LAUSR

Metabolic engineering of isopropyl alcohol (IPA)‐producing Escherichia coli strains was conducted along with 13C‐metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. 13C‐MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen‐starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. 13C‐MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen‐starved condition relative to glucose uptake rate. To prevent CO2 release in the 6‐phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6‐phosphate dehydrogenase (G6PDH) and Entner‐Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen‐starved culture condition with the metabolic redirection. The 13C‐MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that 13C‐MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine‐tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.