Adaptive_control_of_synthetic_metabolic_pathways_alleviates_metabolic_burden_by_stabilizing_E.coli_glycolysis

Expressing synthetic metabolic pathways is a burden for the producing strain because they consume energy and biomass precursors from metabolism. However, engineering feedback control in synthetic pathways can mitigate the metabolic burden and improve production. But how engineered feedback control interacts with metabolism of producer strains is currently not clear. Here, we show that transcriptional feedback control enables robust overproduction of glycolysis-derived chemicals by stabilizing glycolysis in Escherichia coli. Using synthesis of glycerol and β-carotene as case studies, we found that transcriptional regulation of E. coli glycolysis is responsible for the metabolic burden of overproducing glycolysis-derived chemicals. E. coli glycolysis falsely responds transcriptionally to decreased glycolytic intermediates by activating gluconeogenesis and shutting down glycolysis expression. Ensemble modelling predicted new regulatory logics that avoid this miss-regulation. We tested the predicted logics and show by metabolomics and proteomics how they stabilize glycolysis. Our results show that burden emerged from changes in metabolite levels drives a false transcriptional response of the host. An engineered feedback of the synthetic pathway saves from this miss-regulation.