Anaerobic Succinate Production Research Articles

Investigation of exopolysaccharide formation and its impact on anaerobic succinate production with Vibrio natriegens.

Vibrio natriegens is an emerging host for biotechnology due to its high growth and substrate consumption rates. In industrial processes typically fed-batch processes are applied to obtain high space-time yields. In this study, we established an aerobic glucose-limited fed-batch fermentation with the wild type (wt) of V. natriegens which yielded biomass concentrations of up to 28.4 gX L-1 . However, we observed that the viscosity of the culture broth increased by a factor of 800 at the end of the cultivation due to the formation of 157 ± 20 mg exopolysaccharides (EPS) L-1 . Analysis of the genomic repertoire revealed several genes and gene clusters associated with EPS formation. Deletion of the transcriptional regulator cpsR in V. natriegens wt did not reduce EPS formation, however, it resulted in a constantly low viscosity of the culture broth and altered the carbohydrate content of the EPS. A mutant lacking the cps cluster secreted two-fold less EPS compared to the wt accompanied by an overall low viscosity and a changed EPS composition. When we cultivated the succinate producer V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) under anaerobic conditions on glucose, we also observed an increased viscosity at the end of the cultivation. Deletion of cpsR and the cps cluster in V. natriegens Succ1 reduced the viscosity five- to six-fold which remained at the same level observed at the start of the cultivation. V. natriegens Succ1 ΔcpsR and V. natriegens Succ1 Δcps achieved final succinate concentrations of 51 and 46 g L-1 with a volumetric productivity of 8.5 and 7.7 gSuc L-1 h-1 , respectively. Both strains showed a product yield of about 1.4 molSuc molGlc -1 , which is 27% higher compared with that of V. natriegens Succ1 and corresponds to 81% of the theoretical maximum.

Metabolic engineering of Vibrio natriegens for anaerobic succinate production.

SummaryThe biotechnological production of succinate bears serious potential to fully replace existing petrochemical approaches in the future. In order to establish an economically viable bioprocess, obtaining high titre, yield and productivity is of central importance. In this study, we present a straightforward engineering approach for anaerobic succinate production with Vibrio natriegens, consisting of essential metabolic engineering and optimization of process conditions. The final producer strain V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pyc Cg (Succ1) yielded 1.46 mol of succinate per mol of glucose under anaerobic conditions (85% of the theoretical maximum) and revealed a particularly high biomass‐specific succinate production rate of 1.33 gSucc gCDW −1 h−1 compared with well‐established production systems. By applying carbon and redox balancing, we determined the intracellular flux distribution and show that under the tested conditions the reductive TCA as well as the oxidative TCA/glyoxylate pathway contributed to succinate formation. In a zero‐growth bioprocess using minimal medium devoid of complex additives and expensive supplements, we obtained a final titre of 60.4 gSucc l−1 with a maximum productivity of 20.8 gSucc l−1 h−1 and an overall volumetric productivity of 8.6 gSucc l−1 h−1 during the 7 h fermentation. The key performance indicators (titre, yield and productivity) of this first engineering approach in V. natriegens are encouraging and compete with costly tailored microbial production systems.

Transcriptional response of key metabolic and stress response genes of a nuculanid bivalve, Lembulus bicuspidatus from an oxygen minimum zone exposed to hypoxia-reoxygenation.

