An efficient production of bio-succinate in a novel metabolically engineered Klebsiella oxytoca by rational metabolic engineering and evolutionary adaptation.

Klebsiella oxytoca KC004 (ΔadhEΔpta-ackAΔldhAΔbudABΔpflB) was engineered to enhance succinate production. The strain exhibited poor growth without succinate production due to its deficiencies in ATP production and NADH reoxidation. To overcome obstacles, evolutionary adaptation with over 6,000 generations of growth-based selection was conducted. Under anaerobic conditions, enhanced productions of ATP for growth and succinate for NADH reoxidation by the evolved KC004-TF160 strain were coupled to an increased transcript of PEP carboxykinase (pck) while those of genes in the oxidative branch of TCA cycle (gltA, acnAB, and icd), and pyruvate and acetate metabolisms (pykA, acs, poxB and tdcD) were alleviated. The expression of pyruvate dehydrogenase repressor (pdhR) decreased whereas threonine decarboxylase (tdcE) increased. KC004-TF160 produced succinate at 84 g/L (0.84 g/g, 79 % theoretical maximum). KC004-TF160 produced succinate at 0.87 g/g non-pretreated sugarcane molasses without addition of nutrients and buffers. KC004-TF160 may be a microbial platform for commercial production of bio-succinate.

Genome engineering of Kluyveromyces marxianus for high D-( -)-lactic acid production under low pH conditions.

Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.

Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade.

ß-Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+ ). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D-ribose in the medium. The supernatant containing D-ribose was applied in the second module which is composed of EcRbsK-EcPRPS-CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D-ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.

Engineering Escherichia coli for a high yield of 1,3-propanediol near the theoretical maximum through chromosomal integration and gene deletion.

Glycerol dehydratase (gdrAB-dhaB123) operon from Klebsiella pneumoniae and NADPH-dependent 1,3-propanediol oxidoreductase (yqhD) from Escherichia coli were stably integrated on the chromosomal DNA of E. coli under the control of the native-host ldhA and pflB constitutive promoters, respectively. The developed E. coli NSK015 (∆ldhA::gdrAB-dhaB123 ∆ackA::FRT ∆pflB::yqhD ∆frdABCD::cat-sacB) produced 1,3-propanediol (1,3-PDO) at the level of 36.8 g/L with a yield of 0.99 mol/mol of glycerol consumed when glucose was used as a co-substrate with glycerol. Co-substrate of glycerol and cassava starch was also utilized for 1,3-PDO production with the concentration and yield of 31.9 g/L and 0.84 mol/mol of glycerol respectively. This represents a work for efficient 1,3-PDO production in which the overexpression of heterologous genes on the E. coli host genome devoid of plasmid expression systems. Plasmids, antibiotics, IPTG, and rich nutrients were omitted during 1,3-PDO production. This may allow a further application of E. coli NSK015 for the efficient 1,3-PDO production in an economically industrial scale. KEY POINTS:  â€¢ gdrAB-dhaB123 and yqhD were overexpressed in E. coli devoid of a plasmid system • E. coli NSK015 produced a high yield of 1,3-PDO at 99% theoretical maximum • Cassava starch was alternatively used as substrate for economical 1,3-PDO production.

Combining metabolic engineering and evolutionary adaptation in Klebsiella oxytoca KMS004 to significantly improve optically pure D-(-)-lactic acid yield and specific productivity in low nutrient medium.

