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Metabolic Engineering 13 (2011) 76–81 Contents lists available at ScienceDirect Metabolic Engineering journal homepage: www.elsevier.com/locate/ymben Manipulating redox and ATP balancing for improved production of succinate in E. coli Amarjeet Singh a, Keng Cher Soh b, Vassily Hatzimanikatis b, Ryan T. Gill a,n a b Department of Chemical and Biological Engineering, University of Colorado, UCB 424, Boulder, CO 80309, USA Laboratory of Computational Systems Biotechnology, Ecole Polytechnique Federale de Lausanne (EPFL), CH 1015 Lausanne, Switzerland a r t i c l e i n f o a b s t r a c t Article history: Received 19 October 2010 Accepted 21 October 2010 Available online 30 October 2010 Redox and energy balance plays a key role in determining microbial fitness. Efforts to redirect bacterial metabolism often involve overexpression and deletion of genes surrounding key central metabolites, such as pyruvate and acetyl-coA. In the case of metabolic engineering of Escherichia coli for succinate production, efforts have mainly focused on the manipulation of key pyruvate metabolizing enzymes. E. coli AFP111 strain lacking ldhA, pflB and ptsG encoded activities accumulates acetate and ethanol as well as shows poor anaerobic growth on rich and minimal media. To address these issues, we first deleted genes (adhE, ackA-pta) involved in byproduct formation downstream of acetyl-CoA followed by the deletion of iclR and pdhR to activate the glyoxylate pathway. Based on data from these studies, we hypothesized that the succinate productivity was limited by the insufficient ATP generation. Genomescale thermodynamics-based flux balance analysis indicated that overexpression of ATP-forming PEPCK from Actinobacillus succinogenes in an ldhA, pflB and ptsG triple mutant strain could result in an increase in biomass and succinate flux. Testing of this prediction confirmed that PEPCK overexpression resulted in a 60% increase in biomass and succinate formation in the ldhA, pflB, ptsG mutant strain. & 2010 Elsevier Inc. All rights reserved. Keywords: Escherichia coli Redox ratio NADH/NAD + Succinate Fumarate Metabolic engineering NZN111 AFP111 1. Introduction Escherichia coli performs mixed acid fermentation in the absence of exogenous electron acceptors (Neidhardt and Curtiss, 1996). The primary fermentation products are acetate, lactate and formate, while ethanol and succinate are formed in minor quantities (Gupta and Clark, 1989; Matjan et al., 1989). Pyruvate serves as an important branch point for carbon flux distribution (Arita, 2004; Fell and Wagner, 2000). In wild-type strains of E. coli growing anaerobically, most of the pyruvate flux is handled by two primary enzymes of fermentative metabolism—lactate dehydrogenase (ldhA) and pyruvate-formate lyase (pflB); accounting for the production of acetate, ethanol, formate and lactate. E. coli NZN111 (DldhA, DpflB) was created to redirect the carbon flux towards the formation of succinic acid. This mutant, however, is incapable of growth on glucose in rich or minimal media under anaerobic conditions (Bunch et al., 1997; Gupta and Clark, 1989; Stols and Donnelly, 1997) and is known to accumulate high levels of pyruvate and NADH (Vemuri et al., 2002a). Intracellular redox ratios (NADH/NAD + ) as high as three times that of the wild-type E. coli have been observed in NZN111 (Singh et al., 2009). The inability to synthesize acetyl-coA and/or to regenerate NAD + via n Corresponding author: Fax: + 1 303 492 4341. E-mail address: [email protected] (R.T. Gill). 1096-7176/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2010.10.006 pyruvate reduction are cited as the likely causes of the growth defect of this strain (Stols and Donnelly, 1997; Stols et al., 1997). A variety of metabolic engineering strategies including those focused toward the overexpression of pyruvate metabolizing enzymes have been pursued for improving succinate production in E. coli (Chatterjee et al., 2001; Millard et al., 1996; Sanchez et al., 2005; Stols and Donnelly, 1997; Vemuri et al., 2002a). Other strategies include providing additional reducing power (Chatterjee et al., 2001; Hong and Lee, 2002; VanderWerf et al., 1997), as well as creative combinations of aerobic and anaerobic metabolism (Sanchez et al., 2006; Vemuri et al., 2002a, b) to attain optimal succinate yields on