Benthic animals inhabiting the edges of marine oxygen minimum zones (OMZ) are exposed to unpredictable large fluctuations of oxygen levels. Sessile organisms including bivalves must depend on physiological adaptations to withstand these conditions. However, as habitats are rather inaccessible, physiological adaptations of the OMZ margin inhabitants to oxygen fluctuations are not well understood. We therefore investigated the transcriptional responses of selected key genes involved in energy metabolism and stress protection in a dominant benthic species of the northern edge of the Namibian OMZ, the nuculanid clam Lembulus bicuspidatus,. We exposed clams to normoxia (~5.8ml O2 l-1), severe hypoxia (36h at ~0.01ml O2 l-1) and post-hypoxic recovery (24h of normoxia following 36h of severe hypoxia). Using newly identified gene sequences, we determined the transcriptional responses to hypoxia and reoxygenation of the mitochondrial aerobic energy metabolism (pyruvate dehydrogenase E1 complex, cytochrome c oxidase, citrate synthase, and adenine nucleotide translocator), anaerobic glycolysis (hexokinase (HK), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase, and aldolase), mitochondrial antioxidants (glutaredoxin, peroxiredoxin, and uncoupling protein UCP2) and stress protection mechanisms (a molecular chaperone HSP70 and a mitochondrial quality control protein MIEAP) in the gills and the labial palps of L. bicuspidatus. Exposure to severe hypoxia transcriptionally stimulated anaerobic glycolysis (including HK and PEPCK), antioxidant protection (UCP2), and quality control mechanisms (HSP70 and MIEAP) in the gills of L. bicuspidatus. Unlike UCP2, mRNA levels of the thiol-dependent mitochondrial antioxidants were not affected by hypoxia-reoxygenation stress. Transcript levels of marker genes for aerobic energy metabolism were not responsive to oxygen fluctuations in L. bicuspidatus. Our findings highlight the probable importance of anaerobic succinate production (via PEPCK) and mitochondrial and proteome quality control mechanisms in responses to oxygen fluctuations of the OMZ bivalve L.bicuspidatus. The reaction of L.bicuspidatus to oxygen fluctuations implies parallels to that of other hypoxia-tolerant bivalves, such as intertidal species.

Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli

Metabolic engineering of microorganisms for production of succinate from glycerol remains challenging. It was thought that energy supply is a severe problem for anaerobic fermentation of glycerol to produce succinate. In this study, an energy-conserving glycerol utilization pathway was recruited to improve anaerobic succinate production in Escherichia coli. ATP dependent dihydroxyacetone kinase (DhaK) from Klebsiella was used to replace the native phosphoenolpyruvate (PEP) dependent dihydroxyacetone kinase (DhaKLM) of E. coli so that 2 NADH, instead of 1 NADH and 1 menaquinol, can be produced from glycerol to PEP. Besides consumption of 1 NADH and 1 menaquinol for converting PEP to succinate, NADH dehydrogenase I transfers electrons from 1 NADH to 1 menaquinone and pumps 4 protons. Proton-motive force are generated as the additional energy to support cell growth and succinate export. After using this energy-conserving glycerol utilization pathway, succinate titer, productivity and intercellular ATP content increased 282%, 63% and 338% respectively. The best strain YY-GS004 produced 483 mM succinate in 96 h with a yield of 0.92 mol mol-1 glycerol under anaerobic conditions. The specific succinate productivity was 0.47 g g-1 (DCW) h-1, which was the highest one up to date for succinate production from glycerol. This study demonstrated that the energy-conserving glycerol utilization pathway GldA-DhaK can solve the energy problem during anaerobic fermentation of glycerol to produce succinate.

De novo transcriptome assembly and gene expression profile of thermally challenged green abalone (Haliotis fulgens: Gastropoda) under acute hypoxia and hypercapnia

Transcriptional regulation constitutes a rapid response of marine organisms facing stressful environmental conditions, such as the concomitant exposure to warming, ocean acidification and hypoxia under climate change. In previous studies, we investigated whole-organism physiological patterns and cellular metabolism in gill and muscle of the marine gastropod Haliotis fulgens in response to increasing temperature (18 °C to 32 °C at +3 °C per day) under hypoxia (50% air saturation), hypercapnia (1000 μatm pCO2) and both factors combined. Here, we report investigations of the molecular responses of H. fulgens to temperature and identify mechanisms concomitantly affected by hypoxia and hypercapnia. A de novo transcriptome assembly with subsequent quantitative PCR and correlation network analysis of genes involved in the molecular response were used to unravel the correlations between gene expression patterns under the different experimental conditions. The correlation networks identified a shift from the expression of genes involved in energy metabolism (down-regulated) to the up-regulation of Hsp70 during warming under all experimental conditions in gill and muscle, indicating a strong up-regulation of damage prevention and repair systems at sustained cellular energy production. However, a higher capacity for anaerobic succinate production was evicted in gill, matching with observations from our previous studies indicating succinate accumulation in gill but not in muscle. Additionally, warming under hypoxia and hypercapnia kept mRNA levels of citrate synthase in both tissues unchanged following a similar pattern as muscle enzyme capacity from a previous study, suggesting an emphasis on maintaining rather than down-regulating mitochondrial activity.

