Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses

Abstract

To obtain an efficient ethanologenic Escherichia coli strain, we reduced the functional space of the central metabolic network, with eight gene knockout mutations, from over 15,000 pathway possibilities to 6 pathway options that support cell function. The remaining pathways, identified by elementary mode analysis, consist of four pathways with non-growth-associated conversion of pentoses and hexoses into ethanol at theoretical yields and two pathways with tight coupling of anaerobic cell growth with ethanol formation at high yields. Elimination of three additional genes resulted in a strain that selectively grows only on pentoses, even in the presence of glucose, with a high ethanol yield. We showed that the ethanol yields of strains with minimized metabolic functionality closely matched the theoretical predictions. Remarkably, catabolite repression was completely absent during anaerobic growth, resulting in the simultaneous utilization of pentoses and hexoses for ethanol production.

FIG. 1.

Metabolic map of E. coli central metabolic network. Deleted reactions in TCS083 are shown next to the symbol X. Listed are glucose-6-phosphate-1-dehydrogenase (PPP1, zwf), NADH dehydrogenase II (OPM4r, ndh), NAD/NADP-dependent malate enzyme (ANA2, sfcA/maeB), d-lactate dehydrogenase (FEM3, ldhA), fumarate reductase (TCA10, frdA), pyruvate oxidase (FEM2, poxB), and phosphate acetyltransferase (FEM7, pta). ETOH, ethanol.

FIG. 2.

Effect of reaction deletions on the numbers of anaerobic EMs for growth on xylose (A and B) and glucose (C and D). The bars in panels A and C specify the numbers of EMs for strains with deletions of the indicated genes. In each group of bars, the numbers of (i) total modes, (ii) modes that make ethanol, (iii) modes that produce biomass, and (iv) modes that make both biomass and ethanol are listed. The possible maximal and minimal ethanol and biomass yields for xylose (B) and glucose (D) are shown. Note that the minimal yields are pushed toward the upper theoretical limit with increasing numbers of deleted genes. ETOH, ethanol.

FIG. 3.

PCR of deleted genes for TCS083 and CT1101, with the wild type used as a positive control. Panels A and C show deleted genes tested for TCS083, including zwf, ndh, sfcA, maeB, ldhA, frdA, poxB, and pta. Panels B and D display deleted genes tested for CT1101, including zwf, ndh, sfcA, maeB, ldhA, frdA, poxB, pta, glk, manX, and ptsG. For each gene tested, the left lane shows the location of an amplified gene for the wild type and the right lane for the mutant. A shift to a smaller band size occurring in a lane of a mutant indicates that the tested gene is deleted. The arrow in each lane points to the location of the expected band size.

FIG. 4.

Time profiles for glucose, xylose, cell dry weight (cdw), and ethanol for the wild type (upper three panels) and mutant TCS083/pLOI297 (lower three panels). The strains were anaerobically cultivated in controlled 10-liter bioreactors where they were sparged with nitrogen. The initial sugar concentration was 80 g/liter. In an experiment with mixed sugars, 40 g/liter of each sugar was provided. Note that the figure shown is representative of a single-batch bioreactor run of duplicate experiments and that each data point represents the mean ± standard deviation of three independent measurements.

FIG. 5.

Time profiles for glucose, xylose, cell dry weight (cdw), and ethanol for mutant CT1101/pLOI297 growing on a sugar mixture (lower panel) (see the legend to Fig. 4 for growth conditions). The relationship between consumed xylose and consumed glucose is shown for anaerobic growth (upper left panel) and aerobic growth (upper right panel) for the wild-type MG1655 and mutant strains TCS083 and CT1101 and a 1:4 initial mixture of strains TCS083 and CT1101.

FIG. 6.

Growth characteristics of different E. coli strains, including BL21 MDS42, MG1655, TCS062, and TCS083. (A) Specific growth rates. (B) Biomass yields on glucose. (C) Acetate yields on glucose. The experiments were conducted in baffled shake flasks containing defined medium supplied with various pentoses and hexoses under aerobic conditions. Each value represents the mean ± standard deviation of the results of triplicate experiments.