Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO₂-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

Improved Succinic Acid Production in the Anaerobic Culture of an Escherichia coli pflB ldhA Double Mutant as a Result of Enhanced Anaplerotic Activities in the Preceding Aerobic Culture

Escherichia coli NZN111 is a pflB ldhA double mutant which loses its ability to ferment glucose anaerobically due to redox imbalance. In this study, two-stage culture of NZN111 was carried out for succinic acid production. It was found that when NZN111 was aerobically cultured on acetate, it regained the ability to ferment glucose with succinic acid as the major product in subsequent anaerobic culture. In two-stage culture carried out in flasks, succinic acid was produced at a level of 11.26 g/liter from 13.4 g/liter of glucose with a succinic acid yield of 1.28 mol/mol glucose and a productivity of 1.13 g/liter·h in the anaerobic stage. Analyses of key enzyme activities revealed that the activities of isocitrate lyase, malate dehydrogenase, malic enzyme, and phosphoenolpyruvate (PEP) carboxykinase were greatly enhanced while those of pyruvate kinase and PEP carboxylase were reduced in the acetate-grown cells. The two-stage culture was also performed in a 5-liter fermentor without separating the acetate-grown NZN111 cells from spent medium. The overall yield and concentration of succinic acid reached 1.13 mol/mol glucose and 28.2 g/liter, respectively, but the productivity of succinic acid in the anaerobic stage dropped to 0.7 g/liter·h due to cell autolysis and reduced anaplerotic activities. The results indicate the great potential to take advantage of cellular regulation mechanisms for improvement of succinic acid production by a metabolically engineered E. coli strain.     

Succinic acid production from continuous fermentation process using Mannheimia succiniciproducens LPK7

To achieve a higher succinic acid productivity and evaluate the industrial applicability, this study used Mannheimia succiniciproducens LPK7 (knock-out: lahA, pflB, pta-ackA), which was recently designed to enhance the productivity of succinic acid and reduce by-product secretion. Anaerobic continuous fermentation of Mannheimia succiniciproducens LPK7 was carried out at different glucose feed concentrations and dilution rates. After extensive fermentation experiments, a succinic acid yield and productivity of 0.38 mol/mol and 1.77 g/l/h, respectively, were achieved with a glucose feed concentration of 18.0 g/l and 0.2 h-1 dilution rate. A similar amount of succinic acid production was also produced in batch culture experiments. Therefore, these optimal conditions can be industrially applied for the continuous production of succinic acid. To examine the quantitative balance of the metabolism, a flux distribution analysis was also performed using the metabolic network model of glycolysis and the pentose phosphate pathway.     

Genome-scale analysis of Mannheimia succiniciproducens metabolism

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is a capnophilic gram-negative bacterium that efficiently produces succinic acid, an industrially important four carbon dicarboxylic acid. In order to design a metabolically engineered strain which is capable of producing succinic acid with high yield and productivity, it is essential to optimize the whole metabolism at the systems level. Consequently, in silico modeling and simulation of the genome-scale metabolic network was employed for genome-scale analysis and efficient design of metabolic engineering experiments. The genome-scale metabolic network of M. succiniciproducens consisting of 686 reactions and 519 metabolites was constructed based on reannotation and validation experiments. With the reconstructed model, the network structure and key metabolic characteristics allowing highly efficient production of succinic acid were deciphered; these include strong PEP carboxylation, branched TCA cycle, relative weak pyruvate formation, the lack of glyoxylate shunt, and non-PTS for glucose uptake. Constraints-based flux analyses were then carried out under various environmental and genetic conditions to validate the genome-scale metabolic model and to decipher the altered metabolic characteristics. Predictions based on constraints-based flux analysis were mostly in excellent agreement with the experimental data. In silico knockout studies allowed prediction of new metabolic engineering strategies for the enhanced production of succinic acid. This genome-scale in silico model can serve as a platform for the systematic prediction of physiological responses of M. succiniciproducens to various environmental and genetic perturbations and consequently for designing rational strategies for strain improvement.     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