Improving Succinate Productivity by Engineering a Cyanobacterial CO2 Concentrating System (CCM) in Escherichia coli

Biologically fixation of CO2 has great potential as a significant carbon source for biosynthesis, which is also a major way to reduce CO2 accumulation in atmosphere. Phosphoenolpyruvate (PEP) carboxylation is the key step of anaerobic succinate production in Escherichia coli. In this reaction, one mole CO2 is assimilated with PEP to form oxaloacetate by PEP carboxykinase (PCK). The preferred substrate of PCK is CO2 , which is very limited in cytoplasm. In this study, the carbon concentration mechanism (CCM) of cyanobacteria was introduced into Escherichia coli to enhance the intracellular inorganic carbon concentration for improving carboxylation velocity. Overexpression of the bicarbonate transporter (BT) or carbonic anhydrase (CA) gene from Synechococcus sp. PCC7002 led to a 22 or 35% increase in succinate titer at 36 h, respectively. The carboxylation rate of PCK increased from 2.46 to 3.92 µmol min-1 mg-1 protein by overexpression of the CA gene. In addition, co-overexpression of BT and CA genes had a synergetic effect, leading to a 44% increase in succinate titer at 36 h. This work is the first attempt to increase carbon fixation involved in microbial biosynthesis by engineering a biological CO2 delivery system, which provides new direction and strategies for improving industrial fermentations based on biological CO2 assimilation pathways.

Efficient anaerobic production of succinate from glycerol in engineered Escherichia coli by using dual carbon sources and limiting oxygen supply in preceding aerobic culture

Glycerol is an important resource for production of value-added bioproducts due to its large availability from the biodiesel industry as a by-product. In this study, two metabolic regulation strategies were applied in the aerobic stage of a two-stage fermentation to achieve high metabolic capacities of the pflB ldhA double mutant Escherichia coli strain overexpressing phosphoenolpyruvate carboxykinase (PCK) in the subsequent anaerobic stage: use of acetate as a co-carbon source of glycerol and restriction of oxygen supply in the PCK induction period. The succinate concentration achieved 926.7mM with a yield of 0.91mol/mol during the anaerobic stage of fermentation in a 1.5-L reactor. qRT-PCR indicated that the two strategies enhanced transcription of genes related with glycerol metabolism and succinate production. Our results showed this metabolically engineered E. coli strain has a great potential in producing succinate using glycerol as carbon source.

High-yield anaerobic succinate production by strategically regulating multiple metabolic pathways based on stoichiometric maximum in Escherichia coli.

