Reoxidation Of NADH Research Articles

Systematic reengineering of Klebsiella oxytoca KC004-TF160 for enhancing metabolic carbon flux towards succinate production pathway

Klebsiella oxytoca KP001-TF60 (ΔadhEΔpta-ackAΔldhAΔbudABΔpflBΔtdcDΔpmd) was re-engineered to direct more carbon flux towards succinate production with less acetate. Glucose uptake, cell growth, and carbon distribution were restricted by alterations in relative expressions and nucleotide sequences of genes associated with PEP and pyruvate metabolisms. Transcripts of pck, ppc, and frd genes were up-regulated for enhancing NADH reoxidation during succinate production while increased pyk and tdcE transcripts were observed due to maintenance of acetyl-CoA through the oxidative branch of TCA cycle. Based on whole-genome sequencing, several genes in sugars-specific PTS (ptsG, bglF, chbR, fruA, mtlR, and treY), ABC transporters (alsK, and rbsK), Major Facilitator Superfamily (uhpB and setB), and catabolite repression (cyaA and csrB) were found to be mutated. The strain produced succinate yield up to 0.89 g/g (∼80 % theoretical maximum) with acetate < 1 g/L, and may be one of the succinate producers applied in an industrial-production scale with simplified purification processes.

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.

Design and thermodynamic analysis of a pathway enabling anaerobic production of poly-3-hydroxybutyrate in Escherichia coli

Utilizing anaerobic metabolisms for the production of biotechnologically relevant products presents potential advantages, such as increased yields and reduced energy dissipation. However, lower energy dissipation may indicate that certain reactions are operating closer to their thermodynamic equilibrium. While stoichiometric analyses and genetic modifications are frequently employed in metabolic engineering, the use of thermodynamic tools to evaluate the feasibility of planned interventions is less documented. In this study, we propose a novel metabolic engineering strategy to achieve an efficient anaerobic production of poly-(R)-3-hydroxybutyrate (PHB) in the model organism Escherichia coli. Our approach involves re-routing of two-thirds of the glycolytic flux through non-oxidative glycolysis and coupling PHB synthesis with NADH re-oxidation. We complemented our stoichiometric analysis with various thermodynamic approaches to assess the feasibility and the bottlenecks in the proposed engineered pathway. According to our calculations, the main thermodynamic bottleneck are the reactions catalyzed by the acetoacetyl-CoA β-ketothiolase (EC 2.3.1.9) and the acetoacetyl-CoA reductase (EC 1.1.1.36). Furthermore, we calculated thermodynamically consistent sets of kinetic parameters to determine the enzyme amounts required for sustaining the conversion fluxes. In the case of the engineered conversion route, the protein pool necessary to sustain the desired fluxes could account for 20% of the whole cell dry weight.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

Quantification and mitigation of byproduct formation by low-glycerol-producing Saccharomyces cerevisiae strains containing Calvin-cycle enzymes

BackgroundAnaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures.ResultsIn slow-growing anaerobic chemostat cultures (D = 0.05 h−1), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h−1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h−1) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h−1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower.ConclusionsAcetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.

Cr3+-ZnGa2O4@Pt for Light-Triggered Dark Catalytic Regeneration of Nicotinamide Coenzymes without Other Electron Mediators.

Photocatalysts for regeneration of reduced nicotinamide adenine dinucleotide (NADH) usually work with continuous lighting and electron mediators, which causes impracticability under dark conditions, risk of NADH reoxidation, and complex separation. To solve these problems, we present a new catalyst of tiny Pt nanoparticles photodeposited on chromium-doped zinc gallate (CZGO@Pt). Upon being light-triggered, the photogenerated electrons are stored in the traps of CZGO and then gradually released and transferred by Pt to directly reduce NAD+ after stoppage of illumination. Three lighting modes are compared to demonstrate the feasibility and advantage of this light-triggered dark catalysis. Within 4 h of reaction, the in-the-dark NADH yield reaches 75.0% under prelighting CZGO@5%Pt and it reaches 80.0% under prelighting CZGO@5%Pt and triethanolamine (TEOA). However, the NADH yield is only 53.5% under continuous lighting of CZGO@5%Pt, TEOA, and NAD+. Consequently, the light-triggered dark catalytic regeneration of NADH not only saves energy and operates easily but also significantly elevates the NADH yield. It thus would secure wide interests and applications in places where no light or only intermittent light is available.

