Metabolic engineering of isopropyl alcohol‐producing Escherichia coli strains with ¹³C‐metabolic flux analysis

Abstract

Metabolic engineering of isopropyl alcohol (IPA)-producing Escherichia coli strains was conducted along with C-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. C-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. C-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 CO 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 C-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 C-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.

Effect of precise control of flux ratio between the glycolytic pathways on mevalonate production in Escherichia coli

“…Because NADPH for mevalonate production is insufficient when the EMP flux was 50% or more, the excess As for other metabolites that require NADPH as reductive power for their synthesis, the relationship between the flux ratio and their yields were investigated. Isopropyl alcohol (IPA) is a valuable precursor for the synthesis of various chemicals, such as polypropylene (Hanai et al, 2007;Okahashi et al, 2017). IPA is synthesized from three molecules of acetyl-CoA using one molecule of NADPH.…”

13C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in Escherichia coli

“…Concentrations of glucose, acetate, formate, ethanol, lactate, and succinate in the culture were measured using a high-performance liquid chromatography system (Shimadzu, Kyoto, Japan) with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, United States). The detailed method was described in Okahashi et al (2017). The detection limits were 5 mM for ethanol and 1 mM for organic acids including lactate, acetate, formate, and succinate.…”

13C Metabolic Flux Analysis of Escherichia coli Engineered for Gamma‐Aminobutyrate Production

“…The introduction of nitrogen‐starved culture condition to reduce the consumption of NADPH for biomass synthesis increased isopropyl alcohol yield and production. [ 15 ]…”