Anaerobic biosynthesis of intermediates of reductive branch of tricarboxylic acids cycle by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with inactivated <Emphasis Type="Italic">frdAB</Emphasis> and <Emphasis Type="Italic">sdhAB</Emphasis> genes

The genes frdAB and sdhAB, which encode components of fumarate reductase and succinate dehydrogenase, have been deleted in a recombinant E. coli strain with the inactivated pathways of mixed-acid fermentation and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. Under anaerobic conditions, the parental strain efficiently converted glucose to succinic acid without synthesizing notable amounts of fumaric or malic acid. Upon individual deletion of the frdAB genes, the mutant strain fermented glucose to succinic acid less efficiently secreting notable amounts of malic and fumaric acids. Individual deletion of the sdhAB genes in the parental strain did not significantly affect the formation of the main fermentation end-product. The combined inactivation of fumarate reductase and succinate dehydrogenase in the constructed strain enhanced the anaerobic conversion of glucose to fumaric and malic acids with the activation of the glyoxylate bypass and decrease in the contribution of the reductive branch of the TCA cycle to the formation of the target products.     

Peculiarities of C<Subscript>2</Subscript> metabolism and intensification of the synthesis of surface-active substances in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 grown in ethanol

Oxidation of ethanol, acetaldehyde, and acetate in Rhodococcus erythropolis EK-1, producer of surface-active substances (SAS), is catalyzed by N,N-dimethyl-4-nitrosoaniline (DMNA)-dependent alcohol dehydrogenase, NAD⁺/NADP⁺-dependent dehydrogenases (optimum pH 9.5), and acetate kinase/acetyl-CoA-synthetase, respectively. The glyoxylate cycle and complete tricarboxylic acid cycle function in the cells of R. erythropolis EK-1 growing on ethanol; the synthesis of phosphoenolpyruvate (PEP) is provided by the two key enzymes of gluconeogenesis, PEP carboxykinase and PEP synthetase. Introduction of citrate (0.1%) and fumarate (0.2%) into the cultivation medium of R. erythropolis EK-1 containing 2% ethanol resulted in the 1.5-and 3.5-fold increase in the activities of isocitrate lyase and PEP synthetase (the key enzymes of the glyoxylate cycle and gluconeogenesis branch of metabolism, respectively) and of lipid synthesis, as evidenced by the 1.5-fold decrease of isocitrate dehydrogenase activity. In the presence of fumarate and citrate, the indices of SAS synthesis by strain R. erythropolis EK-1 grown on ethanol increased by 40–100%.     

Metabolism of the anaerobic formation of succinic acid bySaccharomyces cerevisiae

1. Succinic acid is formed in amounts of 0.2–1.7 g/l by fermenting yeasts of the genusSaccharomyces during the exponential growth phase. No differences were observed between the various species, respiratory deficient mutants and wild type strains.
2. At low glucose concentrations the formation of succinic acid depended on the amount of sugar fermented. However, the nitrogen source was found to be of greater importance than the carbon source.
3. Of all nitrogen sources, glutamate yielded the highest amounts of succinic acid. Glutamate led to an oxidative and aspartate to a reductive formation of succinic acid.
4. A reductive formation of succinic acid by the citric acid cycle enzymes was observed with malate. This was partially inhibited by malonate. No evidence was obtained that the glyoxylate cycle is involved in succinic acid formation by yeasts.
5. Anaerobically grown cells ofSaccharomyces cerevisiae contained α-ketoglutarate dehydrogenase. Its activity was found in the 175000 x g sediment after fractionated centrifugation. The specific activity increased 6-fold after growth on glutamate as compared with cells grown on ammonium sulfate.
6. The specific activities of malate dehydrogenase, fumarase, succinate dehydrogenase, succinylcoenzymeA synthetase, α-ketoglutarate dehydrogenase and glutamate dehydrogenase (nicotinamide adenine dinucleotide dependent) were determined in yeast cells grown on glutamate or ammonium sulfate. Similar results were obtained with a wild type strain and a respiratory deficient mutant. The latter did not contain succinate dehydrogenase.
7. In fermenting yeasts succinic acid is mainly formed from glutamate by oxidation.

