Metabolic Flux Analysis of Isopropyl Alcohol-Producing Escherichia coli

The replacement of petrochemicals with biofuels produced from renewable resources has attracted growing attention. The dehydration of isopropyl alcohol (IPA), a secondary alcohol, produces a useful material, propylene. Its polymerized form, polypropylene, is widely used to manufacture commodity plastics because of its superior characteristics such as high mechanical strength and heat and chemical resistance. To accomplish the bioproduction of IPA, we developed an IPA-producing strain of Escherichia coli by expressing heterologous acetoacetate decalboxylase and IPA dehydrogenase. To further increase IPA production, we constructed an Entner-Doudoroff (ED) pathway-dependent IPA-producing strain (hereafter termed “ED strain”) by enhancing the NADPH supply required for IPA production. In the ED strain, pgi and gnd were deleted to catabolize glucose via the NADPH-generating oxidative pentose phosphate (PP) pathway. An understanding of the metabolic state of the cell provides information that is valuable in developing metabolic engineering strategies. ¹³C metabolic flux analysis (MFA) is a powerful tool to quantify the intracellular carbon flow. In this study, we performed MFA in the ED strain to elucidate its metabolic state.

The ED strain was aerobically cultured at 30°C in M9 minimal medium containing ¹³C-labeled glucose, which is composed of [1-¹³C] and [U-¹³C] glucose in the ratio of 1:1. For MFA, cells were harvested at the mid-exponential phase and ¹³C enrichments of proteinogenic amino acids were measured by gas chromatograph mass spectrometry. Analysis of  ¹³C enrichment of alanine indicated that glucose was catabolized through the ED pathway. Based on the MFA results, the most of the glucose was estimated to flow into the ED pathway via the PP pathway. Moreover, fluxes of the reactions corresponding to the deleted genes pgi and gnd were very small. This result indicated that the metabolic flux distribution in the upper part functioned as expected. In the TCA cycle, low flux through the glyoxylate shunt and high flux through the TCA cycle were estimated, in agreement with previous MFA results for wild type E. coli grown under aerobic culture conditions.