The quantitative knowledge of these fluxes is of high importance in deciphering cellular functions and guiding rational strain engineering for industrial biotechnology. 13C metabolic flux analysis is currently the most sophisticated and reliable method for determining intracellular reaction rates and has become a widely used tool in systems bio(techno)logy. Because the demand for quantitative metabolic flux data is increasing, the quality and quantity of analytical results increases, too. Especially Inhibitors,research,lifescience,medical new protocols for cell cultivation,

sample handling, and sample analysis by mass spectroscopy are driving these developments [1]. While early publications rarely presented more than six flux distributions, the first examples exist that include 30 [2] or even more than 150 data sets [3,4]. Currently, available software tools for 13C-based flux Inhibitors,research,lifescience,medical analysis, such as FiatFlux [5], OpenFlux [6], 13CFLUX [7] and the updated version 13CFLUX2 [8] require (intensive) user interactions and expert knowledge, as GC-MS data quality and relevance have to be assessed. Yet, these interactive data evaluation workflows can become limiting when hundreds of data sets have to be handled. Ideally, automated software versions would be available that calculate high quality flux distributions in the metabolic BIX 1294 network under study Inhibitors,research,lifescience,medical using labeling and physiological data with a minimal need of user interaction.

Consequently, in this study we aimed to translate the user interactions and expert knowledge required for the analysis into quantifiable criteria Inhibitors,research,lifescience,medical suited for the automated determination of intracellular flux distributions. 1.1. Metabolic Flux Analysis Metabolic flux analysis (MFA) is applicable for systems that are in a pseudo-steady state. Under this condition, the differential Inhibitors,research,lifescience,medical equation system of metabolite mass balances reduces to a linear equation system, which relies solely on the known stoichiometry of the biochemical reaction network. However, the system is often underdetermined if only constrained by extracellular uptake and secretion rates and the growth rate of the cell, with the consequence that not all fluxes, especially

those of parallel pathways and cyclic fluxes in the network, can be resolved. Additional constraints can be gained from growth experiments, in which and cellular growth substrates labeled with stable isotope tracers like 13C are fed to the biological system [9]. The labeled (carbon) atoms are then distributed over the metabolic network by incorporation into intracellular metabolites and conserved in amino acids located in proteins, whose labeling patterns can be measured by nuclear magnetic resonance (NMR) [10] or mass spectrometry (MS) instruments [11]. The rationale behind these 13C tracer experiments is that the carbon backbones of the metabolites are often manipulated differently by alternative pathways, resulting in distinct 13C labeling patterns of the metabolites.