     

Redirecting carbon flux through <Emphasis Type="Italic">pgi</Emphasis>-deficient and heterologous transhydrogenase toward efficient succinate production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is particularly known for its potentiality in succinate production. We engineered C. glutamicum for the production of succinate. To enhance C₃–C₄ carboxylation efficiency, chromosomal integration of the pyruvate carboxylase gene pyc resulted in strain NC-4. To increase intracellular NADH pools, the pntAB gene from Escherichia coli, encoding for transhydrogenase, was chromosomally integrated into NC-4, leading to strain NC-5. Furthermore, we deleted pgi gene in strain NC-5 to redirect carbon flux to the pentose phosphate pathway (PPP). To solve the drastic reduction of PTS-mediated glucose uptake, the ptsG gene from C. glutamicum, encoding for the glucose-specific transporter, was chromosomally integrated into pgi-deficient strain resulted in strain NC-6. In anaerobic batch fermentation, the production of succinate in pntAB-overexpressing strain NC-5 increased by 14% and a product yield of 1.22 mol/mol was obtained. In anaerobic fed-batch process, succinic acid concentration reached 856 mM by NC-6. The yields of succinate from glucose were 1.37 mol/mol accompanied by a very low level of by-products. Activating PPP and transhydrogenase in combination led to a succinate yield of 1.37 mol/mol, suggesting that they exhibited a synergistic effect for improving succinate yield.     

Studies on l-Glutamic Acid Fermentation

In the previous paper, most of the enzymes of the Embden-Meyerhof-Parnas pathway and glucose-6-phosphate dehydrogenase have been demonstrated to be present in cell-free extracts of Brevibacterium divaricatum, No. 1627. In this paper, the presence of condensing enzyme, aconitase, TPN-linked isocitric dehydrogenase, succinic dehydrogenase, fumarase, DPN-linked malic dehydrogenase, TPN-linked malic enzyme, oxalacetic carboxylase, isocitritase and malate synthetase in cell-free extracts of this bacterium was also demonstrated. From these results it was concluded that a strain of Brevibacterium divaricatum which has been found to contain all of the enzymes of the tricarboxylic acid cycle, would be capable of forming the key enzymes of the glyoxylate bypass as well. It suggests that the accumulation of α-ketoglutarate involves the glyoxylate bypass besides the tricarboxylic acid cycle in this bacterium.     

Bio-Based Succinate Production from <Emphasis Type="Italic">Arundo donax</Emphasis> Hydrolysate with the New Natural Succinic Acid-Producing Strain <Emphasis Type="Italic">Basfia succiniciproducens</Emphasis> BPP7

Bio-based succinic acid production from lignocellulosic biomass is one of the attractive and prominent alternative technologies to overcome issues associated with the utilization of fossil sources. In this context, it is necessary to find new microorganisms that are able to efficiently ferment this recalcitrant feedstock. The ecological approach developed in this study enabled the isolation of Basfia succiniciproducens BPP7 from a complex rumen ecosystem. This new wild-type strain was able to synthesize up to 6.06 ± 0.05 g/L of succinate (corresponding to 0.84 ± 0.017 g of succinate per gram of consumed glucose + xylose and to 0.14 ± 0.001 g of succinate per gram of glucans + xylans present in the biomass before hydrolysis) from Arundo donax hydrolysate in separate hydrolysis and fermentation (SHF) experiments. Higher titers of succinic acid were obtained through the optimization of growth conditions. The optimal medium composition identified on the smaller scale was then used for 2.5-L batch experiments, which used A. donax hydrolysate and yeast extract as the main C and N sources, respectively. A maximal titer of 9.4 ± 0.4 g/L of succinic acid was obtained after 24 h. The overall results clearly demonstrate the potential of B. succiniciproducens BPP7 for succinate production.     

Regulation of carbon flows in the tricarboxylic acid cycle-glyoxylate bypass system in <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> under different growth conditions

The functional roles of the malate dehydrogenase (MDH) tetrameric and dimeric isoforms in the metabolism of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris, strain f-8pt was studied with the use of specific inhibitors. It was shown that the enzyme tetrameric form allows the functioning of the glyoxylate cycle and the dimeric form provides for the operation of the tricarboxylic acid cycle.     

