasing the [1-13C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected 13C 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. |
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