Анализ метаболических потоков с использованием 13 С изотопов ( 13 C-MFA): 1. Экспериментальные основы метода и состояние исследований

Abstract

Abs tract — A quantitatively characterized picture of intracellular metabolite fluxes provides useful information for both fundamental and applied investigations of various metabolic pathways and individual reactions, for the description of the cell metabolic status, metabolic differences between strains, and developing of the strategy for improving characteristics of strains producing biologically active compounds. A kit of methods aimed at the cell metabolic state characterization by determining the intracellular metabolite fluxes has got a common name of Metabolic Flux Analysis (MFA), or fluxomics. Together with so-called X-omic technologies (genomics, transcriptomics, proteomics, and metabolomics), fluxomics is a part of the recent arsenal of system biology methods. 13 C-based Metabolic Flux Analysis ( 13 C-MFA) dealing with 13 C(heavy carbon)-labeled substrates is one of the most developed approaches to the analysis of intracellular metabolic fluxes in vivo under steady-state conditions. The application of 13 C-MFA requires a combined functioning of experts in biochemistry, applied mathematics and NMR/mass-spectrometry. In view of this feature, authors prepared a three-part review to highlight various, but equally important fluxomic aspects. In the first part represented bellow, the attention is paid to the basic principles of 13 C-MFA like: design of stoichiometric model of target organism; experiments with labeled substrate which permits to obtain the basic portion of information for intracellular fluxes calculation; and experimental data extraction. Principles of mathematical modeling of experiments with labeled substrates together with quantitative carbon flux estimation and assessment of their statistic reliability are discussed in the second part. The final part reviews the recent achievements in fundamental and applied investigations of bacterial metabolism reached with the 13 C-MFA assistance.