BackgroundSuccinate has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals, which can be used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Escherichia coli are now one of the most important succinate producing candidates. However, the stoichiometric maximum succinate yield under anaerobic conditions through the reductive branch of the TCA cycle is restricted by NADH supply in E. coli.ResultsIn the present work, we report a rational approach to increase succinate yield by regulating NADH supply via pentose phosphate (PP) pathway and enhancing flux towards succinate. The deregulated genes zwf243 (encoding glucose-6-phosphate dehydrogenase) and gnd361 (encoding 6-phosphogluconate dehydrogenase) involved in NADPH generation from Corynebacterium glutamicum were firstly introduced into E. coli for succinate production. Co-expression of beneficial mutated dehydrogenases, which removed feedback inhibition in the oxidative part of the PP pathway, increased succinate yield from 1.01 to 1.16 mol/mol glucose. Three critical genes, pgl (encoding 6-phosphogluconolactonase), tktA (encoding transketolase) and talB (encoding transaldolase) were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose. Furthermore, introducing Actinobacillus succinogenes pepck (encoding phosphoenolpyruvate carboxykinase) together with overexpressing sthA (encoding soluble transhydrogenase), further increased succinate yield to 1.31 mol/mol glucose. In addition, removing byproduct formation through inactivating acetate formation genes ackA-pta and heterogenously expressing pyc (encoding pyruvate carboxylase) from C. glutamicum led to improved succinate yield to 1.4 mol/mol glucose. Finally, synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield to 1.54 mol/mol glucose, representing 52 % increase relative to the parent strain and amounting to 90 % of the strain-specific stoichiometric maximum (1.714 mol/mol glucose).ConclusionsIt’s the first time to rationally regulate pentose phosphate pathway to improve NADH supply for succinate synthesis in E. coli. 90 % of stoichiometric maximum succinate yield was achieved by combining further flux increase towards succinate and engineering its export. Regulation of NADH supply via PP pathway is therefore recommended for the production of products that are NADH-demanding in E. coli.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0536-1) contains supplementary material, which is available to authorized users.

Model-assisted formate dehydrogenase-O (fdoH) gene knockout for enhanced succinate production in Escherichia coli from glucose and glycerol carbon sources

Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H+ conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l−1 (41.2-fold in 7 days) from glycerol and .39 g l−1 (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.

Improved succinate production in Corynebacterium glutamicum by engineering glyoxylate pathway and succinate export system

A dual route for anaerobic succinate production was engineered into Corynebacterium glutamicum. The glyoxylate pathway was reconstructed by overexpressing isocitrate lyase, malate synthase and citrate synthase. The engineered strain produced succinate with a yield of 1.34 mol (mol glucose)(-1). Further overexpression of succinate exporter, SucE, increased succinate yield to 1.43 mol (mol glucose)(-1). Metabolic flux analysis revealed that the glyoxylate pathway was further activated by engineering succinate export system. Using an anaerobic fed-batch fermentation process, the final strain produced 926 mM succinate (= 109 g l(-1)) with an overall volumetric productivity of 9.4 mM h(-1) and an average yield of 1.32 mol (mol glucose)(-1).

Succinate production by metabolically engineered Escherichia coli using sugarcane bagasse hydrolysate as the carbon source

Efficient biosynthesis of succinate from a renewable biomass resource by engineered Escherichia coli is reported in this paper. Fermentation of sugarcane bagasse hydrolysate by engineered E. coli BA204, a pflB, ldhA, and ppc deletion strain overexpressing the ATP-forming phosphoenolpyruvate carboxykinase from Bacillus subtilis 168, produced a final succinate concentration of 15.85gL−1 with a high yield of 0.89gL−1 total sugar under anaerobic conditions. During dual-phase fermentations, initial aerobic growth facilitated subsequent anaerobic succinate production, with a final succinate concentration of 18.88gL−1 and a yield of 0.96gg−1 total sugar after 24h of anaerobic fermentation. The high succinate yield from sugarcane bagasse hydrolysate demonstrated a great potential application of renewable biomass as a feedstock for the economical production of succinate using metabolically engineered E. coli.

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

Previous studies have demonstrated the capability of Corynebacterium glutamicum for anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δcat Δpqo Δpta-ackA ΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase gene pyc(P458S) into BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, the fdh gene from Mycobacterium vaccae, coding for an NAD(+)-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene gapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gap accumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).

Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization

Phosphoenolpyruvate (PEP) is an important precursor for anaerobic production of succinate and malate. Although inactivating PEP/carbohydrate phosphotransferase systems (PTS) could increase PEP supply, the resulting strain had a low glucose utilization rate. In order to improve anaerobic glucose utilization rate for efficient production of succinate and malate, combinatorial modulation of galactose permease (galP) and glucokinase (glk) gene expression was carried out in chromosome of an Escherichia coli strain with inactivated PTS. Libraries of artificial regulatory parts, including promoter and messenger RNA stabilizing region (mRS), were firstly constructed in front of β-galactosidase gene (lacZ) in E. coli chromosome through λ-Red recombination. Most regulatory parts selected from mRS library had constitutive strengths under different cultivation conditions. A convenient one-step recombination method was then used to modulate galP and glk gene expression with different regulatory parts. Glucose utilization rates of strains modulated with either galP or glk all increased, and the rates had a positive relation with expression strength of both genes. Combinatorial modulation had a synergistic effect on glucose utilization rate. The highest rate (1.64 g/L h) was tenfold higher than PTS(-) strain and 39% higher than the wild-type E. coli. These modulated strains could be used for efficient anaerobic production of succinate and malate.

Attitude of Baikal and Palearctic amphipods to oxygen as a factor of environment and mechanism of adaptation to its lowered level

The paper presents the materials of a comparative study of characteristics of anaerobic mechanisms use under the conditions of lowered oxygen content by Baikal amphipods Eulimnogammarus vittatus (Dyb.), E. cyaneus (Dyb.), as well as by Palearctic Gammarus lacustris Sars. It is shown that the system of anaerobic metabolism works most effectively for Palearctic G. lacustris. This species is characterized by the least degree of use of low-efficient way of energy production—anaerobic lipolysis, and the highest degree of use of the most efficient way of energy production under the conditions of low oxygen content-anaerobic succinate production. For the Baikal species that are less resistant to the lowered level of oxygen we show more expressed induction of anaerobic lipolysis and lower degree of engagement of anaerobic succinate production.

Metabolic impact of the level of aeration during cell growth on anaerobic succinate production by an engineered Escherichia coli strain

The metabolic impact of two different aeration conditions during the growth phase on anaerobic succinate production by the high succinate producer Escherichia coli SBS550MG (pHL413) was investigated. Gene expression profiles, metabolites concentrations and metabolic fluxes were analyzed. Different oxygen levels are known to induce or repress transcription, synthesis of different enzymes, or both, affecting cell metabolism and thus product yield and productivity. The succinate yield was 1.55 and 1.25 mol succinate/mol glucose, and the productivity was 1.3 and 0.9 g L −1 h −1) for the low aeration experiment and high aeration experiment, respectively. Changes in the level of aeration during the cells growth phase significantly modified gene expression profiles and metabolic fluxes in this system. Pyruvate was accumulated during the anaerobic phase in the high aeration experiment, which could be explained by a lower pflAB expression during the transition time and a lower flux towards acetyl-CoA during the anaerobic phase compared to the low aeration case. The higher PflAB flux and the higher expression of genes related to the glyoxylate shunt ( aceA, aceB, acnA, acnB) during the transition time, anaerobic phase, or both, improved succinate yield in the low aeration case, allowing the system to attain the maximum theoretical succinate yield for E. coli SBS550MG (pHL413).

Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.

Effect of CO 2 on succinate production in dual-phase Escherichia coli fermentations

Succinate production under different concentrations of carbon dioxide (CO 2) was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase ( pfl), lactate dehydrogenase ( ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II ( ptsG). A series of two-phase fermentations were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using several constant concentrations of CO 2. As the concentration of CO 2 in the gas phase increased from 0% to 50%, the succinate specific productivity increased from 1.9 mg/g h to 225 mg/g h, and the succinate yield increased from 0.04 g/g to 0.75 g/g. Above 50% CO 2, succinate production did not increase further. Intracellular fluxes were determined at three different CO 2 concentrations (3%, 10%, and 50%) using 13C-label tracing coupled with LC–MS analysis. The fraction of carbon flux into the pentose phosphate pathway increased from 0.04 at 3% CO 2 to 0.17 at 50% CO 2. Also, the fractional flux through anaplerotic carboxylation at the phosphoenolpyruvate (PEP) node increased slightly from 0.53 at 3% CO 2 to 0.63 at 50% CO 2. The increased flux into the pentose phosphate pathway is attributed to an increased demand for reduced cofactors with elevated CO 2. A four-process explicit model to describe the CO 2 transfer and utilization was proposed. The model predicted that at CO 2 concentrations below about 30–40% the system becomes limited by gas phase CO 2, while at higher CO 2 concentrations the system is limited by PEP carboxylase enzyme kinetics.

