Metabolic evolution of energy-conserving pathways for succinate production in Escherichia coli

Abstract

During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzyme-dominated CO(2) fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.

Fig. 1.

Comparison of mixed acid pathway for glucose fermentation in native E. coli and energy-conserving succinate production pathway in recombinant E. coli. (A) Mixed acid pathway: Phosphoenolpyruvate (PEP) is the primary source of energy for glucose uptake and phosphorylation. Succinate production is primarily determined by partitioning of carbon at the PEP node. In native E. coli, half of the pyruvate produced from PEP is used for this glucose uptake. The four solid stars indicate metabolic steps that have been blocked by constructed deletions. Dotted arrows indicate steps that are either nonfunctional or expected to have reduced carbon flow as a result of these deletions. Pyruvate (oval) is shown twice but represents a single metabolic pool. (B) Energy-conserving succinate production pathway: An acquired mutation in ptsI inactivating the PEP-dependent glucose uptake systems was functionally replaced by increased expression of the galP permease and glucokinase (glk). Additional mutations increased the expression of phosphoenolpyruvate carboxykinase (pck) sufficiently to make this enzyme the dominant route for carboxylation, increasing net production of ATP and increasing the pool of PEP available for succinate production. Bold solid arrows indicate the primary route for succinate production in KJ060 and related strains. Some intermediate steps in glycolysis have been omitted for clarity. G6P, glucose 6-phosphate; PEP, phosphoenolpyruvate.

Fig. 2.

Carboxylation pathways potentially available for succinate production in E. coli. (A) PEP carboxylase (primary fermentative route). (B) NADH-linked malic enzyme (gluconeogenic). (C) NADPH-linked malic enzyme (gluconeogenic). (D) PEP carboxykinase (gluconeogenic). Genes encoding carboxylation activities are shown in bold. The energy in phosphoenolpyruvate is lost in the first pathway but conserved as ATP in the other three pathways. Pathway D is the dominant route for carboxylation in our succinate-producing strains of E. coli and in succinate-producing rumen bacteria. PEP, phosphoenolpyruvate; OAA, oxaloacetic acid.