Homeostatic regulation of NAD(H) and NADP(H) in cells.

Nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) and nicotinamide adenine dinucleotide phosphate (NADP+)/reduced NADP+ (NADPH) are essential metabolites involved in multiple metabolic pathways and cellular processes. NAD+ and NADH redox couple plays a vital role in catabolic redox reactions, while NADPH is crucial for cellular anabolism and antioxidant responses. Maintaining NAD(H) and NADP(H) homeostasis is crucial for normal physiological activity and is tightly regulated through various mechanisms, such as biosynthesis, consumption, recycling, and conversion between NAD(H) and NADP(H). The conversions between NAD(H) and NADP(H) are controlled by NAD kinases (NADKs) and NADP(H) phosphatases [specifically, metazoan SpoT homolog-1 (MESH1) and nocturnin (NOCT)]. NADKs facilitate the synthesis of NADP+ from NAD+, while MESH1 and NOCT convert NADP(H) into NAD(H). In this review, we summarize the physiological roles of NAD(H) and NADP(H) and discuss the regulatory mechanisms governing NAD(H) and NADP(H) homeostasis in three key aspects: the transcriptional and posttranslational regulation of NADKs, the role of MESH1 and NOCT in maintaining NAD(H) and NADP(H) homeostasis, and the influence of the circadian clock on NAD(H) and NADP(H) homeostasis. In conclusion, NADKs, MESH1, and NOCT are integral to various cellular processes, regulating NAD(H) and NADP(H) homeostasis. Dysregulation of these enzymes results in various human diseases, such as cancers and metabolic disorders. Hence, strategies aiming to restore NAD(H) and NADP(H) homeostasis hold promise as novel therapeutic approaches for these diseases.

Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors.

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

Nicotinic acid availability impacts redox cofactor metabolism in Saccharomyces cerevisiae during alcoholic fermentation.

Anaerobic alcoholic fermentation, particularly in high-sugar environments, presents metabolic challenges for yeasts. Crabtree-positive yeasts, including Saccharomyces cerevisiae, prefer fermentation even in the presence of oxygen. These yeasts rely on internal NAD+ recycling and extracellular assimilation of its precursor, nicotinic acid (vitamin B3), rather than de novo NAD+ production. Surprisingly, nicotinic acid assimilation is poorly characterized, even in S. cerevisiae. This study elucidated the timing of nicotinic acid uptake during grape juice-like fermentation and its impact on NAD(H) levels, the NAD+/NADH ratio, and metabolites produced. Complete uptake of extracellular nicotinic acid occurred premid-exponential phase, thereafter small amounts of vitamin B3 were exported back into the medium. Suboptimal levels of nicotinic acid were correlated with slower fermentation and reduced biomass, disrupting redox balance and impeding NAD+ regeneration, thereby affecting metabolite production. Metabolic outcomes varied with nicotinic acid concentrations, linking NAD+ availability to fermentation efficiency. A model was proposed encompassing rapid nicotinic acid uptake, accumulation during cell proliferation, and recycling with limited vitamin B3 export. This research enhances the understanding of nicotinic acid uptake dynamics during grape juice-like fermentation. These insights contribute to advancing yeast metabolism research and have profound implications for the enhancement of biotechnological practices and the wine-making industry.

Differences in the management of intracellular redox state between wine yeast species dictate their fermentation performances and metabolite production.

