Metabolic engineering of Escherichia coli for efficient conversion of glycerol to ethanol

Abstract

Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.

FIG. 1.

Effects of deletion of multiple reactions on the number of EMs (A) and the biomass and ethanol yields (B). ETOH, ethanol.

FIG. 2.

(A) 3-D solution space of mutant TCS099/pLOI297 with the ethanol yield expressed as a function of the biomass yield and oxygen consumption on glycerol. This solution space of the mutant is enclosed in the area constructed from the lines connecting clusters of EMs and situated on the shaded plane. (B) Projection of the 3-D solution space of the mutant on the 2-D space of the ethanol and biomass yields. The inverse relationship of the ethanol and biomass yields on glycerol of the mutant is indicated by a perfect-fit line connecting clusters of EMs as follows: Y_EtOH/GLY = −0.64·Y_X/GLY + 0.50 (R² = 1), where Y_EtOH/GLY is the ethanol yield on glycerol (in g ethanol/g glycerol) and Y_X/GLY is the biomass yield on glycerol (in g biomass/g glycerol). (C) Projection of the 3-D solution space of the mutant on the 2-D space of the ethanol yield and the oxygen consumption on glycerol. In each panel, the circles, filled triangles, and squares indicate clusters of EMs, while the open triangles and open diamonds indicate experimental data for mutant TCS099/pLOI297 and TCS099/pLOI297 evolved mutants, respectively, for different k_La values. The open diamond located at the point with the lowest ethanol yield and highest biomass yield indicates the result of the experiment conducted with an elevated k_La (2/min).

FIG. 3.

Dynamic changes in the specific growth rates of wild-type strain MG1655/pLOI297 and TCS099/pLOI297 through metabolic evolution.

FIG. 4.

Percentages of DO for the wild type and the mutant and their evolved derivatives in batch bioreactors during the first 24 h at a k_La of 0.3/min. Line 1, MG1655/pLOI297; line 2, TCS099/pLOI297; line 3, MG1655 e50rep3/pLOI297; line 4, TCS099 e50rep1/pLOI297.

FIG. 5.

Time profiles for glycerol concentration, ethanol concentration, and cell dry weight for the wild type (A), evolved wild-type strain MG1655 e50rep3/pLOI297 (B), mutant TCS099/pLOI297 (C), and evolved mutant TCS099 e50rep1/pLOI297 (D). The dashed vertical line in each panel indicates the time that the DO level in the batch bioreactor reached zero. The strains were cultivated in controlled batch bioreactors using defined medium containing 40 g/liter of glycerol. The growth conditions were microaerobic with a k_La of 0.3/min. cdw, cell dry weight.

FIG. 6.

(A) Biomass production for wild-type strain MG1655/pLOI297, evolved wild-type strain MG1655 e50rep3/pLOI297, mutant TCS099/pLOI297, and evolved mutant TCS099 e50rep1/pLOI297 at a k_La of 0.3/min. (B) Local ethanol yield. The local ethanol yield [] was determined by using the following formula: where C_EtOH(t_i) and C_glycerol(t_i) are the concentrations of ethanol and glycerol (in g/liter) at time t_i, respectively.

FIG. 7.

Ethanol yields (A) and ethanol volumetric productivities (B) of variants of evolved derivatives at a k_La of 0.3/min.

FIG. 8.

Effect of k_La on the biomass production rate (A), on the ethanol production rate (B), on the glycerol consumption rate (C), and on the oxygen consumption rate (D) for MG1655/pLOI297, TCS099/pLOI297, and TCS099 e50rep1/pLOI297.

FIG. 9.

Effect of k_La on the ethanol yield (A) and on the specific ethanol productivity (B) for MG1655/pLOI297, TCS099/pLOI297, and TCS099 e50rep1/pLOI297.

FIG. 10.

Profiles of cell dry weight for wild-type strain MG1655/pLOI297 (A), mutant TCS099/pLOI297 (B), and evolved mutant TCS099 e50rep1/pLOI297 (C) at k_La values of 0.15/min (open circles), 0.2/min (open triangles), and 0.3/min (open squares).

FIG. 11.

Concentrations of accumulated by-products of the wild type and the mutant and their evolved derivatives for microaerobic growth at a k_La of 0.3/min.