In this study, K. oxytoca KMS004 (ΔadhE Δpta-ackA) was further reengineered by the deletion of frdABCD and pflB genes to divert carbon flux through D-(-)-lactate production. During fermentation of high glucose concentration, the resulted strain named K. oxytoca KIS004 showed poor in growth and glucose consumption due to its insufficient capacity to generate acetyl-CoA for biosynthesis. Evolutionary adaptation was thus employed with the strain to overcome impaired growth and acetate auxotroph. The evolved K. oxytoca KIS004-91T strain exhibited significantly higher glucose-utilizing rate and D-(-)-lactate production as a primary route to regenerate NAD+. D-(-)-lactate at concentration of 133 g/L (1.48 M), with yield and productivity of 0.98 g/g and 2.22 g/L/h, respectively, was obtained by the strain. To the best of our knowledge, this strain provided a relatively high specific productivity of 1.91 g/gCDW/h among those of other previous works. Cassava starch was also used to demonstrate a potential low-cost renewable substrate for D-(-)-lactate production. Production cost of D-(-)-lactate was estimated at $3.72/kg. Therefore, it is possible for the KIS004-91T strain to be an alternative biocatalyst offering a more economically competitive D-(-)-lactate production on an industrial scale. KEY POINTS: • KIS004-91T produced optically pure D-(-)-lactate up to 1.48 M in a low salts medium. • It possessed the highest specific D-(-)-lactate productivity than other reported strains. • Cassava starch as a cheap and renewable substrate was used for D-(-)-lactate production. • Costs related to media, fermentation, purification, and waste disposal were reduced.

Evaluation of inhibitory effect and feasible utilization of dilute acid-pretreated rice straws on succinate production by metabolically engineered Escherichia coli AS1600a.

This work demonstrated a pioneer work in the pre-treatment of rice straw by phosphoric acid (H3PO4) for succinate production. The optimized pre-treatment condition of rice straw was at 121 °C for 30 min with 2 N H3PO4. With this condition, total sugar concentration of 31.2 g/L with the highest hemicellulose saccharification yield of 94% was obtained. The physicochemical analysis of the pre-treated rice straw showed significant changes in its structure thus enhancing enzymatic saccharification. Succinate concentrations of 78.5 and 63.8 g/L were produced from hydrolysate liquor (L) and solid fraction (S) of the pre-treated rice straw respectively, with a comparable yield of 86% by E. coli AS1600a. Use of a combined L + S fraction in simultaneous saccharification and fermentation (LS + SSF) further improved succinate production at a concentration and yield of 85.6 g/L and 90% respectively. The results suggested that H3PO4 pre-treated rice straw may be utilized for economical succinate production by E. coli AS1600a.

Optimization of sodium hydroxide pretreatment and enzyme loading for efficient hydrolysis of rice straw to improve succinate production by metabolically engineered Escherichia coli KJ122 under simultaneous saccharification and fermentation.

Rice straw was pretreated with sodium hydroxide (NaOH) before subsequent use for succinate production by Escherichia coli KJ122 under simultaneous saccharification and fermentation (SSF). The NaOH pretreated rice straw was significantly enhanced lignin removal up to 95%. With the optimized enzyme loading of 4% cellulase complex + 0.5% xylanase (endo-glucanase 67 CMC-U/g, ß-glucosidase 26 pNG-U/g and xylanase 18 CMC-U/g dry biomass), total sugar conversion reached 91.7 ±â€¯0.8% (w/w). The physicochemical analysis of NaOH pretreated rice straw indicated dramatical changes in its structure, thereby favoring enzymatic saccharification. In batch SSF, succinate production of 69.8 ±â€¯0.3 g/L with yield and productivity of 0.84 g/g pretreated rice straw and 0.76 ±â€¯0.02 g/L/h, respectively, was obtained. Fed-batch SSF significantly improved succinate concentration and productivity to 103.1 ±â€¯0.4 g/L and 1.37 ±â€¯0.07 g/L/h with a comparable yield. The results demonstrated a feasibility of sequential saccharification and fermentation of rice straw as a promising process for succinate production in industrial scale.

Re-engineering Escherichia coli KJ122 to enhance the utilization of xylose and xylose/glucose mixture for efficient succinate production in mineral salt medium.