Gamma-aminobutyric acid production through GABA shunt by synthetic scaffolds introduction in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nylon 4 is a biodegradable polymer which can be produced from the monomer of pyrrolidone. Gammaaminobutyric acid (GABA) is a precursor of pyrrolidone used for the production of bioplastics. In this study, Escherichia coli were engineered to produce gammaaminobutyric acid from glucose via an alternative novel pathway by the introduction of synthetic scaffolds. The GABA pathway constructed contained succinate dehydrogenase, succinate-semialdehyde dehydrogenase and GABA aminotransferase to redirect the Krebs cycle flux to GABA production. By introduction of a synthetic scaffold, production of 0.64 g/L GABA was achieved at 30°C and pH 6.5. Final GABA concentration was increased by 11.3% via the inactivation of competing pathways, and higher initial glucose concentration led to the enhanced final GABA concentration of 1.01 g/L.     

Enhanced succinic acid production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> with increasing the available NADH supply and glucose consumption rate by decreasing H<Superscript>+</Superscript>-ATPase activity

Objectives

To enhance succinic acid production in Corynebacterium glutamicum by increasing the supply of NADH and the rate of glucose consumption by decreasing H⁺-ATPase activity.

Results

A mutant of C. glutamicum NC-3-1 with decreased H⁺-ATPase activity was constructed. This increased the rate of glycolysis and the supply of NADH. Fermentation of C. glutamicum NC-3-1 gave 39 % higher succinic acid production (113 and 81 g/l), a 29 % higher succinic acid yield (0.94 and 0.73 g succinic acid/g glucose) and decreased by-products formation compared to that of C. glutamicum NC-3 in 5 l bioreactor.

Conclusion

The point mutation in C. glutamicum NC-3-1 increased the rate of glycolysis and resulted in higher succinic acid production, higher succinic acid yield and significantly decreased formation of by-products.

     

Metabolic engineering of Escherichia coli to produce (2S, 3R, 4S)-4-hydroxyisoleucine

The stereo-specific l-isoleucine-4-hydroxylase (l-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to “shunt” destroyed TCA, thereby coupling l-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of l-isoleucine into 4-HIL with an 82% yield.     

Features of structural organization and expression regulation of malate dehydrogenase isoforms from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> strain 2R

Two isoforms of malate dehydrogenase (MDH), dimeric and tetrameric, have been found in the purple non-sulfur bacterium Rhodobacter sphaeroides strain 2R, devoid of the glyoxylate shunt, which assimilate acetate via the citramalate cycle. Inhibitory analysis showed that the 74-kDa protein is involved in tricarboxylic acid cycle, while the 148-kDa MDH takes part in the citramalate pathway. A single gene encoding synthesis of the isologous subunits of the MDH isoforms was found during molecular-biological investigations. The appearance in the studied bacterium of the tetrameric MDH isoform during growth in the presence of acetate is probably due to the increased level of mdh gene expression, revealed by the real-time PCR, the product of which in cooperation with the citramalate cycle enzymes plays an important role in acetate assimilation.     

Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

Performance and mechanism analysis of succinate production under different transporters in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Succinic acid is a platform chemical with potential for bio-based synthesis. However, the production of bio-based succinate is limited because of insufficient succinate efflux capacity in the late stage of fermentation. In the present study, three different transporters, which have been reported to be responsible for C₄-dicarboxylates transport, were employed for investigation of the transport capacity of succinate in Escherichia coli. After engineered strains were constructed, the fermentative production of succinic acid was studied in serum bottles and 3 L of fermentor. The results demonstrated that engineered strain showed better efflux capacity than control strain under high concentration of succinate. The highest production of succinate was 68.66 g/L, while the NCgl2130 transporter may be the best candidate for succinate export in E. coli. Further research showed that the expression levels and relative enzyme activities involved in the metabolic pathway all increased markedly, and the maximum activities of PPC, PCK, PYK, and MDH increased by 1.50, 1.38, 1.28, and 1.27-fold in recombinant E. coli AFP111/pTrc99a-NCgl2130, respectively. Moreover, the maximum level of intracellular ATP increased by 23.79% in E. coli AFP111/pTrc99a-NCgl2130. Taken together, these findings indicated that engineered transporters can improve succinate production by increasing key enzyme activities and intracellular ATP levels. To the best of thew authors’ knowledge, this is the first report on a mechanism to improve succinate production by engineered transporters. This strategy set up a foundation for improving the biosynthesis of other C₄-dicarboxylates, such as fumaric acid and malic acid.     