PH and base counterion affect succinate production in dual-phase Escherichia coli fermentations

Succinate production was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). Two-phase fermentations using a defined medium at several controlled levels of pH were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using 100% (v/v) CO(2). A pH of 6.4 yielded the highest specific succinate productivity. A metabolic flux analysis at a pH of 6.4 using (13)C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate, while 93% of the succinate was formed via the reductive arm of the TCA cycle. The flux distribution at a pH of 6.8 was also analyzed and was not significantly different compared to that at a pH of 6.4. Ca(OH)(2) was superior to NaOH or KOH as the base for controlling the pH. By maintaining the pH at 6.4 using 25% (w/v) Ca(OH)(2), the process achieved an average succinate productivity of 1.42 g/l h with a yield of 0.61 g/g.

Fed‐batch culture of a metabolically engineered Escherichia coli strain designed for high‐level succinate production and yield under aerobic conditions

An aerobic succinate production system developed by Lin et al. (Metab Eng, in press) is capable of achieving the maximum theoretical succinate yield of 1.0 mol/mol glucose for aerobic conditions. It also exhibits high succinate productivity. This succinate production system is a mutant E. coli strain with five pathways inactivated: DeltasdhAB, Delta(ackA-pta), DeltapoxB, DeltaiclR, and DeltaptsG. The mutant strain also overexpresses Sorghum vulgare pepc. This mutant strain is designated HL27659k(pKK313). Fed-batch reactor experiments were performed for the strain HL27659k(pKK313) under aerobic conditions to determine and demonstrate its capacity for high-level succinate production. Results showed that it could produce 58.3 g/l of succinate in 59 h under complete aerobic conditions. Throughout the entire fermentation the average succinate yield was 0.94+/-0.07 mol/mol glucose, the average productivity was 1.08+/-0.06 g/l-h, and the average specific productivity was 89.77+/-3.40 mg/g-h. Strain HL27659k (pKK313) is, thus, capable of large-scale succinate production under aerobic conditions. The results also showed that the aerobic succinate production system using the designed strain HL27659k(pKK313) is more practical than conventional anaerobic succinate production systems. It has remarkable potential for industrial-scale succinate production and process optimization.

Effects of glutamic acid on cardiac function and energy metabolism of rat heart during ischaemia and reperfusion

The effects of exogenous glutamate (20 m m) on myocardial energy metabolism and cardiac function during low-flow ischaemia and subsequent reperfusion were studied in isolated working rat hearts. Hearts were made severely ischaemic for 60 min by reducing the perfusion rate to 0.17 ml/min, and then reperfused for 30 min. Low-flow ischaemia resulted in a 50% reduction of myocardial ATP, a 70% reduction of both creatine phosphate (CP) and GTP, and a 250% rise in AMP. After reperfusion, CP was restored to normal levels but ATP and GTP remained significantly low. All hearts failed completely to recover cardiac pump function. The addition of glutamate to the perfusate during low-flow ischaemia had no significant effect on myocardial highenergy phosphates (HEP) but slightly increased succinate production. Subsequent reperfusion without added glutamate resulted in the recovery of 62% of pre-ischaemic aortic flow rate, as well as restoration of myocardial ATP and GTP to 70% of their control values and of creatine phosphate to supranormal levels. Reperfusion with added glutamate did not raise HEP levels any further but did increase recovery of cardiac function to 92% or more of pre-ischaemic values. Thus, by mechanism(s) which are not yet clear but which may include an increase in HEP via anaerobic succinate production, elevated levels of exogenous glutamate exert a highly beneficial effect on the post-ischaemic recovery of cardiac function.