Escherichia coli KJ122 was previously engineered to produce high concentration and yield of succinate in mineral salt medium containing glucose and sucrose under anaerobic conditions. However, this strain does not efficiently utilize xylose. To improve the xylose uptake and utilization in the strain KJ122, xylFGH and xylE genes were individually and simultaneously deleted. E. coli KJ12201 (KJ122::ΔxylFGH) exhibited superior abilities in growth, xylose consumption, and succinate production compared to those of the parental strain KJ122. However, E. coli KJ12202 (KJ122::ΔxylE) lessened xylose consumption due to an ATP deficit for metabolizing xylose thus making succinate production from xylose not preferable. Moreover, E. coli KJ12203 (KJ122::ΔxylFGHΔxylE) exhibited an impaired growth on xylose due to lacking of xylose transporters. After performing metabolic evolution, the evolved KJ12201-14T strain exhibited a great improvement in succinate production from pure xylose with higher concentration and productivity about 18 and 21%, respectively, compared to KJ12201 strain. During fed-batch fermentation, KJ12201-14T also produced succinate from xylose at a concentration, yield, and overall productivity of 84.6 ± 0.7 g/L, 0.86 ± 0.01 g/g and 1.01 ± 0.01 g/L/h, respectively. KJ12201 and KJ12201-14T strains co-utilized glucose/xylose mixture without catabolite repression. Both strains produced succinate from glucose/xylose mixture at concentration, yield, and overall and specific productivities of about 85 g/L, 0.85 g/g, 0.70 g/L/h, and 0.44 g/gCDW/h, respectively. Based on our results, KJ12201 and KJ12201-14T strains exhibited a greater performance in succinate production from xylose containing medium than those of other published works. They would be potential strains for the economic bio-based succinate production from xylose.

Genome analysis of food-processing stressful-resistant probiotic Bifidobacterium animalis subsp. lactis BF052, and its potential application in fermented soymilk.

In this study, Bifidobacterium animalis subsp. lactis BF052 was demonstrated the growth capability in soymilk and could be thus supplemented as a probiotic starter that employed soymilk as one of its food vehicles. The complete genome sequence of BF052 was therefore determined to understand the genetic basis of BF052 as a technological and functional probiotic starter. The whole genome sequence of BF052 consists of a circular genome of 1938 624 bp with a G+C content of 60.50%. This research highlights relevant genes involving in its adaptive responses to industrial and/or environmental stresses and utilization of α-galacto-oligosaccharides in BF052 strain compared with other representative bifidobacterial genomes.

Process Optimization on Micro-Aeration Supply for High Production Yield of 2,3-Butanediol from Maltodextrin by Metabolically-Engineered Klebsiella oxytoca.

An optimization process with a cheap and abundant substrate is considered one of the factors affecting the price of the production of economical 2,3-Butanediol (2,3-BD). A combination of the conventional method and response surface methodology (RSM) was applied in this study. The optimized levels of pH, aeration rate, agitation speed, and substrate concentration (maltodextrin) were investigated to determine the cost-effectiveness of fermentative 2,3-BD production by metabolically-engineered Klebsiella oxytoca KMS005. Results revealed that pH, aeration rate, agitation speed, and maltodextrin concentration at levels of 6.0, 0.8 vvm, 400 rpm, and 150 g/L respectively were the optimal conditions. RSM also indicated that the agitation speed was the most influential parameter when either agitation and aeration interaction or agitation and substrate concentration interaction played important roles for 2,3-BD production by the strain from maltodextrin. Under interim fed-batch fermentation, 2,3-BD concentration, yield, and productivity were obtained at 88.1±0.2 g/L, 0.412±0.001 g/g, and 1.13±0.01 g/L/h respectively within 78 h.

Effect of physicochemical pre-treatment of rice straw on its digestibility by Clostridium cellulolyticum.

