Robustifying Experimental Tracer Design for¹³C-Metabolic Flux Analysis

¹³C 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 the¹³C 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 for¹³C-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.