The maintenance of the balance between oxidised and reduced redox cofactors is essential for the functioning of many cellular processes in all living organisms. While the electron transport chain plays a key role in maintaining this balance under respiratory conditions, its inactivity in the absence of oxygen poses a challenge that yeasts such as Saccharomyces cerevisiae overcome through the production of various metabolic end-products during alcoholic fermentation. In this study, we investigated the diversity occurring between wine yeast species in their management of redox balance and its consequences on the fermentation performances and the formation of metabolites. To this aim, we quantified the changes in NAD(H) and NADP(H) concentrations and redox status throughout the fermentation of 6 wine yeast species. While the availability of NADP and NADPH remained balanced and stable throughout the process for all the strains, important differences between species were observed in the dynamics of NAD and NADH intracellular pools. A comparative analysis of these data with the fermentation capacity and metabolic profiles of the strains revealed that Saccharomyces cerevisiae, Torulaspora delbrueckii and Lachancea thermotolerans strains were able to reoxidise NADH to NAD throughout the fermentation, mainly by the formation of glycerol. These species exhibited good fermentation capacities. Conversely, Starmerella bacillaris and Metschnikowia pulcherrima species were unable to regenerate NAD as early as one third of sugars were consumed, explaining at least in part their poor growth and fermentation performances. The Kluyveromyces marxianus strain exhibited a specific behaviour, by maintaining similar levels of NAD and NADH throughout the process. This balance between oxidised and reduced redox cofactors ensured the consumption of a large part of sugars by this species, despite a low fermentation rate. In addition, the dynamics of redox cofactors affected the production of by-products by the various strains either directly or indirectly, through the formation of precursors. Major examples are the increased formation of glycerol by S. bacillaris and M. pulcherrima strains, as a way of trying to reoxidise NADH, and the greater capacity to produce acetate and derived metabolites of yeasts capable of maintaining their redox balance. Overall, this study provided new insight into the contribution of the management of redox status to the orientation of yeast metabolism during fermentation. This information should be taken into account when developing strategies for more efficient and effective fermentation.

Over-expressing NadA quinolinate synthase in Escherichia coli enhances the bioelectrochemistry in microbial fuel cells.

The microbial fuel cell (MFC), which converts biomass energy into electricity through microbial metabolism, is one of the important devices for generating new bioenergy. However, low power production efficiency limits the development of MFCs. One possible method to solve this problem is to genetically modify the microbial metabolism pathways to enhance the efficiency of MFCs. In this study, we over-expressed the nicotinamide adenine dinucleotide A quinolinate synthase gene (nadA) in order to increase the NADH/+ level in Escherichia coli and obtain a new electrochemically active bacteria strain. The following experiments showed an enhanced performance of the MFC, including increased peak voltage output (70.81 mV) and power density (0.29 µW/cm2), which increased by 361% and 20.83% compared to the control group, respectively. These data suggest that genetic modification of electricity producing microbes could be a potential way to improve MFC performance.

Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors.

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD+/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.

Redox cofactor metabolism in Saccharomyces cerevisiae and its impact on the production of alcoholic fermentation end-products.

The alcoholic fermentation of organic carbon sources by Saccharomyces cerevisiae produces many by-products, with the most abundant originating from central carbon metabolism. The production of these metabolites involves redox reactions and largely depends on the maintenance of redox homeostasis. Despite the metabolic pathways being mostly conserved across strains of S. cerevisiae, their production of various amounts of metabolic products suggests that their intracellular concentration of redox cofactors and/or redox balance differ. This study explored the redox status dynamics and NAD(H) and NADP(H) cofactor ratios throughout alcoholic fermentation in four S. cerevisiae strains that exhibit different carbon metabolic fluxes. This study focussed on the molecular end-products of fermentation, redox cofactor ratios and the impact thereof on redox homeostasis. Strain-dependent differences were identified in the redox cofactor levels, with NADP(H) ratios and levels remaining stable while NAD(H) levels decreased drastically as the fermentation progressed. Changes in the NAD+/NADH ratio were also observed. Total levels of NAD(H) decreased drastically as the fermentation progressed despite the cells remaining viable until the end of fermentation. NAD+ was found to be favoured initially while NADH was favoured towards the end of the fermentation. The change in the NAD+/NADH redox cofactor ratio during fermentation was linked with the production of end-products. The findings in this study could steer further research in the selection of S. cerevisiae wine strains for desirable aroma contributions based on their intracellular redox balance management.

Optical Redox Imaging Is Responsive to TGFß Receptor Signalling in Triple-Negative Breast Cancer Cells.