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis.ResultsConsistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD+-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high Km for CO2 of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO2. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway.ConclusionsCombination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

Engineering an acetoacetyl-CoA reductase from Cupriavidus necator toward NADH preference under physiological conditions

The coupling of PHB generation with NADH reoxidation is required to generate PHB as a fermentation product. A fundamental trait to accomplish this feature is to express a functional NADH-preferring acetoacetyl-CoA reductase, engaged in PHB accumulation. One way to obtain such a reductase is by engineering the cofactor preference of the acetoacetyl-CoA reductase encoded by the phaB1 gene from Cupriavidus necator (AARCn1). Aiming to have a deeper understanding of the structural determinants of the cofactor preference in AARCn1, and to obtain an NADH-preferring acetoacetyl-CoA reductase derived from this protein, some engineered enzymes were expressed, purified and kinetically characterized, together with the parental AARCn1. One of these engineered enzymes, Chimera 5, experimentally showed a selectivity ratio ((kcat/KM)NADH/(kcat/KM)NADPH) ≈ 18, which is 160 times higher than the selectivity ratio experimentally observed in the parental AARCn1. A thermodynamic-kinetic approach was employed to estimate the cofactor preference and flux capacity of Chimera 5 under physiological conditions. According to this approach, Chimera 5 could prefer NADH over NADPH between 25 and 150 times. Being a derivative of AARCn1, Chimera 5 should be readily functional in Escherichia coli and C. necator. Moreover, with the expected expression level, its activity should be enough to sustain PHB accumulation fluxes similar to the fluxes previously observed in these biotechnologically relevant cell factories.

Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the Saccharomyces cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.

Another Form of Modified, Highly-Active 6-Phosphofructo-1-Kinase in Cancer Cells

Enhanced glycolytic flux is a hallmarks of cancer cells. Posttranslational modification of the key regulatory enzyme of glycolysis, 6-Phosphofructo-1- Kinase (Pfk1) might trigger metabolic flux deregulation. In the cancer cells the human 85 kDa muscle type nPfk-M enzyme can be proteolytically cleaved to form highly-active 47 kDa shorter fragments that retain activity but become resistant to feed-back inhibition. In several tumorigenic cell lines, no native 85 kDa liver type nPfk-L isoforms could be either found and only 70 kDa shorter fragments were detected by immune-blotting. To learn more about the cancer-specific modified sfPfk-L enzyme, the truncated human sfPfk-L gene encoding 70 kDa fragments was inserted into the pfk null yeast S.cerevisiae cell. The recombinant modified enzyme showed higher affinity toward the substrate fructose-6-phosphate, reduced sensitivity toward the citrate and ATP inhibition in respect to the recombinant native PFK-L enzyme. Partially purified cancer-specific sfPfk-L fragments lacking the C-portion of the enzyme showed some instability under the diluted conditions in the buffer in respect to the tetrameric native nPfk-L enzyme. Growth characteristics of the yeast transformant encoding short sfPfk-L enzymes were similar to those encoding shorter sfPfk-M enzymes. No growth of the transformant with the sfPfk-L gene was observed on glucose but it grew faster than the transformant with the native human nPfk-L enzyme in a narrow ecological niche with low maltose concentration and 10 mM of ethanol in the medium. Similar to modified 47 kDa sfPfk-M fragments, also the short 70 kDa nPfk- Lfragments might cause deregulation of the glycolytic flux in the yeast and in the cancer cells. In yeast, deregulated metabolic flux unbalances redox potential that results in reduced growth rate. However, the cancer cells beat the redox unbalance by rapid re-oxidation of redundant NADH that results in lactate formation while the growth rate remains high.

The fructose-dependent acceleration of ethanol metabolism

The aim of the present study was to elucidate how fructose is able to increase the rate of ethanol metabolism in the liver, an observation previously termed the fructose effect. Previous studies suggest that an increase in ATP consumption driven by glucose synthesis from fructose stimulates the oxidation of NADH in the mitochondrial respiratory chain, allowing faster oxidation of ethanol by alcohol dehydrogenase; however, this idea has been frequently challenged. We tested the effects of fructose, sorbose and tagatose both in vitro and in vivo. Both ethanol and each sugar were either added to isolated hepatocytes or injected intraperitoneally in the rat. In the in vitro experiments, samples were taken from the hepatocyte suspension in a time-dependent manner and deproteinized with perchloric acid. In the in vivo experiments, blood samples were taken every 15 min and the metabolites were determined in the plasma. These metabolites include ethanol, glucose, glycerol, sorbitol, lactate, fructose and sorbose. Ethanol oxidation by rat hepatocytes was increased by more than 50% with the addition of fructose. The stimulation was accompanied by increased glucose, glycerol, lactate and sorbitol production. A similar effect was observed with sorbose, while tagatose had no effect. The same pattern was observed in the in vivo experiments. This effect was abolished by inhibiting alcohol dehydrogenase with 4-methylpyrazole, whereas inhibition of the respiratory chain with cyanide did not affect the fructose effect. In conclusion, present results provide evidence that, by reducing glyceraldehyde and glycerol and fructose to sorbitol, respectively, NADH is consumed, allowing an increase in the elimination of ethanol. Hence, this effect is not linked to a stimulation of mitochondrial re-oxidation of NADH driven by ATP consumption.