Recovery of succinic acid produced by fermentation of a metabolically engineered Mannheimia succiniciproducens strain

There have recently been much advances in the production of succinic acid, an important four-carbon dicarboxylic acid for many industrial applications, by fermentation of several natural and engineered bacterial strains. Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce succinic acid with high efficiency, but also produces acetic, formic and lactic acids just like other anaerobic succinic acid producers. We recently reported the development of an engineered M. succiniciproducens LPK7 strain which produces succinic acid as a major fermentation product while producing much reduced by-products. Having an improved succinic acid producer developed, it is equally important to develop a cost-effective downstream process for the recovery of succinic acid. In this paper, we report the development of a simpler and more efficient method for the recovery of succinic acid. For the recovery of succinic acid from the fermentation broth of LPK7 strain, a simple process composed of a single reactive extraction, vacuum distillation, and crystallization yielded highly purified succinic acid (greater than 99.5% purity, wt%) with a high yield of 67.05wt%. When the same recovery process or even multiple reactive extraction steps were applied to the fermentation broth of MBEL55E, lower purity and yield of succinic acid were obtained. These results suggest that succinic acid can be purified in a cost-effective manner by using the fermentation broth of engineered LPK7 strain, showing the importance of integrating the strain development, fermentation and downstream process for optimizing the whole processes for succinic acid production.     

Production of succinate by a <Emphasis Type="Italic">pfl</Emphasis>B <Emphasis Type="Italic">ldh</Emphasis>A double mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing malate dehydrogenase

The gene encoding malate dehydrogenase (MDH) was overexpressed in a pflB ldhA double mutant of Escherichia coli, NZN111, for succinic acid production. With MDH overexpression, NZN111/pTrc99A-mdh restored the ability to metabolize glucose anaerobically and 0.55 g/L of succinic acid was produced from 3 g/L of glucose in shake flask culture. When supplied with 10 g/L of sodium bicarbonate (NaHCO₃), the succinic acid yield of NZN111/pTrc99A-mdh reached 1.14 mol/mol glucose. Supply of NaHCO₃ also improved succinic acid production by the control strain, NZN111/pTrc99A. Measurement of key enzymes activities revealed that phosphoenolpyruvate (PEP) carboxykinase and PEP carboxylase in addition to MDH played important roles. Two-stage culture of NZN111/pTrc99A-mdh was carried out in a 5-L bioreactor and 12.2 g/L of succinic acid were produced from 15.6 g/L of glucose. Fed-batch culture was also performed, and the succinic acid concentration reached 31.9 g/L with a yield of 1.19 mol/mol glucose.     

Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source

Succinic acid was produced by fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. When cells were anaerobically cultured in a medium containing 6.5 g/L glycerol, a high succinic acid yield (133%) was obtained while avoiding the formation of by-product acetic acid. The gram ratio of succinic acid to acetic acid was 25.8:1, which is 6.5 times higher than that obtained using glucose (ca. 4:1) as a carbon source. Therefore, succinic acid can be produced with much less by-product formation by using glycerol as a carbon source, which will facilitate its purification. When glucose and glycerol were cofermented with the increasing ratio of glucose to glycerol, the gram ratio of succinic acid to acetic acid and succinic acid yield decreased, suggesting that glucose enhanced acetic acid formation irrespective of the presence of glycerol. Glycerol consumption by A. succiniciproducens required unidentified nutritional components present in yeast extract. By intermittently feeding yeast extract along with glycerol, a high succinic acid yield (160%) could be obtained while still avoiding acetic acid formation. This resulted in the highest ratio of succinic acid to acetic acid (31.7:1).     

Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source

Escherichia coli strain NZN111 could convert glucose to succinic acid efficiently in anaerobic conditions after the induction of gluconeogenic carbon sources in aerobic conditions. Acetate shows a strong effect on both yield and productivity of succinic acid. In this study, the fed-batch process of succinic acid production by NZN111 using acetate in a chemically defined medium in the aerobic stage was investigated and developed. Increasing cell density could increase succinic acid with a productivity of 3.97 g/(L h) in the first 8 h of the anaerobic phase with an overall yield of 1.42 mol/mol glucose in a 5 L fermentor. However, there was strong repression from succinic acid in the later anaerobic stage. When succinic acid exceeded 30 g/L, the glucose consumption rate began to drop sharply along with the succinic acid production rate. Supplementation with glucose from 30 to 70 g/L in the anaerobic stage showed little effect on succinic acid production. Acetic acid and pyruvic acid accumulated had no effect on succinic acid formation because of their low concentration. With acetate as the sole carbon source for aerobic cultivation in the following scale-up, 60.09 g/L of succinic acid was produced with a yield of 1.37 mol/mol in a 50 L bioreactor.     

外源添加代谢中间体对产琥珀酸放线杆菌厌氧发酵制备丁二酸的影响

考察了外源添加中间代谢产物对菌体生长及发酵产酸的影响,结果表明添加0.5g/L磷酸烯醇式丙酮酸(PEP)时丁二酸产量最高。围绕产琥珀酸放线杆菌NJ113厌氧发酵产丁二酸的代谢网络进行代谢通量分析,发现添加PEP后己糖磷酸途径(HMP)与糖酵解途径(EMP)的通量比由39.4∶60.3提高至76.8∶22.6,解决了丁二酸合成过程中还原力不足的矛盾,导致PEP生成草酰乙酸的通量提高了23.8%,丁二酸代谢通量从99.8mmol/(gDCW·h)增至124.4mmol/(gDCW·h),而副产物乙酸及甲酸的代谢通量分别降低了22.9%、15.4%;关键酶活分析结果表明,添加0.5g/LPEP后PEP羧化激酶比酶活达到1910U/mg,与对照相比提高了74.7%,而丙酮酸激酶的比酶活降低了67.5%。最终丁二酸浓度为29.1g/L,收率达到76.2%,比未添加PEP时提高了11.0%。     

Proteome-based physiological analysis of the metabolically engineered succinic acid producer Mannheimia succiniciproducens LPK7

Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry, the proteome of a metabolically engineered succinic acid-overproducing bacterium, Mannheimia succiniciproducens LPK7, was examined and compared with that of its wild type strain, MBEL55E, to elucidate the physiological and metabolic changes responsible for succinic acid overproduction and cell growth. Comparative proteomic studies clearly showed that the expression levels of enzymes involved in the ATP formation and consumption (AtpD, Ppa, SerS, ProS, Pnp, PotD, MalK, RbsB, and TbpA), pyruvate metabolism (AceF and Lpd), glycolysis (GapA, Pgk, Fba, and TpiA), and amino acid biosynthesis (Asd, DapA, DapD, Gdh, ArgD, and ArgG) varied significantly in the LPK7 strain compared with those in the MBEL55E strain. Based on the comparative proteome profiling, the formation of pyruvic acid, a newly formed byproduct in the engineered LPK7 strain, could be reduced by adding into the culture medium pantothenate and l-cysteine, which serve as precursors of CoA biosynthesis.     

The effect of protectants and pH changes on the cellular growth and succinic acid yield of Mannheimia succiniciproducens LPK7

The harmful effects of succinic acid and oxidative stress on cell growth were determined during batch fermentation with Mannheimia succiniciproducens LPK7, a powerful succinic acid-producing strain, and conditions were optimized to minimize these effects. In terms of toxicity, the cell concentration decreased as the concentration of succinic acid increased. By changing the pH from 6.5 to 7 during fermentation, the cell concentration increased by about 10%, and the level of succinic acid production was 6% higher than that of the control. In addition, by introducing protectants, the cell concentration increased by about 10%, and the level of succinic acid produced was increased by 3%.     