Co-enzyme nicotinamide adenine dinucleotide NAD(H) regulates hundreds of biochemical reactions within the cell. We previously reported that NAD(H) redox status may have prognostic value for predicting breast cancer metastasis. However, the mechanisms of NAD(H) involvement in metastasis remain elusive. Given the important roles of TGFß signalling in metastatic processes, such as promoting the epithelial-to-mesenchymal transition, we aimed to investigate the involvement of the mitochondrial NAD(H) redox status in TGFß receptor signalling. Here we present the initial evidence that NAD(H) redox status is responsive to TGFß receptor signalling in triple-negative breast cancer cells in culture. The mitochondrial NAD(H) redox status was determined by the optical redox imaging (ORI) technique. Cultured HCC1806 (less aggressive) and MDA-MB-231 (more aggressive) cells were subjected to ORI after treatment with exogenous TGFß1 or LY2109761, which stimulates or inhibits TGFß receptor signalling, respectively. Cell migration was determined with the transwell migration assay. Global averaging quantification of the ORI images showed that 1) TGFß1 stimulation resulted in differential responses between HCC1806 and MDA-MB-231 lines, with HCC1806 cells having a significant change in the mitochondrial redox status, corresponding to a larger increase in cell migration; 2) HCC1806 cells acutely treated with LY2109761 yielded immediate increases in ORI signals. These preliminary data are the first evidence that suggests the existence of a cell line-dependent shift of the mitochondrial NAD(H) redox status in the TGFß receptor signalling induced migratory process of breast cancer cells. Further research should be conducted to confirm these results as improved understanding of the underlying mechanisms of metastatic process may contribute to the identification of prognostic biomarkers and therapeutic targets.

E. coli 6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides.

The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S RNA 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for noncanonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the conserved 6S RNA template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions.

Luciferase-Based Determination of ATP/NAD(H) Pools in a Marine (Environmental) Bacterium.

In all living organisms, adenosine triphosphate (ATP) and NAD(H) represent universal molecular currencies for energy and redox state, respectively, and are thus widely applicable molecular proxies for an organism's viability and activity. To this end, corresponding luciferase-based assays in combination with a microplate reader were established with the marine model bacterium Phaeobacter inhibens DSM 17395 (Escherichia coli K12 served as reference). Grey multiwell plates best balanced sensitivity and crosstalk, and optimal incubation times were 5 min and 30 min for the ATP and NAD(H) assay, respectively, together allowing limits of detection of 0.042, 0.470 and 0.710 nM for ATP, NAD+, and NADH, respectively. Quenching of bacterial cell samples involved Tris-EDTA-DTAB and bicarbonate base-DTAB for ATP and NAD(H) assays, respectively. The ATP and NAD(H) yields determined for P. inhibens DSM 17395 at » ODmax were found to reside well within the range previously reported for E. coli and other bacteria, e.g., 3.28 µmol ATP (g cellsdry)-1. Thus, the here described methods for luciferase-based determination of ATP/NAD(H) pools open a promising approach to investigate energy and redox states in marine (environmental) bacteria.

Monitoring NAD(H) and NADP(H) dynamics during organismal development with genetically encoded fluorescent biosensors.

Cell metabolism plays vital roles in organismal development, but it has been much less studied than transcriptional and epigenetic control of developmental programs. The difficulty might be largely attributed to the lack of in situ metabolite assays. Genetically encoded fluorescent sensors are powerful tools for noninvasive metabolic monitoring in living cells and in vivo by highly spatiotemporal visualization. Among all living organisms, the NAD(H) and NADP(H) pools are essential for maintaining redox homeostasis and for modulating cellular metabolism. Here, we introduce NAD(H) and NADP(H) biosensors, present example assays in developing organisms, and describe promising prospects for how sensors contribute to developmental biology research.

NADH/NAD+ binding and linked tetrameric assembly of the oncogenic transcription factors CtBP1 and CtBP2.

The activation of oncogenic C-terminal binding Protein (CtBP) transcriptional activity is coupled with NAD(H) binding and homo-oligomeric assembly, although the level of CtBP assembly and nucleotide binding affinity continues to be debated. Here, we apply biophysical techniques to address these fundamental issues for CtBP1 and CtBP2. Our ultracentrifugation results unambiguously demonstrate that CtBP assembles into tetramers in the presence of saturating NAD+ or NADH with tetramer to dimer dissociation constants about 100 nm. Isothermal titration calorimetry measurements of NAD(H) binding to CtBP show dissociation constants between 30 and 500 nm, depending on the nucleotide and paralog. Given cellular levels of NAD+ , CtBP is likely to be fully saturated with NAD under physiological concentrations suggesting that CtBP is unable to act as a sensor for NADH levels.

Inborn disorders of the malate aspartate shuttle.

Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+ /NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox-dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3-phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.

NAD(H) phosphates mediate tetramer assembly of human C-terminal binding protein (CtBP).