glucose by activating the glyoxylate pathway and the reductive TCA cycle (Vemuri et al., 2002a). Evolutionary engineering strategies have also been successful. By employing a dual metabolic engineering and evolutionary approach, Jantama et al. (2008) constructed an E. coli strain capable of overproducing succinic acid on minimal media in single batch fermentation. A spontaneous ptsG mutant of NZN111, with improved fitness and restored fermentation capability, was reported by Donnelly et al. (1998) as E. coli AFP111. Inactivation of ptsG is thought to increase the PEP pool, which is then diverted to the reductive TCA cycle via the action of PEP-carboxylase (PPC) (Chatterjee et al., 2001; Lin et al., 2005; Vemuri et al., 2002a). The redistribution of the carbon flux resulting from the ptsG mutation in AFP111, however, came at the cost of succinate yield. AFP111 produces succinate, ethanol and acetate in a 1:0.5:0.5 ratios, presumably to maintain redox cofactor balances (Chatterjee et al., 2001; Donnelly et al., 1998). The succinate yield and productivity was A. Singh et al. / Metabolic Engineering 13 (2011) 76–81 increased by the overexpression of the anaplerotic pyruvate carboxylase (pyc) gene from Rhizobium etli in AFP111, although the formation of acetate and ethanol could not be completely eliminated (Vemuri et al., 2002a, b). In the studies reported herein, we observed that deletion of the acetate forming enzymes acetate kinase and phosphotransacetylase (ackA-pta) completely abolished growth in the initial microaerobic phase in microaerobic–anaerobic dual-phase fermentation in our AFP111 equivalent strain AD32 (a pflB, ldhA, ptsG triple mutant). PTA mutants have widely been reported to not grow under anaerobic conditions and show reduced growth rates in aerobic conditions (Wolfe, 2005). Formation of acetate also results in ATP generation, which we suspected could be limiting anaerobic growth in this strain as the formation of succinate via reductive TCA cycle does not generate any ATP. PEP is converted to OAA as the first step in succinate production. In E. coli, this step is catalyzed by PEP-carboxylase (ppc), while in Actinobacillus succinogenes, this step is catalyzed by the ATP-generating PEP-carboxykinase (Kim et al., 2004; Laivenieks et al., 1997; VanderWerf et al., 1997). Prior studies have shown that PEP-carboxylase overexpression can significantly enhance succinate production in E. coli (Millard et al., 1996), while PEP-carboxykinase overexpression only enhances succinate production in an E. coli ppc mutant(Kim et al., 2004; Millard et al., 1996). These prior studies suggested that this is explained by the roughly 100-fold lower Km towards bicarbonate of the PEP-carboxylase relative to PEP-carboxykinase enzyme. These results also suggested that the PEP to OAA reaction can operate under the physiological conditions in E. coli, thus promoting this strategy for the use in succinate production strains. Here, we further assessed the feasibility of this reaction using Thermodynamics-based flux balance analysis (TFBA) with the iAF1260 E. coli genome-scale metabolic model (Feist et al., 2007b). The model confirmed the reaction could proceed in the ATP-generating direction at elevated extracellular CO2 concentrations. The model also predicted higher biomass and succinate yields as a result of increased ATP formation. Based on these data, we investigated the effect of PEPCK overexpression in E. coli strains engineered for succinic acid production. Overexpression of PEPCK was indeed observed to improve growth, glucose consumption, and succinic acid production in an ldhA, pflB and ptsG triple mutant strain. To further improve succinate yield, we deleted the ethanol forming alcohol dehydrogenase (adhE) enzyme and observed an increase in succinate yield. However, byproduct formation could not be completely eliminated as the deletion of ackA-pta completely abolished microaerobic growth. 77 University, New Haven, CT. Plasmid pAsPCK carrying the PEPCK gene from A. succinogenes under the control of the inducible lac promoter was provided by the Dr. C. Vielle’s laboratory at the Michigan State University (MSU), Lansing, MI. E.coli phosphoenol-carboxylase (ppc) was PCR amplified and cloned under the control of its natural promoter into a low copy vector pSmart-LC-Kan from Lucigen Corp. The ASKA clone plasmid carrying the E.coli PPC under the control of inducible lac promoter (JW3928) was also utilized for the overexpression studies (Kitagawa et al., 2005). 