Elementary mode analysis for the rational design of efficient succinate conversion from glycerol by Escherichia coli

Abstract

By integrating the restriction of oxygen and redox sensing/regulatory system, elementary mode analysis was used to predict the metabolic potential of glycerol for succinate production by E. coli under either anaerobic or aerobic conditions. It was found that although the theoretical maximum succinate yields under both anaerobic and aerobic conditions are 1.0 mol/mol glycerol, the aerobic condition was considered to be more favorable for succinate production. Although increase of the oxygen concentration would reduce the succinate yield, the calculation suggests that controlling the molar fraction of oxygen to be under 0.65 mol/mol would be beneficial for increasing the succinate productivity. Based on the elementary mode analysis, the rational genetic modification strategies for efficient succinate production under aerobic and anaerobic conditions were obtained, respectively. Overexpressing the phosphoenolpyruvate carboxylase or heterogeneous pyruvate carboxylase is considered to be the most efficient strategy to increase the succinate yield.

Figure 1

Central metabolic network of glycerol in wildtype E. coli. The dashed arrows represent the particular pathways in anaerobic conditions. Reversible reactions are represented by a double-headed arrow. Key genes associated with the pathway are included.

Figure 2

Relationship between the yields of biomass and byproducts for the obtained elementary modes of E. coli under anaerobic conditions. (a) Succinate, (b) Lactate, (c) Acetate, (d) 1,2-Propanediol, (e) Formate, and (f) Ethanol. The enclosed regions represent the possible solution space. The fluxes were normalized by glycerol uptake rate and expressed as mol/mol (glycerol).

Figure 3

The optimum flux distribution of glycerol metabolism for succinate production in E. coli under anaerobic conditions when only ATP-dependent DHA kinase plays a function.

Figure 4

The alternative optimum flux distribution of glycerol metabolism for succinate production in E. coli under anaerobic conditions when only PEP-dependent DHA kinase plays a function and heterogeneous PEP carboxylase (pyc) are introduced and overexpressed in E. coli.

Figure 5

Relationship between the yields of biomass and byproducts for the obtained elementary modes of E. coli under aerobic conditions. (a) Succinate, (b) Lactate, (c) Acetate, and (d) Ethanol. The enclosed regions represent the possible solution space. The fluxes were normalized by glycerol uptake rate and expressed as mol/mol (glycerol).

Figure 6

The optimum flux distribution of glycerol metabolism for succinate production in E. coli under aerobic conditions.

Figure 7

Sensitivity of succinate yield to the relative fluxes at the (a) PEP node and (b) AcCoA node under aerobic conditions. The PEP node involves the catabolic reactions of R40 and R17 which are catalyzed by PEP carboxylase and pyruvate kinase, respectively. The AcCoA node involves the catabolic reactions of R22, R50, R44, and R53 which are catalyzed by citrate synthase, aldehyde dehydrogenase, malate synthase, and phosphate acetyltransferase, respectively.

Figure 8

Effect of oxygen consumption on the succinate production and biomass formation under aerobic conditions. The enclosed regions represent the possible solution space. The fluxes were normalized by glycerol uptake rate and expressed as mol/mol (glycerol).