title: Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS.
abstract: Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.
authors.affiliation: Systems Biology Group, Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki 210-8681, Japan. shintaro_iwatani@ajinomoto.com
authors.forename: Shintaro
authors.initials: S
authors.lastname: Iwatani
authors.forename: Stephen
authors.initials: S
authors.lastname: Van Dien
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Shimbo
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kubota
authors.forename: Naoko
authors.initials: N
authors.lastname: Kageyama
authors.forename: Daigo
authors.initials: D
authors.lastname: Iwahata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyano
authors.forename: Kazuo
authors.initials: K
authors.lastname: Hirayama
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Usuda
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Shimizu
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Matsui
mesh_terms: D000596:Amino Acids / Q000096:biosynthesis* / Q000378:metabolism*; D019149:Bioreactors; D053898:Biosynthetic Pathways; D002247:Carbon Isotopes; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000378:metabolism; D008954:Models, Biological; D011506:Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D011506:Proteins