2.2. Microaerobic–anaerobic dual-phase fermentation Single bacterial colonies were grown in a 15 ml plastic tube containing 5 ml of Luria-Bertani (LB) medium until it reached its exponential growth at an OD600 nm of 0.4–0.6. The culture was then diluted to a new OD600 nm of 0.005–0.01 to be used as an inoculum for the fermentation studies in LB medium supplemented with 50 mM of sugar source. Glucose, sorbitol and gluconate were purchased from the Fisher scientific. 10 g/L NaHCO3 was added to the media for the dual purpose of carbon dioxide source and buffering agent. The cells were grown in the absence of air headspace by filling the culturing tubes with the media to the top to enable rapid anaerobiosis. The cultures were grown at 37 1C and at an agitation rate of 200 rpm. Separate tubes were run for each data point to avoid oxygen exposure during sampling. 2.3. Analytical techniques Cell growth was monitored by measuring the optical density (OD) at 600 nm (UV–vis spectrophotometer, Shimadzu Corp.). To analyze the fermentation culture, 1 mL aliquots were centrifuged at 13000 rpm for 2 min and the supernatant was filtered through a 0.2 mm syringe filter. Samples were analyzed using HPLC Agilent 1100 series Chemstation equipped with ICSep Coregel 64H HPLC column (Transgenomics, Omaha, NE). 50 ml of sample was loaded into the column operated at 50 1C and ran isocratically with 8 mM H2SO4 sat a flow rate of 0.6 ml/min. In the analysis, the concentrations of organic acids such as succinate, lactate, acetate and formate were quantified from the signals obtained at 210 nm by the UV–vis detector (Eiteman and Chastain, 1997). The concentrations of the metabolites were determined using a standard curve constructed using HPLC grade reagents purchased from Sigma-Aldrich Co. Glucose concentrations were monitored by YSI 2700 Select Biochemistry Analyzer. 2. Materials and methods 2.1. Strains and plasmids All mutations were made in E. coli BW25113 (D(araD-araB)567, DlacZ4787(::rrnB-3), lambda , rph-1, D(rhaDrhaB)568, hsdR514). Deletion strains were constructed following the method developed earlier (Datsenko and Wanner, 2000). The kanamycin resistance cassette was amplified from plasmid pKD13 by PCR using primers with flanking homologous regions for the target gene. The purified PCR product was electroporated into host E. coli strain harboring l-Red recombinase induced off the plasmid pKD46 using 10 mM arabinose. The resulting kanamycin resistant colonies were screened for the desired gene knockout by the PCR amplification and subsequent sequencing. Primers for this confirmation step were designed to bind 300–400 by upstream and downstream respectively of the target gene. Plasmid pCP20 carrying the FLP-recombinase was subsequently used to excise the kanamcin selection marker from the knockouts strain. All plasmids were cured by propagating the strains at 43 1C before preparing for the deletion of the next target gene. Strains and plasmid stocks were obtained from the E. coli Genetic Stock Center at Yale 2.4. Thermodynamics-based flux balance analysis using iAF1260 E. coli genome-scale metabolic model The latest genome-scale E. coli metabolic model, iAF1260 (Feist et al., 2007a) was used to perform the analysis reported here. This model was defined as the wild-type (WT), while the AD32 strain was simulated by constraining the reactions for ldhA, pflB, ptsG to zero. Thermodynamics-based Flux Balance Analysis (TFBA) generates thermodynamically feasible flux distributions in a given metabolic network as compared with conventional Flux Balance Analysis (FBA) which can produce distributions that violate thermodynamics (Henry et al., 2007). A minimal set of metabolites were allowed to be exported out of system (in this case: acetate, formate, malate, hydrogen gas, ethanol, lactate and succinate). The in-silico media was set to glucose minimal media under anaerobic conditions. Model assumptions include (i) maintenance energy remains the same as per aerobic conditions at 8.39 mmol-ATP/ gDW hr, (ii) biomass composition for anaerobic conditions simulated is the same as per aerobic condition, (iii) glucose uptake was fixed at 10 mmol/gDW hr for ease of comparison of yield and 78 A. Singh et al. / Metabolic Engineering 13 (2011) 76–81 Table 1 Redox balance and the theoretical succinate and ATP yields for the engineered succinic acid production strains of E. coli. Strains AD12 AD32 AD32 AD216 AD483 AD346 AD568 AD725 a b c Relevant deletions ldhA, ldhA, ldhA, ldhA, ldhA, ldhA, ldhA, ldhA, pflB pflB, pflB, pflB, pflB, pflB, pflB, pflB, ptsG ptsG, ppc ptsG, ppc ptsG, ppc, ackA-pta ptsG, ppc, adhE ptsG, ppc, adhE, ackA-pta ptsG adhE, ack-ptA pdhR, iclR pAsPCK PEPCK No No No Yes Yes Yes Yes Yes Theoretical yield (mol / mol glucose) Experimental data (g/L h) ATP generation Net NADH Growth rate Glucose consumption Succinate generation 0 0.5 1 2 2 2 2 1.4 2a 0b 0 2c 2c 2c 2c 0 0.074 0.091 0.078 0.093 0.012 0.067 0.013 0.107 No growth 0.104 0.008 0.056 0 0.083 0.09 No growth No growth 0.08 It is assumped that only the succinate pathway is active consistent with the reported data. 1:0.5:05 ratio for succinate:acetate:ethanol is assumed consistent with the reported experimental data. It is assumed that the presence of PEPCK would drive the carbon flux solely through the succinate branch. (iv) cytoplasmic pH was fixed at 7.2, with periplasmic and extracellular pH fixed at 7. 3. Results and discussion E. coli fitness is closely tied to its ability to balance reducing equivalents across various pathways while simultaneously generating all required biosynthetic precursors. E. coli strains lacking ldhA and pflB encoded activities suffer from an inability to synthesize sufficient acetyl-coA, accumulation of pyruvate, and formation of undesired by-products acetate and ethanol, all of which lead to incomplete flux through the desired succinate pathway (Bunch et al., 1997; Matjan et al., 1989; Stols and Donnelly, 1997; Vemuri et al., 2002a). An additional mutation in the ptsG restores fermentative growth on glucose in complex media. This strain produces succinate, acetate and ethanol in a molar ratio of 1:0.5:0.5. It is thought that pyruvate dehydrogenase complex maintains a low level of activity under laboratory anaerobic conditions, thus enabling the conversion of pyruvate to acetyl-CoA. Ethanol is then produced via adhE with acetate arising from ackA-pta. With a ratio of 1:0.5:0.5, this pathway is NADH balanced and nets 0.5 moles of ATP/mol of glucose consumed (Table 1). To maximize succinate yield, it is essential to remove byproduct formation; however, sole production of succinate through the reductive TCA pathway is neither NADH balanced nor does it lead to ATP generation (Fig. 1). Thus, removal of byproduct formation requires a complementary strategy that yields ATP. Our strategy was to the use the ATP-generating PEPCK enzyme in combination with deletions in ethanol and acetate forming pathways to improve succinate production. Using TFBA, conversion of PEP to OAA and ATP by PEPCK enzyme was found to thermodynamically feasible at extracellular CO2 concentrations greater than 1.96 mM. Moreover, the model also predicted a 15–20% increase in succinate productivity and yield. The fermentation media was thus supplemented with 10 g/L sodium bicarbonate to favor the PEPCK reaction. 3.1. ATP production limits fermentative growth of E. coli AD32 (DldhA, DpflB, DptsG) Despite a positive overall ATP balance (Table 1), the deletion of either adhE or ackA-pta (or both; AD483) was observed to completely abolish fermentative growth of AD32 (BW25113 ldhA , pflB , ptsG ) (Fig. 2a). This result suggests that approximately 0.5 mol ATP/mol glucose is required for maintenance under these conditions and in this genetic background. To test this inference, we deleted the ppc gene required for anaerobic succinate production. Fig. 1. Central carbon metabolism of E. coli. Target reactions for the improved succinic acid production are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). This strain has the ability to be redox balanced as well as ATP positive, which is not possible for the succinate production strain alone. As expected, we observed that the growth was maintained in the Dppc strain albeit at a lower final cell density (Fig. 2a). A. Singh et al. / Metabolic Engineering 13 (2011) 76–81 Fig. 2. (a) Effect of various gene deletions on growth profile on LB media supplemented with 50 mM glucose in microaerobic–anaerobic conditions. (b) Effect of the carbon sources of differing oxidation state on the growth of AD 12 (BW25113 ldhA , pflB ) and AD32 (BW25113 ldhA , pflB , ptsG ) after 24 h on LB media supplemented with 50 mM glucose. To further explore the interrelationship between redox and ATP balancing, we tested the growth of these mutants on sorbitol (Oxidation state¼ 1), glucose (Ox state¼ 0) and gluconate (Ox state¼1). Biochemically, sorbitol metabolism results in 3 mol/mol NADH compared with 2 mol/mol for glucose and 1 mol/mol for gluconate, respectively. The substitution of glucose with sorbitol lead to a 44% decrease in cell growth for AD32 (BW25113 ldhA , pflB , ptsG ) as opposed to AD12 (BW25113 ldhA , pflB ) which grew to over four fold higher cell density (Fig. 2b). Since succinate appears to be the primary fermentative pathway in AD12, excess NADH would lead to redox balancing and higher growth. The cause of lower cell growth of AD32 on sorbitol is unclear; we speculate that excess NADH would upset the flux distribution among the three end-products succinate, acetate and ethanol. Higher cell density of AD12 in sorbitol also suggests that its growth is not limited by acetyl-coA requirement for biosynthesis. 3.2. Effect of the overexpression of A. succinogenes PEPCK Our observations suggest that at least 0.5 moles of ATP per mole of glucose is required for the growth of AD32, which is apparently accomplished through conversion of 50% of the carbon to acetate 79 Fig. 3. (a) Effect of pAsPCK on growth and fermentation profile in the microaerobic– anaerobic dual-phase fermentation. (a) Open and closed squares represent anaerobic growth profiles for AD32 (BW25113 ldhA , pflB , ptsG ) and AD216 (AD32 pAsPCK) in LB + 10 g/L glucose, respectively. (b) Glucose consumption (squares), succinate (triangles) and acetate (circles) production for AD32 (open symbols) and AD216 (closed symbols). and ethanol as opposed to succinate. We reasoned that overexpression of an enzyme that could tie ATP production to succinate formation would increase the fitness of succinate production strains. PEP-carboxykinase (PEPCK) from A. succinogenes, which catalyzes the formation of oxaloacetate (OAA) from PEP along with the generation of an ATP, has been previously cloned into an expression plasmid (pAsPCK) and shown to complement the an E.coli K12 PEP-carboxylase (Dppc) mutant (Kim et al., 2004). The transformation of the pAsPCK in AD12 (BW25113 ldhA , pflB ) and AD32 (BW25113 ldhA , pflB , ptsG ) did not significantly increase cell growth (data not shown), presumably due to high activity of the native phoephoenolpyruvate carboxylase (ppc), as previously reported (Kim et al., 2004; Millard et al., 1996). It has been shown that the kinetic properties of the E. coli PPC are superior to those of PEPCK with respect to bicarbonate. Specifically, E. coli PPC has a Km of 0.19 and 0.1 towards PEP and bicarbonate anion, respectively, (Kai et al., 1999). In comparison, the Km towards bicarbonate for PEPCK is expected to be closer to that of PEP carboxykinases from E. coli (13 mM) and Anaerobiospirillum succiniciproducens, which have been measured at about two orders of magnitude greater (17 mM) than E. coli PPC (Millard et al., 1996). To address this issue, we deleted the native E. coli ppc and transformed the pAsPCK plasmid into strain AD32 (designated AD216). Indeed, we observed that overexpression of A. succinogenes PEPCK in the absence of the native ppc gene increased growth and 80 A. Singh et al. / Metabolic Engineering 13 (2011) 76–81 Fig. 4. Growth of AD32 (BW25113 ldhA , pflB , ptsG ) derivatives harboring pAsPCK plasmid on LB media supplemented with 50 mM glucose after 24 h in microaerobic– anaerobic conditions. succinate production in the microaerobic–anaerobic dual-phase fermentation (Fig. 3a). AD216 consumed 50% more glucose in 72 h and produced 60% more succinate (5.97 g/L) titers compared to AD32 (Fig. 3b) although the yield remained roughly the same at 0.78 (70% of the theoretical maximum, note that this improvement is significant as AD32 has the same disrupted activity in ldhA, pflB, and ptsG as previously described AFP111). Acetate constituted 20% of the fermentation product. This observation suggests that ATP limits cell growth but not the flux distribution in this strain, as would be expected based on the redox and ATP balance. 