(562d) Design Principles for 13C-Metabolic Flux Analysis Using Elementary Metabolite Unit Basis Vectors (EMU-BV)

Metabolic flux analysis (MFA) is a powerful tool for elucidating in vivo fluxes in microbial and mammalian systems. A key step in ¹³C-MFA is the selection of an appropriate isotopic tracer to observe fluxes in a proposed network model. Despite the importance of MFA in metabolic engineering and beyond, current approaches for tracer experiment design are still largely based on trial-and-error. The lack of a rational methodology for selecting isotopic tracers prevents MFA from achieving its full potential. Here, we introduce a new technique for tracer experiment design based on the concept of elementary metabolite unit basis vectors (EMU-BV). We demonstrate that any metabolite in a network model can be described as a linear combination of so-called EMU basis vectors, where the corresponding coefficients indicate the fractional contribution of the EMU-BV to the product metabolite. The strength of this approach is the complete decoupling of substrate labeling, i.e. the EMU-BV, from the dependence on free fluxes, i.e. the coefficients. In this work, we demonstrate that flux observability depends inherently on the number of independent EMU-BV and the sensitivities of coefficients with respect to free fluxes. Specifically, the number of independent EMU-BV places hard limits on how many free fluxes in a model can be determined. This constraint is used as a guide for selecting feasible substrate labeling. In three example models, we demonstrate that by maximizing the number of independent EMU-BV the observability of a system is optimized. Inspection of sensitivities of the coefficients with respect to free fluxes provides additional constraints for a proper selection of tracers. In summary, this work provides a fresh perspective on an important topic in metabolic engineering and gives practical guidelines and design principles for a priori selection of isotopic tracers for ¹³C-MFA studies.