Rice straw (RS) may serve as a low-cost biomass for the production of biofuels and biochemicals, but its native structure is resistant to enzymatic and microbial deconstruction. Therefore, an efficient pre-treatment method is required to modify crystalline cellulose to a more reactive amorphous form. This work investigated pre-treatments of rice straw involving size reduction (S) followed by either sodium hydroxide (NaOH) or diluted sulfuric acid (H2SO4) and liquid hot water (LHW). The shrinkage of the vascular bundles in the rice straw structure pre-treated with NaOH-LHW-S was higher than that with LHW-S and H2SO4-LHW-S pre-treatments. The highest levels of total fermentative products and residual sugars were obtained at the concentrations of 7.8 ± 0.2 and 2.1 ± 0.3 g/L, respectively, after fermentation by Clostridium cellulolyticum for NaOH-LHW-S pre-treated rice straw at 121 °C for 120 min. Overall, the combined physicochemical pre-treatment of RS led to improved microbial hydrolysis during cellulose degradation at the percentage of 85.5 ± 0.5.

Effects of the Food Manufacturing Chain on the Viability and Functionality of Bifidobacterium animalis through Simulated Gastrointestinal Conditions.

The viability and functionality of probiotics may be influenced by industrial production processes resulting in a decrease in probiotic efficiency that benefit the health of humans. This study aimed to investigate the probiotic characteristics of Bifidobacterium strains isolated from fecal samples of healthy Thai infants. In the present work, three local strains (BF014, BF052, and BH053) belonging to Bifidobacterium animalis showed a great resistance against conditions simulating the gastrointestinal tract. Among these, B. animalis BF052 possessed considerable probiotic properties, including high acid and bile tolerance, strong adhesion capability to Caco-2 cells, and inhibitory activity against pathogens including Salmonella typhimurium and Vibrio cholerae. This strain also exhibited a high survival rate compared to commercial strains during storage in a wide variety of products, including pasteurized milk, soy milk, drinking yogurt, and orange juice. The impact of food processing processes as well as the freeze-drying process, storage of freeze-dried powders, and incorporation of freeze-dried cells in food matrix on probiotic properties was also determined. The stability of the probiotic properties of the BF052 strain was not affected by food processing chain, especially its resistance in the simulated gastrointestinal conditions and its adherence ability to Caco-2 cells. It indicates that it satisfies the criteria as a potential probiotic and may be used as an effective probiotic starter in food applications.

Mutation in galP improved fermentation of mixed sugars to succinate using engineered Escherichia coli AS1600a and AM1 mineral salts medium.

Escherichia coli KJ122 was engineered to produce succinate from glucose using the wild type GalP for glucose uptake instead of the native phosphotransferase system (ptsI mutation). This strain now ferments 10% xylose poorly. Mutants were selected by serial transfers in AM1 mineral salts medium with 10% xylose. Clones from this population all exhibited a similar improvement, co-fermentation of an equal mixture of xylose and glucose. One of these, AS1600a, produced 84.26 ± 1.37 g/L succinate, equivalent to that produced by the parent (KJ122) from 10% glucose (85.46 ± 1.78 g/L). AS1600a was sequenced and found to contain a mutation in galactose permease (GalP, G236D). This mutation was shown to be responsible for the improvement in fermentation using KJΔgalP as the host and expression vectors with native galP and with mutant galP(∗). Strain AS1600a and KJΔgalP(pLOI5746; galP(∗)) also co-fermented a mixture of glucose, xylose, arabinose, and galactose in sugarcane bagasse hydrolysate using mineral salts medium.

Efficient reduction of the formation of by-products and improvement of production yield of 2,3-butanediol by a combined deletion of alcohol dehydrogenase, acetate kinase-phosphotransacetylase, and lactate dehydrogenase genes in metabolically engineered Klebsiella oxytoca in mineral salts medium.

