Robustifying Experimental Tracer Design for(13)C-Metabolic Flux Analysis.

(13)C metabolic flux analysis (MFA) has become an indispensable tool to measure metabolic reaction rates (fluxes) in living organisms, having an increasingly diverse range of applications. Here, the choice of the(13)C labeled tracer composition makes the difference between an information-rich experiment and an experiment with only limited insights. To improve the chances for an informative labeling experiment, optimal experimental design approaches have been devised for(13)C-MFA, all relying on some a priori knowledge about the actual fluxes. If such prior knowledge is unavailable, e.g., for research organisms and producer strains, existing methods are left with a chicken-and-egg problem. In this work, we present a general computational method, termed robustified experimental design (R-ED), to guide the decision making about suitable tracer choices when prior knowledge about the fluxes is lacking. Instead of focusing on one mixture, optimal for specific flux values, we pursue a sampling based approach and introduce a new design criterion, which characterizes the extent to which mixtures are informative in view of all possible flux values. The R-ED workflow enables the exploration of suitable tracer mixtures and provides full flexibility to trade off information and cost metrics. The potential of the R-ED workflow is showcased by applying the approach to the industrially relevant antibiotic producer Streptomyces clavuligerus, where we suggest informative, yet economic labeling strategies.