Low concentrations of furfural are formed as a side product during the dilute acid hydrolysis of hemicellulose. Growth is inhibited by exposure to furfural but resumes after the complete reduction of furfural to the less toxic furfuryl alcohol. Growth-based selection was used to isolate a furfural-resistant mutant of ethanologenic Escherichia coli LY180, designated strain EMFR9. Based on mRNA expression levels in the parent and mutant in response to furfural challenge, genes encoding 12 oxidoreductases were found to vary by more than twofold (eight were higher in EMFR9; four were higher in the parent). All 12 genes were cloned. When expressed from plasmids, none of the eight genes in the first group increased furfural tolerance in the parent (LY180). Expression of three of the silenced genes (yqhD, dkgA, and yqfA) in EMFR9 was found to decrease furfural tolerance compared to that in the parent. Purified enzymes encoded by yqhD and dkgA were shown to have NADPH-dependent furfural reductase activity. Both exhibited low K m values for NADPH (8 M and 23 M, respectively), similar to those of biosynthetic reactions. Furfural reductase activity was not associated with yqfA. Deleting yqhD and dkgA in the parent (LY180) increased furfural tolerance, but not to the same extent observed in the mutant EMFR9. Together, these results suggest that the process of reducing furfural by using an enzyme with a low K m for NADPH rather than a direct inhibitory action is the primary cause for growth inhibition by low concentrations of furfural.

Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.

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...