Klebsiella oxytoca KMS005 (∆adhE∆ackA-pta∆ldhA) was metabolically engineered to improve 2,3-butanediol (BDO) yield. Elimination of alcohol dehydrogenase E (adhE), acetate kinase A-phosphotransacetylase (ackA-pta), and lactate dehydrogenase A (ldhA) enzymes allowed BDO production as a primary pathway for NADH re-oxidation, and significantly reduced by-products. KMS005 was screened for the efficient glucose utilization by metabolic evolution. KMS005-73T improved BDO production at a concentration of 23.5±0.5 g/L with yield of 0.46±0.02 g/g in mineral salts medium containing 50 g/L glucose in a shake flask. KMS005-73T also exhibited BDO yields of about 0.40-0.42 g/g from sugarcane molasses, cassava starch, and maltodextrin. During fed-batch fermentation, KMS005-73T produced BDO at a concentration, yield, and overall and specific productivities of 117.4±4.5 g/L, 0.49±0.02 g/g, 1.20±0.05 g/Lh, and 27.2±1.1 g/gCDW, respectively. No acetoin, lactate, and formate were detected, and only trace amounts of acetate and ethanol were formed. The strain also produced the least by-products and the highest BDO yield among other Klebsiella strains previously developed.

Efficient utilization of cassava pulp for succinate production by metabolically engineered Escherichia coli KJ122.

A metabolically engineered Escherichia coli KJ122 was efficiently utilized for succinate production from cassava pulp during batch separate hydrolysis and fermentation (SHF) under simple anaerobic conditions. Succinate concentration of 41.46 ± 0.05 g/L with yield and productivity of 82.33 ± 0.14 g/100 g dry pulp and 0.84 ± 0.02 g/L/h was obtained. In batch simultaneous saccharification and fermentation (SSF), hydrolysis of 12 % (w/v) cassava pulp with an enzyme loading of 2 % AMG + 3 % Cel (v/w) at pH 6.5 was optimized at 39 °C. Succinate concentration of 80.86 ± 0.49 g/L with a yield of 70.34 ± 0.37 g/100 g dry pulp and a productivity of 0.84 ± 0.01 g/L/h was attained using E. coli KJ122. Fed-batch SSF significantly enhanced succinate concentration to 98.63 ± 0.12 g/L at yield and productivity of 71.64 ± 0.97 g/100 g dry pulp and 1.03 ± 0.01 g/L/h. This result indicated an efficient and economical succinate production from cassava pulp using SHF and SSF by the use of E. coli KJ122.

Metabolic engineering of Klebsiella oxytoca M5a1 to produce optically pure D-lactate in mineral salts medium.

Klebsiella oxytoca strains were constructed to produce optical pure d-lactate by pH-controlled batch fermentation in mineral salts medium. The alcohol dehydrogenase gene, adhE, and the phospho-transacetylase/acetate kinase A genes, pta-ackA, were deleted from the wild type. KMS002 (ΔadhE) and KMS004 (ΔadhE Δpta-ackA) exhibited d-lactate production as a primary pathway for the regeneration of NAD(+). Both strains produced 11-13 g/L of d-lactate in medium containing 2% (w/v) glucose with yields of 0.64-0.71 g/g glucose used. In sugarcane molasses, KMS002 and KMS004 produced 22-24 g/L of d-lactate with yields of 0.80-0.87 g/g total sugars utilized. Both strains also utilized maltodextrin derived from cassava starch and produced d-lactate at a concentration of 33-34 g/L with yields of 0.91-0.92 g/g maltodextrin utilized. These d-lactate yields are higher than those reported for engineered E. coli strains.

Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli.

Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selection and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51 g/L (productivity equal to 1.05 g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47 g/L were produced in 10L bioreactor within 48 h. Antibiotics had no effect on the succinate production by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62 g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56 g/L in 10 L bioreactor within 72 h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses.

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

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.

Effect of periplasmic nitrate reductase on diauxic lag of Paracoccus pantotrophus.

Paracoccus pantotrophus expresses two nitrate reductases-membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap(-) mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions.

Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C.

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.