3.3. Efforts to increase theoretical yield of succinic acid production We expected that diverting the flux towards succinate formation via ATP-forming PEPCK would eliminate the need for the ATP producing acetate pathway, and thus could be used to further increase succinate formation. The effect of the presence of PEPCK was thus studied in strains lacking ethanol and acetate pathways. As expected, the presence of PEPCK had no effect on the strains carrying a native PPC, thus the studies were continued in a ppc deletion background. The strain carrying the adhE deletion, AD346 was able to grow anaerobically upon transformation with pAsPCK (Fig. 4). The growth of AD346 was similar to AD32, thus channeling the carbon flux towards succinate formation could eliminate the need for NADH oxidation via the ethanol pathway. However, the deletion of acetate forming genes, ackA-pta in AD483 abolished microaerobic and anaerobic growth even in the presence of pAsPCK plasmid (Fig. 4). As the derivatives of AD32 (BW25113 ldhA , pflB , ptsG ) appear to have pyruvate to acetyl-coA conversion activity, we suspected that acetate formation would be essential in such strains in the absence of a functional glyoxylate pathway to avoid acetyl-coA accumulation. A 71:29 ratio split in the carbon flux towards reductive TCA cycle and the glyoxylate pathway respectively would lead to redox balanced succinate production with a molar yield of 1.71 mol/mol glucose (Vemuri et al., 2002a). Since the glyoxylate pathway is typically active during aerobic conditions, optimized dual-phase aerobic/anaerobic fermentations have been successfully employed to attain high yields of succinate formation at impressive productivities (Sanchez et al., 2006; Vemuri et al., 2002b). With these studies in mind, we sought to investigate if the deletions of pdhR and iclR would increase cellular fitness of our engineered strains. pdhR is the anaerobic repressor of the pyruvate dehydrogenase complex while iclR is the repressor of the glyoxylate pathway genes. The deletions of pdhR and iclR leads to the constitutive expression of the PDHc (Haydon et al., 1993) and the glyoxylate pathways genes (Sunnarborg et al., 1990), respectively. However, these two deletions had no effect on the fitness on the AD32 and its derivatives, suggesting that neither the conversion of pyruvate to acetyl-CoA nor the glyoxylate pathway is limiting in our strains. In summary, cellular fitness of the succinate production strains appears to be limited by sub-optimal acetyl-coA metabolism. While the formation of acetyl-coA from pyruvate is essential for biosynthesis, subsequent formation of acetate decreases the overall yield. The engineered strains were observed to be sensitive to ackA-pta mutation, underscoring the rigid control of acetyl-coA metabolism. Future engineering efforts should focus on modulating the pyruvate to acetyl-coA conversion to enable biosynthesis as well as achieve the delicate split of the carbon flux between the reductive TCA and glyoxylate pathways. Acknowledgments KCS was supported by the Swiss National Science Foundation. VH was supported by funding from Ecole Polytechnique Fédérale de Lausanne (EPFL), SystemsX.ch and DuPont. References Arita, M., 2004. The metabolic world of Escherichia coli is not small. Proceedings of the National Academy of Sciences of the United States of America 101, 1543–1547. Bunch, P.K., MatJan, F., Lee, N., Clark, D.P., 1997. The IdhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology-UK 143, 187–195. Chatterjee, R., Millard, C.S., Champion, K., Clark, D.P., Donnelly, M.I., 2001. Mutation of the ptsC gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Applied and Environmental Microbiology 67, 148–154. A. Singh et al. / Metabolic Engineering 13 (2011) 76–81 Datsenko, K.A., Wanner, B.L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the national academy of sciences of the United States of America 97, 6640–6645. Donnelly, M.I., Millard, C.S., Clark, D.P., Chen, M.J., Rathke, J.W., 1998. A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol. Applied Biochemistry and Biotechnology 70-2, 187–198. Eiteman, M.A., Chastain, M.J., 1997. Optimization of the ion-exchange analysis of organic acids from fermentation. Analytica Chimica Acta 338, 69–75. Feist, A., Henry, C., Reed, J., Krummenacker, M., Joyce, A., Karp, P., Broadbelt, L., Hatzimanikatis, V., Palsson, B., 2007a. A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Molecular Systems Biology 3, 