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 energyconserving 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 enzymedominated 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.biocatalyst ͉ metabolic engineering ͉ succinic acid S uccinate, a four-carbon dicarboxylic acid, is currently used as a specialty chemical in the food, agricultural, and pharmaceutical industries (1). Succinic acid can also serve as a starting point for the synthesis of many commodity chemicals used in plastics and solvents with a potential global market of $15 billion (2). Although succinate is primarily produced from petroleum, recent increases in costs have generated considerable interest in the fermentative production of succinate from sugars using Escherichia coli and other biocatalysts (2, 3).The yield of succinate from glucose fermentation is primarily determined by carbon partitioning at the phosphoenolpyruvate (PEP) node (Fig. 1A). In rumen bacteria such as Anaerobiospirillum succiniciproducens (4), Actinobacillus succinogenes (5, 6) and Mannheimia succiniciproducens (7,8), more than half of the phosphoenolpyruvate formed from glucose is carboxylated to oxaloacetate and converted to succinate, the primary fermentation product. However, requirements for complex nutrients by these bacteria increase both the cost and process complexity. Native strains of E. coli ferment glucose effectively in simple mineral salts medium but produce succinate only as a minor product (9). In E. coli, half of the PEP from glucose is metabolized directly to pyruvate by the PEP-dependent phosphotransferase system for glucose uptake. Most of the remaining PEP is used for ATP produc...