Enzymes of the citramalate cycle in <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis>

Rhodospirillum rubrum is among the bacteria that can assimilate acetate in the absence of isocitrate lyase, the key enzyme of glyoxylate shunt. Previously we have suggested the functioning of a new anaplerotic cycle of acetate assimilation in this bacterium: citramalate cycle, where acetyl-CoA is oxidized to glyoxylate. This work has demonstrated the presence of all the key enzymes of this cycle in R. rubrum extracts: citramalate synthase catalyzing condensation of acetyl-CoA and pyruvate with the formation of citramalate, mesaconase forming mesaconate from L-citramalate, and the enzymes catalyzing transformation of propionyl-CoA + glyoxylate 3-methylmalyl-CoA ? mesaconyl-CoA. At the same time, R. rubrum synthesizes crotonyl-CoA carboxylase/reductase, which is the key enzyme of ethylmalonyl-CoA pathway discovered recently in Rhodobacter sphaeroides. Physiological differences between the citramalate cycle and the ethylmalonyl-CoA pathway are discussed.     

High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Enhanced succinic acid productivity by expression of <Emphasis Type="Italic">mgtCB</Emphasis> gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> mutant

In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg²⁺ concentration and mgtB to enhance the transport of Mg²⁺ into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)₂ and NH₃·H₂O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97 g L^?1 h^?1) compared with that by using the engineering strain with the overexpression of mgtA gene.     

Impact of Lead and Cadmium on Enzyme of Citric Acid Cycle in Germinating Pea Seeds

The present investigations were conducted to ascertain the influence of Pb₂₊ and Cd₂₊ both individually and in combination on selected enzymes of tricarboxylic acid (TCA) cycle. All the enzymes of TCA cycle examined (-ketoglutarate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase) were affected deleteriously by Pb₂₊ as well as Cd₂₊ and these metals in combination gave more or less an additive effect.     

Activity of NAD-dependent isocitrate dehydrogenase,isocitrate lyase,and malate dehydrogenase in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of γ-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of γ-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

Characterization of two β-decarboxylating dehydrogenases from <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg²⁺, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.     

Construction of glycerol synthesis pathway in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> for bioconversion of glucose into 1,3-propanediol

1,3-Propanediol (1,3-PDO) is an important three-carbon compound widely used in new polyester polymer materials. Natural organisms that can produce 1,3-PDO from glycerol were well studied. However, no natural microorganisms found could directly convert glucose to 1,3-PDO due to its insufficient glycerol synthesis pathway. In this study, two essential glycerol synthesis genes, CgGPD gene (encoding glycerol-3-phosphate dehydrogenase from Candida glycerinogenes) and ScGPP2 gene (encoding glycerol-3-phosphatase from Saccharomyces cerevisiae), were expressed in wild-type Klebsiella pneumoniae, a natural 1,3-PDO producers with reduction pathway for 1,3-PDO synthesis from glycerol. The results of fermentation, key enzyme activities, and metabolites analysis confirmed that recombinant K. pneumoniae now possessed a metabolic pathway capable of converting glucose to 1,3-PDO. The strain could produce 1,3-PDO from glucose with a final titer of 17.27 g/L with 40 g/L glucose in the medium, showing a 1.26-fold increase compared with 30 g/L glucose. Also, adding certain concentrations of glycerol could quickly initiate the 1,3-PDO synthetic pathway and promote the accumulation of 1,3-PDO, which could shorten the fermentation cycle. These results have important implications for further studies involving the use of one strain for bioconversion of glucose to 1,3-PDO.