Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.
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