Analyzing the effects of metabolic state and perturbations on genetic device performance by metabolomics and 13C-labeled metabolic flux analysis

Characterization of engineered cellular systems is required for understanding the metabolic state and associated metabolic burden that cells endure from the implementation of genetic devices and circuits. Competition for shared resources, such as cellular macromolecules, in genetic circuits can impair essential cellular functions and thus limits the robustness of engineered cellular systems to be implemented in therapeutic and medicinally relevant synthetic biology applications. To characterize the metabolic state and burden of genetic circuits at the systems-level, metabolomics and 13C-labeled metabolic flux analysis (13CMFA), with a focus on bioenergetics and central carbon metabolism, can be performed. Changes in the concentrations of key metabolites, such as G6P and ATP, measured by GC-MS and LC-MS/MS and analyzed in metabolomics, point directly to the metabolic state of a cell. Similarly, 13CMFA estimates the intracellular metabolic fluxes using the measured concentrations of key metabolites obtained through cellular metabolism and by feeding cells isotopically labeled substrates. The estimated changes in metabolic fluxes in response to competition for shared resources can shed light on the complex interplay among gene expression, protein expression, and the environment. Genetic variants designed to probe the tolerance of central endogenous genes to metabolic perturbation are being tested and analyzed using both metabolomics and 13C-MFA. Finally, strategies to engineer autoregulation on shared resources are being investigated for minimizing metabolic burden.