121. Feist, A.M., Henry, C.S., Reed, J.L., Krummenacker, M., Joyce, A.R., Karp, P.D., Broadbelt, L.J., Hatzimanikatis, V., Palsson, B.O., 2007b. A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Molecular Systems Biology 3, 121. Fell, D.A., Wagner, A., 2000. The small world of metabolism. Nature Biotechnology 18, 1121–1122. Gupta, S., Clark, D.P., 1989. Escherichia Coli derivatives lacking both alcoholdehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation. Journal of Bacteriology 171, 3650–3655. Haydon, D.J., Quail, M.A., Guest, J.R., 1993. A mutation causing constitutive synthesis of the pyruvate-dehydrogenase complex in Escherichia Coli is located within the Pdhr gene. FEBS Letters 336, 43–47. Henry, C., Broadbelt, L.J., Hatzimanikatis, V., 2007. Thermodynamics-based metabolic flux analysis. Biophysical Journal 92, 1792–1805. Hong, S.H., Lee, S.Y., 2002. Importance of redox balance on the production of succinic acid by metabolically engineered Escherichia coli. Applied Microbiology and Biotechnology 58, 286–290. Jantama, K., Haupt, M.J., Svoronos, S.A., Zhang, X.L., Moore, J.C., Shanmugam, K.T., Ingram, L.O., 2008. Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate. Biotechnology and Bioengineering 99, 1140–1153. Kai, Y., Matsumura, H., Inoue, T., Terada, K., Nagara, Y., Yoshinaga, T., Kihara, A., Tsumura, K., Izui, K., 1999. Three-dimensional structure of phosphoenolpyruvate carboxylase: A proposed mechanism for allosteric inhibition. Proceedings of the National Academy of Sciences of the United States of America 96, 823–828. Kim, P., Laivenieks, M., Vieille, C., Zeikus, J.G., 2004. Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Applied and Environmental Microbiology 70, 1238–1241. Kitagawa, M., Ara, T., Arifuzzaman, M., Ioka-Nakamichi, T., Inamoto, E., Toyonaga, H., Mori, H., 2005. Complete set of ORF clones of Escherichia coli ASKA library 81 (A complete Set of E. coli K-12 ORF archive): unique resources for biological research. DNA Research 12, 291–299. Laivenieks, M., Vieille, C., Zeikus, J.G., 1997. Cloning, sequencing and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene. Applied and Environmental Microbiology 63, 2273–2280. Lin, H., Bennett, G.N., San, K.Y., 2005. Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli. Journal of Industrial Microbiology & Biotechnology 32, 87–93. Matjan, F., Alam, K.Y., Clark, D.P., 1989. Mutants of Escherichia coli deficient in the fermentative lactate-dehydrogenase. Journal of Bacteriology 171, 342–348. Millard, C.S., Chao, Y.P., Liao, J.C., Donnelly, M.I., 1996. Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Applied and Environmental Microbiology 62, 1808–1810. Neidhardt, F.C., and Curtiss, R. (1996). Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. (Washington, D.C., ASM Press). Sanchez, A.M., Bennett, G.N., San, K.Y., 2005. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metabolic Engineering 7, 229–239. Sanchez, A.M., Bennett, G.N., San, K.Y., 2006. Batch culture characterization and metabolic flux analysis of succinate-producing Escherichia coli strains. Metabolic Engineering 8, 209–226. Singh, A., Lynch, M.D., Gill, R.T., 2009. Genes restoring redox balance in fermentation-deficient E. coli NZN111. Metabolic Engineering 11, 347–354. Stols, L., Donnelly, M.I., 1997. Production of succinic acid through overexpression of NAD(+ )-dependent malic enzyme in an Escherichia coli mutant. Applied and Environmental Microbiology 63, 2695–2701. Stols, L., Kulkarni, G., Harris, B.G., Donnelly, M.I., 1997. Expression of Ascaris suum malic enzyme in a mutant Escherichia coli allows production of succinic acid from glucose. Applied Biochemistry and Biotechnology 63-5, 153–158. Sunnarborg, A., Klumpp, D., Chung, T., Laporte, D.C., 1990. Regulation of the glyoxylate bypass operon—cloning and characterization of Iclr. Journal of Bacteriology 172, 2642–2649. VanderWerf, M.J., Guettler, M.V., Jain, M.K., Zeikus, J.G., 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Archives of Microbiology 167, 332–342. Vemuri, G.N., Eiteman, M.A., Altman, E., 2002a. Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Applied and Environmental Microbiology 68, 1715–1727. Vemuri, G.N., Eiteman, M.A., Altman, E., 2002b. Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions. Journal of Industrial Microbiology & Biotechnology 28, 325–332. Wolfe, A.J., 2005. The acetate switch. Microbiology and Molecular Biology Reviews 69, 12–50.