Fig. 1: Overproduction of glycerol causes a growth burden in E. coli.

a Metabolic map of E. coli glycolysis and the synthetic glycerol pathway (orange). The synthetic pathway consists of two enzymes from S. cerevisiae: glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate phosphohydrolase 2 (GPP2). The genes encoding the two enzymes (gpd1 and gpp2) were expressed from a plasmid using the arabinose-inducible pBAD promoter for gpd1 and the constitutive promoter pJ23101 for gpp2. b Activity of the pBAD promoter at different arabinose levels. GFP fluorescence and OD₆₀₀ were measured in n = 2 plate reader cultures, and promoter activity was calculated as dGFP/dt/OD by regression analysis between 7 and 9 h. c Schematic of the control strategy for the synthetic glycerol pathway. GPP2 is expressed in excess to ensure that GPD1 is the rate-limiting step. GPD1 levels are varied by inducing the pBAD promoter with different amounts of arabinose. Size of boxes indicates enzyme levels, size of arrows indicate flux through the pathway. d gpd1 and gpp2 were expressed from a plasmid in an E. coli strain lacking glpK (base strain). The base strain was cultured in 96-well plates. Growth was measured in a plate reader at different induction levels of GPD1 (0, 0.1, 0.3, 0.5, 1, and 2% ara). Glycerol in the medium was measured after 24 h. Growth rates were determined by regression analysis between 5 and 10 h. Growth curves and dots show the means of n = 2 plate reader cultures. e Theoretical relationship between glycerol flux and growth rate based on flux balance analysis with a genome-scale model of E. coli metabolism (iML1515). Dots are growth rates and glycerol production rates measured in shake flask cultures of the base strain at 0, 0.1, and 0.5% ara. Source data are provided in the Source Data file.