A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity

A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.     

Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniciproducens

Acetic acid is by-product from fermentation processes for producing succinic acid using Mannheimia succiniciproducens . To obtain pure succinic acid from the final fermentation broth, acetic acid was selectively removed based on the different extractability of succinic acid and acetic acid with pH using tri-n-octylamine (TOA) as extractant. When successive batch extractions were performed using 0.25 mol TOA kg(-1) dissolved in 1-octanol at pH 5, the mol ratio of succinic acid to acetic acid before extraction was 4.9 and the final ratio after the fourth batch was 9.4.     

Feed-control development for succinic acid production with Anaerobiospirillum succiniciproducens

The aim of this work was the development of a feed-control for a succinic acid production fed-batch process. The performed batch trials indicated a correlation between succinic acid production and base consumption pH control. Based on the metabolism of Anaerobiospirillum succiniciproducens, a theoretical correlation between base consumption and glucose feed was established and proved in cultivation trials. With the established fed-batch process, the succinic acid yield could be increased to 0.875 (g/g glucose) in comparison to batch processes (0.60) with similar glucose concentrations. Additionally, the results indicate that the osmolarity of the medium has a significant influence on succinic acid production.     

Model-aided atpE gene knockout strategy in Escherichia coli for enhanced succinic acid production from glycerol

Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H⁺ conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l^?1 of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.     

Production of 4-hydroxybutyric acid by metabolically engineered Mannheimia succiniciproducens and its conversion to γ-butyrolactone by acid treatment

γ-Butyrolactone (GBL) is an important four carbon (C4) chemical, which has a wide range of industrial applications. GBL can be produced by acid treatment of 4-hydroxybutyric acid (4-HB), which is a derivative of succinic acid. Heterologous metabolic pathways were designed and established in succinic acid overproducing Mannheimia succiniciproducens LPK7 (ldhA pflD pta ackA mutant) by the introduction of heterologous genes that encode succinyl-CoA synthetase, CoA-dependent succinate semialdehyde dehydrogenase, and either 4-hydroxybutyrate dehydrogenase in LPK7 (p3S4CD) or succinate semialdehyde reductase in LPK7 (p3SYCD). Fed-batch cultures of LPK7 (p3S4CD) and LPK7 (p3SYCD) resulted in the production of 6.37 and 6.34 g/L of 4-HB (molar yields of 0.143 and 0.139), respectively. Finally, GBL was produced by acid treatment of the 4-HB obtained from the fermentation broth with molar yield of 0.673. This study demonstrates that 4-HB, and potentially other four carbon platform chemicals, can be produced by the engineered rumen bacterium M. succiniciproducens.     

Importance of redox balance on the production of succinic acid by metabolically engineered<Emphasis Type="Italic"> Escherichia coli</Emphasis>

We had previously shown that succinic acid production in a pfl ldhA double mutant strain of Escherichia coli could be enhanced by amplifying the malic enzyme activity. However, recombinant E. coli NZN111 (F- Apfl::Cam ldhA::Kan) harboring pTrcML, a plasmid containing the E. coli malic enzyme gene, produced a considerable amount of malic acid along with the desired product, succinic acid. To have an insight into the intracellular metabolism, metabolic control analysis was carried out. From the results of a simulation, it was predicted that supplying additional reducing power could enhance succinic acid production. More reduced carbon substrate sorbitol was thus examined for the possibility of matching the potential during succinic acid production. When NZN111 (pTrcML) was cultured in LB medium containing 20 g sorbitol/l under a CO2 atmosphere, 10 g succinic acid/l was produced. The apparent yield of succinic acid was 1.1 g succinic acid/g sorbitol, which is 85% of the maximum theoretical yield. Therefore, it was found that redox balancing was important for the enhanced production of succinic acid in metabolically engineered E. coli.     

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).