Metabolic Engineering for Upgrading Isopropyl Alcohol Production By Escherichia coli Based on 13c-Metabolic Flux Data

[Introduction] Isopropyl alcohol (IPA) is a valuable precursor for the bioproduction of polypropylene. The metabolically engineered Escherichia coli strain producing IPA has been constructed by heterologous expression of IPA biosynthesis-related genes of Clostridium. Although the productivity has been improved by the optimization of culture conditions and the regulation of gene expressions, metabolic bottlenecks in the IPA production are remains unclear. In this study, ¹³C-metabolic flux analysis (MFA) was performed for the IPA-producing E. coli strains to improve the IPA productivity.

[Materials and methods]

The engineered E.coli strains were constructed by introducing pUC119 plasmid harboring the GAPDH promoter and target genes. Cells were aerobically cultured at 30°C in Sakaguchi-flask with 100 mL of M9 medium containing [1-¹³C]glucose : [U-¹³C]glucose = 7 : 3 at growth phase. Non-growth associated IPA production was performed by inoculating grown cells to nitrogen-free medium containing [1-¹³C]glucose : [U-¹³C]glucose = 6 : 4. ¹³C-MFA of growth and non-growth phase was performed by measuring ¹³C-labeling of proteinogenic amino acids and intermediate metabolites, respectively. Flux distributions and 95% confidence intervals were calculated by “OpenMebius”. 

[Results and Discussion]

The E.coli strain expressing IPA biosynthesis pathway was constructed by the overexpression of endogenous atoDAB genes, adc gene from C. acetobutylicum, and IPAdh gene from C. beijerinckii (Ref strain). The fermentation test by the flask cultivation showed that acetate instead of IPA was produced at mid-log phase (OD₆₀₀~1). The metabolic bottlenecks was investigated by the ¹³C-MFA. Proteinogenic amino acids were obtained from the E. coli cells cultured in the medium containing ¹³C-glucose. A metabolic flux distribution was estimated from the ¹³C-labeling patterns determined by GC-MS. The results indicated that NADPH regeneration rate was insufficient for IPA production since 1 mol of IPA is synthesized from 2 mol of acetyl-CoA (AcCoA) and 1 mol of NADPH. In this study, the NADPH supply was improved by two approaches. The first approach was the activation of NADPH regeneration by the glycolysis via the ED pathway. ED strain was constructed by the deletion of pgi, gnd, and gntR and overexpression of zwf. The ED strain produced IPA at mid-log phase with the yield of 0.13 mol/mol glucose. The result of ¹³C-MFA confirmed that an over-regeneration of NADPH was attained by the ED pathway-dependent glucose catabolism. The second approach was IPA production under the stationary phase to reduce NADPH demand for cell growth. The cultivation of Ref strain under a nitrogen-starving condition successfully increased the IPA yield to 0.20 mol/mol glucose. The metabolic flux distribution at the non-growing state revealed that NAPDH for IPA synthesis was supplied by the oxidative pentose phosphate pathway. Furthermore, IPA production with the yield of 0.55 mol/mol glucose was attained by the combination of two approaches. The level of flux through pyruvate to AcCoA was reached 95% of the theoretical maximal level. The analysis of intracellular NADPH levels showed that there is a positive correlation between specific IPA production rate and NADPH pool size, suggesting that the large NADPH pool size was a driving force for the IPA biosynthesis.