Effects of CO2 limitation on the metabolism of Pseudoclostridium thermosuccinogenes

BackgroundBio-based succinic acid holds promise as a sustainable platform chemical. Its production through microbial fermentation concurs with the fixation of CO2, through the carboxylation of phosphoenolpyruvate. Here, we studied the effect of the available CO2 on the metabolism of Pseudoclostridium thermosuccinogenes, the only known succinate producing thermophile. Batch cultivations in bioreactors sparged with 1 and 20% CO2 were conducted that allowed us to carefully study the effect of CO2 limitation.ResultsFormate yield was greatly reduced at low CO2 concentrations, signifying a switch from pyruvate formate lyase (PFL) to pyruvate:ferredoxin oxidoreductase (PFOR) for acetyl-CoA formation. The corresponding increase in endogenous CO2 production (by PFOR) enabled succinic acid production to be largely maintained as its yield was reduced by only 26%, thus also maintaining the concomitant NADH re-oxidation, essential for regenerating NAD+ for glycolysis. Acetate yield was slightly reduced as well, while that of lactate was slightly increased. CO2 limitation also prompted the formation of significant amounts of ethanol, which is only marginally produced during CO2 excess. Altogether, the changes in fermentation product yields result in increased ferredoxin and NAD+ reduction, and increased NADPH oxidation during CO2 limitation, which must be linked to reshuffled (trans) hydrogenation mechanisms of those cofactors, in order to keep them balanced. RNA sequencing, to investigate transcriptional effects of CO2 limitation, yielded only ambiguous results regarding the known (trans) hydrogenation mechanisms.ConclusionsThe results hinted at a decreased NAD+/NADH ratio, which could ultimately be responsible for the stress observed during CO2 limitation. Clear overexpression of an alcohol dehydrogenase (adhE) was observed, which may explain the increased ethanol production, while no changes were seen for PFL and PFOR expression that could explain the anticipated switch based on the fermentation results.

Engineering Escherichia coli for respiro-fermentative production of pyruvate from glucose under anoxic conditions

An Escherichia coli K-12 MG1655-derived strain was engineered for respiro-fermentative production of pyruvate from glucose under anoxic conditions, which is preferred for industrial-scale microbial synthesis of valuable chemicals. The pathways of anaerobic pyruvate dissimilation were blocked in the strain by the deletion of the ackA, pta, poxB, ldhA, adhE, and pflB genes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was substituted by an alternative ATP-dependent system resulting from the overexpression of galP and glk upon deletion of ptsG. The channelling of pyruvate towards the oxidative branch of the TCA cycle under respiratory conditions was prevented in the strain due to the deletion of aceEF genes, encoding components of pyruvate dehydrogenase, while the operation of the entire reductive branch of the TCA cycle was interrupted by knocking out frdAB and sdhAB. Reoxidation of glycolytic NADH was ensured via anaerobic respiration with nitrate serving as an external electron acceptor. To enforce anaerobic ATP hydrolysis, an ATP-consuming futile cycle of pyruvate-oxaloacetate-malate-pyruvate was established in the strain by expressing the Bacillus subtilis pycA gene, encoding pyruvate carboxylase. In the presence of sufficient amounts of an external electron acceptor and CO2 source, the engineered strain was able to efficiently utilise glucose and convert it to pyruvate anaerobically with a yield of 1.73 mol/mol, amounting to 87% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient processes of bio-based pyruvate production.

Understanding the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation based on integration of RNA-Seq and metabolite data.

The thermotolerant Kluyveromyces marxianus is a potential candidate for high-temperature ethanol fermentation. Although K. marxianus exhibited high ethanol productivity at 45°C during the early fermentation stage, we observed a fermentation arrest due to the accumulated inhibitors. The stress responses of K. marxianus during high-temperature fermentation were revealed based on integration of RNA sequencing (RNA-Seq) and metabolite data. High temperature stimulated mitochondrial respiration but repressed the tricarboxylic acid (TCA) cycle, leading to increased generation of reactive oxygen species (ROS) and a lowered ratio of reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+). Glycerol production was enhanced during the early fermentation stage, which might contribute to NADH reoxidation and ROS generation. Excess ROS could be neutralized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) that might be reserved in the following ways: (1) decreased biosynthesis of branched-chain amino acids (BCAAs) reduced NADPH consumption; (2) enhanced acetic acid production increased NADPH regeneration. The degree of fatty acid unsaturation was also reduced to adapt to high temperature. In addition, stress responses were also observed after the fermentation arrest at 45°C. Genes related to peroxidase activity, iron-sulfur cluster assembly, and flavin mononucleotide (FMN) binding were downregulated, while genes associated with DNA repair and lipid composition of the plasma were upregulated. The yeast also produced more ergosterol to deal with ethanol stress. This study gains comprehensive insights into the K. marxianus transcriptome under various stresses during high-temperature ethanol fermentation, providing rich information for further metabolic engineering towards improved stress tolerance and ethanol production.

Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae.

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported in Saccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid β-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3-NAD+ complex and the MDH3-NAD+-OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

High oxygen tension increases itaconic acid accumulation, glucose consumption, and the expression and activity of alternative oxidase in Aspergillus terreus

Itaconic acid is a five-carbon dicarboxylic acid with an unsaturated alkene bond, frequently used as a building block for the industrial production of a variety of synthetic polymers. It is also one of the major products of fungal "overflow metabolism" which can be produced in submerged fermentations of the filamentous fungus Aspergillus terreus. At the present, molar yields of itaconate are lower than those obtained in citric acid production in Aspergillus niger. Here, we have studied the possibility that the yield may be limited by the oxygen supply during fermentation and hence tested the effect of the dissolved oxygen concentration on the itaconic acid formation rate and yield in lab-scale bioreactors. The data show that a dissolved oxygen concentration of 2% saturation was sufficient for maximal biomass formation. Raising it to 30% saturation had no effect on biomass formation or the growth rate, but the itaconate yield augmented substantially from 0.53 to 0.85mol itaconate/mol glucose. Furthermore, the volumetric and specific rates of itaconic acid formation ameliorated by as much as 150% concurrent with faster glucose consumption, shortening the fermentation time by 48h. Further increasing the dissolved oxygen concentration over 30% saturation had no effect. Moreover, we show that this increase in itaconic acid production coincides with an increase in alternative respiration, circumventing the formation of surplus ATP by the cytochrome electron transport chain, as well as with increased levels of alternative oxidase transcript. We conclude that high(er) itaconic acid accumulation requires a dissolved oxygen concentration that is much higher than that needed for maximal biomass formation, and postulate that the induction of alternative respiration allows the necessary NADH reoxidation ratio without surplus ATP production to increase the glucose consumption and the flux through overflow metabolism.

Mitochondrial complex I dysfunction increases CO2 efflux and reconfigures metabolic fluxes of day respiration in tobacco leaves.

Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (Rd ) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on Rd in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. Rd was higher in mutants and the inhibition of leaf respiration by light was lower. Higher Rd values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) -catalyzed) activity. Denovo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild-type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re-oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re-orchestration of fluxes in day respiratory metabolism, leading to an increased CO2 efflux.

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

BackgroundReduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD+ balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.ResultsMulti-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD+-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.ConclusionsThe metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.

Syntrophomonas wolfei Uses an NADH-Dependent, Ferredoxin-Independent [FeFe]-Hydrogenase To Reoxidize NADH.

ABSTRACTSyntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase (hyd1ABC; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli, and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD+ and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism.IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by Syntrophomonas wolfei would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that S. wolfei has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in E. coli, demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms.

Thymoquinone ameliorates diabetic phenotype in Diet-Induced Obesity mice via activation of SIRT-1-dependent pathways.

Thymoquinone, a natural occurring quinone and the main bioactive component of plant Nigella sativa, undergoes intracellular redox cycling and re-oxidizes NADH to NAD+. TQ administration (20 mg/kg/bw/day) to the Diet-Induced Obesity (DIO) mice reduced their diabetic phenotype by decreasing fasting blood glucose and fasting insulin levels, and improved glucose tolerance and insulin sensitivity as evaluated by oral glucose and insulin tolerance tests (OGTT and ITT). Furthermore, TQ decreased serum cholesterol levels and liver triglycerides, increased protein expression of phosphorylated Akt, decreased serum levels of inflammatory markers resistin and MCP-1, and decreased NADH/NAD+ ratio. These changes were paralleled by an increase in phosphorylated SIRT-1 and AMPKα in liver and phosphorylated SIRT-1 in skeletal muscle. TQ also increased insulin sensitivity in insulin-resistant HepG2 cells via a SIRT-1-dependent mechanism. These findings are consistent with the TQ-dependent re-oxidation of NADH to NAD+, which stimulates glucose and fatty acid oxidation and activation of SIRT-1-dependent pathways. Taken together, these results demonstrate that TQ ameliorates the diabetic phenotype in the DIO mouse model of type 2 diabetes.