C-terminal binding proteins (CtBPs) are cotranscriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multiangle light scattering (MALS), we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H)-linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog-specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge that connects the substrate with coenzyme-binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with cotranscriptional activity of CtBP in cancer.

Alcohol dehydrogenase 1 and NAD(H)-linked methylglyoxal oxidoreductase reciprocally regulate glutathione-dependent enzyme activities in Candida albicans.

Glutathione reductase (Glr1) activity controls cellular glutathione and reactive oxygen species (ROS). We previously demonstrated two predominant methylglyoxal scavengers-NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase 1 (Adh1)-in glutathione-depleted γ-glutamyl cysteinyl synthetase-disrupted Candida albicans. However, experimental evidence for Candida pathophysiology lacking the enzyme activities of Mgd1 and Adh1 on glutathione-dependent redox regulation remains unclear. Herein, we have aimed to demonstrate that glutathione-dependent enzyme activities coupled with cellular ROS changes is regulated by methylglyoxal accumulation in Δmgd1/Δadh1 double disruptants. Δmgd1/Δadh1 showed severe growth defects and G1-phase cell cycle arrest. The observed complementary and reciprocal methylglyoxal-oxidizing and methylglyoxalreducing activities between Δmgd1 and Δadh1 were not always exhibited in Δmgd1/Δadh1. Although intracellular accumulation of methylglyoxal and pyruvate was shown in all disruptants, to a greater or lesser degree, methylglyoxal was particularly accumulated in the Δmgd1/Δadh1 double disruptant. While cellular ROS significantly increased in Δmgd1 and Δadh1 as compared to the wild-type, Δmgd1/Δadh1 underwent a decrease in ROS in contrast to Δadh1. Despite the experimental findings underlining the importance of the undergoing unbalanced redox state of Δmgd1/Δadh1, glutathione-independent antioxidative enzyme activities did not change during proliferation and filamentation. Contrary to the significantly lowered glutathione content and Glr1 enzyme activity, the activity staining-based glutathione peroxidase activities concomitantly increased in this mutant. Additionally, the enhanced GLR1 transcript supported our results in Δmgd1/Δadh1, indicating that deficiencies of both Adh1 and Mgd1 activities stimulate specific glutathione-dependent enzyme activities. This suggests that glutathione-dependent redox regulation is evidently linked to C. albicans pathogenicity under the control of methylglyoxal-scavenging activities.

Methylglyoxal-Scavenging Enzyme Activities Trigger Erythroascorbate Peroxidase and Cytochrome c Peroxidase in Glutathione-Depleted Candida albicans.

γ-Glutamylcysteine synthetase (Gcs1) and glutathione reductase (Glr1) activity maintains minimal levels of cellular methylglyoxal in Candida albicans. In glutathione-depleted Δgcs1, we previously saw that NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase (Adh1) are the most active methylglyoxal scavengers. With methylglyoxal accumulation, disruptants lacking MGD1 or ADH1 exhibit a poor redox state. However, there is little convincing evidence for a reciprocal relationship between methylglyoxal scavenger genes-disrupted mutants and changes in glutathione-(in)dependent redox regulation. Herein, we attempt to demonstrate a functional role for methylglyoxal scavengers, modeled on a triple disruptant (Δmgd1/Δadh1/Δgcs1), to link between antioxidative enzyme activities and their metabolites in glutathione-depleted conditions. Despite seeing elevated methylglyoxal in all of the disruptants, the result saw a decrease in pyruvate content in Δmgd1/Δadh1/Δgcs1 which was not observed in double gene-disrupted strains such as Δmgd1/Δgcs1 and Δadh1/Δgcs1. Interestingly, Δmgd1/Δadh1/Δgcs1 exhibited a significantly decrease in H2O2 and superoxide which was also unobserved in Δmgd1/Δgcs1 and Δadh1/Δgcs1. The activities of the antioxidative enzymes erythroascorbate peroxidase and cytochrome c peroxidase were noticeably higher in Δmgd1/Δadh1/Δgcs1 than in the other disruptants. Meanwhile, Glr1 activity severely diminished in Δmgd1/Δadh1/Δgcs1. Monitoring complementary gene transcripts between double gene-disrupted Δmgd1/Δgcs1 and Δadh1/Δgcs1 supported the concept of an unbalanced redox state independent of the Glr1 activity for Δmgd1/Δadh1/Δgcs1. Our data demonstrate the reciprocal use of Eapx1 and Ccp1 in the absence of both methylglyoxal scavengers; that being pivotal for viability in non-filamentous budding yeast.

