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

Abstract

Phenol is an important chemical product that can be used in a wide variety of applications, and it is currently produced from fossil resources. Fermentation production of phenol from renewable biomass resources by microorganisms is highly desirable for sustainable development. However, phenol toxicity hampers phenol production in industrial microorganisms such as Escherichia coli. In the present study, it was revealed that culturing E. coli in the presence of phenol not only decreased growth rate, but also biomass yield. This suggests that phenol affects the carbon flow of the metabolism, but the mechanism is unknown. To investigate the effect of phenol on the flux distribution of central carbon metabolism, ¹³C-metabolic flux analysis (¹³C-MFA) was performed on cells grown under different phenol concentrations (0, 0.1, and 0.15%). ¹³C-MFA revealed that the TCA cycle flux reduced by 25% increased acetate production from acetyl-CoA by 30% in the presence of 0.1% phenol. This trend of flux changes was emphasized at a phenol concentration of 0.15%. Although the expression level of citrate synthase, which catalyzes the first reaction of the TCA cycle, does not change regardless of phenol concentrations, the in vitro enzyme activity assay shows that the reaction was inhibited by phenol. These results suggest that the TCA cycle flux decreased due to phenol inhibition of citrate synthase; therefore, ATP could not be sufficiently produced by respiration, and growth rate decreased. Furthermore, since carbon was lost as acetate due to overflow metabolism, the biomass yield became low in the presence of phenol.

Keywords: 13C-metabolic flux analysis; Escherichia coli; acetate overflow metabolism; citrate synthase; enzymatic assay; phenol toxicity.

FIGURE 1

Relationships between phenol concentration and Specific growth rate (A), and Maximum OD660 (B) on wild type E. coli BW25113. Closed circles and squares represent the maximum specific growth rate (h^-1) and the maximum OD₆₆₀ during the 24 h period.

FIGURE 2

Batch growth profiles of wild type E. coli under different phenol concentrations. Time courses of biomass (A), glucose (B), and acetate (C) concentrations. Closed circles, open squares, and closed triangles represent measurement data series during 24 h by 0, 0.1, and 0.15% of phenol concentrations, respectively.

FIGURE 3

Effect of pta gene deletion on phenol tolerance in E. coli. The open and closed symbols represent the OD₆₀₀ of wild type and Δpta strains at 0% (circle) and 0.15% (triangle) phenol existence, respectively.

FIGURE 4

Flux distributions of the downstream pathways at 0% (A), 0.1% (B), and 0.15% (C) phenol existence. The unit of fluxes is shown as mmol gDCW^-1 h^-1. Values in the boxes: values in the upper boxes, and values in the lower boxes are best fit values and 95% confidence interval. Color of arrows indicates relative comparison of best fit values of metabolic fluxes to those in the 0% phenol condition. The detailed flux distributions of the overall pathways were described in Supplementary Table S4.

FIGURE 5

Inhibition of citrate synthase (A) and PGI (B) activities by the phenol in vitro reaction.