Abstract
13C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct 13C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal 13C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using 13C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs). Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution.
Highlights
Metabolic flux analysis (MFA) is a tool driving metabolic engineering through a detailed understanding of intracellular carbon flux distributions in various organisms [1,2,3,4,5]
The E. coli cells were repeatedly collected from the culture, and the 13C enrichment of proteinogenic amino acids (PAAs) and free amino acids (FAAs) were determined using the gas chromatography-mass spectrometry (GC-MS) analysis
The faster turnover rate for FAAs is preferable for an MFA study of batch and fed-batch culture [12,13,14,15], it remains unclear whether a reliable result can be produced by FAAs-based MFA
Summary
Metabolic flux analysis (MFA) is a tool driving metabolic engineering through a detailed understanding of intracellular carbon flux distributions in various organisms [1,2,3,4,5]. The smaller pool sizes and large compositional variations of FAAs are expected to affect the design of an MFA experiment [18]. This is because a confidence interval of flux estimation depends on a set of measureable FAAs. The relationship between the set of measurable data and resulting confidence intervals are not well investigated from the aspect of experimental data. The results of MFA demonstrated that the 13C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution
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