Sex and SP-A2 Dependent NAD(H) Redox Alterations in Mouse Alveolar Macrophages in Response to Ozone Exposure: Potential Implications for COVID-19.

Co-enzyme nicotinamide adenine dinucleotide (NAD(H)) redox plays a key role in macrophage function. Surfactant protein (SP-) A modulates the functions of alveolar macrophages (AM) and ozone (O3) exposure in the presence or absence of SP-A and reduces mouse survival in a sex-dependent manner. It is unclear whether and how NAD(H) redox status plays a role in the innate immune response in a sex-dependent manner. We investigated the NAD(H) redox status of AM from SP-A2 and SP-A knockout (KO) mice in response to O3 or filtered air (control) exposure using optical redox imaging technique. We found: (i) In SP-A2 mice, the redox alteration of AM in response to O3 showed sex-dependence with AM from males being significantly more oxidized and having a higher level of mitochondrial reactive oxygen species than females; (ii) AM from KO mice were more oxidized after O3 exposure and showed no sex differences; (iii) AM from female KO mice were more oxidized than female SP-A2 mice; and (iv) Two distinct subpopulations characterized by size and redox status were observed in a mouse AM sample. In conclusions, the NAD(H) redox balance in AM responds to O3 in a sex-dependent manner and the innate immune molecule, SP-A2, contributes to this observed sex-specific redox response.

Metabolic engineering of Escherichia coli for L-malate production anaerobically.

BACKGROUND: L-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce L-malate from renewable resources has attracted significant attention. RESULTS: A potential L-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in L-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final L-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied. CONCLUSIONS: The availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of L-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of L-malate.

Metabolic Responses to Reductive Stress.

Significance: Reducing equivalents (NAD(P)H and glutathione [GSH]) are essential for maintaining cellular redox homeostasis and for modulating cellular metabolism. Reductive stress induced by excessive levels of reduced NAD+ (NADH), reduced NADP+ (NADPH), and GSH is as harmful as oxidative stress and is implicated in many pathological processes. Recent Advances: Reductive stress broadens our view of the importance of cellular redox homeostasis and the influences of an imbalanced redox niche on biological functions, including cell metabolism. Critical Issues: The distribution of cellular NAD(H), NADP(H), and GSH/GSH disulfide is highly compartmentalized. Understanding how cells coordinate different pools of redox couples under unstressed and stressed conditions is critical for a comprehensive view of redox homeostasis and stress. It is also critical to explore the underlying mechanisms of reductive stress and its biological consequences, including effects on energy metabolism. Future Directions: Future studies are needed to investigate how reductive stress affects cell metabolism and how cells adapt their metabolism to reductive stress. Whether or not NADH shuttles and mitochondrial nicotinamide nucleotide transhydrogenase enzyme can regulate hypoxia-induced reductive stress is also a worthy pursuit. Developing strategies (e.g., antireductant approaches) to counteract reductive stress and its related adverse biological consequences also requires extensive future efforts.

Acetate formation during recombinant protein production in Escherichia coli K-12 with an elevated NAD(H) pool.

Acetate formation is a disadvantage in the use of Escherichia coli for recombinant protein production, and many studies have focused on optimizing fermentation processes or altering metabolism to eliminate acetate accumulation. In this study, E. coli MEC697 (MG1655 nadR nudC mazG) maintained a larger pool of NAD(H) compared to the wild-type control, and also accumulated lower concentrations of acetate when grown in batch culture on glucose. In steady-state cultures, the elevated total NAD(H) found in MEC697 delayed the threshold dilution rate for acetate formation to a growth rate of 0.27 h-1. Batch and fed-batch processes using MEC697 were examined for the production of ß-galactosidase as a model recombinant protein. Fed-batch culture of MEC697/pTrc99A-lacZ compared to MG1655/pTrc99A-lacZ at a growth rate of 0.22 h-1 showed only a modest increase of protein formation. However, 1 L batch growth of MEC697/pTrc99A-lacZ resulted in 50% lower acetate formation compared to MG1655/pTrc99A-lacZ and a two-fold increase in recombinant protein production.