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1.
Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae 总被引:5,自引:0,他引:5
Anaerobic fermentation was relatively difficult to optimize due to lack of monitoring parameters. In this paper, a new method
was reported using extracellular oxidoreduction potential (ORP) to monitor 1,3-propanediol (1,3-PD) biosynthesis process by
Klebsiella pneumoniae. In batch fermentation, cell growth, 1,3-propanediol production and by-products distribution were studied at four different
ORP levels: 10, −140, −190 and −240 mV. From the results, the ORP level of −190 mV was preferable, which resulted in fast
cell growth and high 1,3-propanediol concentration. The NAD+/NADH ratio was determined at different ORP levels, and a critical NAD+/NADH ratio of 4 was defined to divide fermentation environments into two categories: relatively oxidative environment (NAD+/NADH>4) and relatively reductive environment (NAD+/NADH<4). The former was correlative with high 1,3-propanediol productivity and high specific growth rate. The mechanism of
ORP regulation was discussed. It is suggested that ORP regulation of fermentation might be due to its influence on the ratio
of NAD+/NADH, which determined metabolic flux. Furthermore, a batch fermentation of modulating ORP following a profile in different
levels corresponding to different fermentation stage was tested. The 1,3-PD concentration was 22.3% higher than that of constant
ORP fermentation at −190 mV. Therefore, ORP is a valuable parameter to monitor and control anaerobic fermentation production. 相似文献
2.
Enterobacter aerogenes NBRC12010 was able to ferment glycerol to ethanol and hydrogen gas. Fermentation of glycerol ceased in the stationary phase
of growth, and it was activated by electrochemical reactions using thionine as an electron transfer mediator from bacterial
cells to an electrode. Using resting cells of E. aerogenes NBRC12010 in only citrate buffer solution, the cells did not consume glycerol at all, but they could metabolize glucose.
These results suggest that the regulation of glycerol metabolism occurred at enzymatic steps before glycolysis. In E. aerogenes NBRC12010, glycerol was metabolized via glycerol dehydrogenase (GDH) and then dehydroxyacetone kinase. The GDH-catalyzed
reaction mainly depended on the ratio of NAD+/NADH. At a NAD+/NADH ratio of nearly 1 or less, it was substantially suppressed and glycerol metabolism stopped. When the ratio was higher
than 1, GDH was activated and glycerol was metabolized. Thus, the reaction of glycerol metabolism depended on the balance
of cellular NAD+/NADH. Exogenous NADH was oxidized to NAD+ by electrochemical reactions with thionine. We proposed the activation mechanism of glycerol metabolism under electrochemical
conditions. 相似文献
3.
Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To regulate NAD(H) pool size and NADH/NAD+ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD+ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD+ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD+ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased. 相似文献
4.
Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber 总被引:2,自引:2,他引:0
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The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O2 consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD+ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD+ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD+ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD+ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD+-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites. 相似文献
5.
6.
Sandra K. Hartmann Yvonne Stockdreher Georg Wandrey Hamed Hosseinpour Tehrani Thiemo Zambanini Andreas J. Meyer Jochen Büchs Lars M. Blank Markus Schwarzländer Nick Wierckx 《BBA》2018,1859(10):1015-1024
Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD+ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD+ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts. 相似文献
7.
Caspar Elo Christensen Magnus Karlsson Jakob R. Winther Pernille Rose Jensen Mathilde H. Lerche 《The Journal of biological chemistry》2014,289(4):2344-2352
Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD+]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments. 相似文献
8.
Ionica Masgras Giuseppe Cannino Francesco Ciscato Carlos Sanchez-Martin Fereshteh Babaei Darvishi Francesca Scantamburlo Marco Pizzi Alessio Menga Dolores Fregona Alessandra Castegna Andrea Rasola 《Cell death and differentiation》2022,29(10):1996
Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.Subject terms: Cancer metabolism, Cell biology 相似文献
9.
Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD+ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD+/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD+/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD+/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD+/NADH, and pH. These distributions were calculated using physiological NAD+/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms. 相似文献
10.
3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD+-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B12, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD+ and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD+ regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD+ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor. 相似文献
11.
Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms. In this study, 7-dehydrocholesterol (7-DHC, a crucial precursor of vitamin D3) biosynthesis pathway was constructed in Saccharomyces cerevisiae BY4742 with endogenous ergosterol synthesis pathway blocked by knocking out the erg5 gene (encoding C-22 desaturase). The deletion of erg5 led to redox imbalance with higher ratio of cytosolic free NADH/NAD+ and more glycerol and ethanol accumulation. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) and an alternative oxidase (AOX1) were employed in our system based on cofactor regeneration strategy. Consequently, the production of 7-dehydrocholesterol was increased by 74.4% in shake flask culture. In the meanwhile, the ratio of free NADH/NAD+ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L. This study provides a reference to increase the production of some desired compounds that are restricted by redox imbalance. 相似文献
12.
Fei Chen Xiao-Hai Feng Jin-Feng Liang Hong Xu Ping-Kai Ouyang 《Bioprocess and biosystems engineering》2013,36(9):1165-1176
The effects of oxidoreduction potential (ORP) regulation on the process of propionic acid production by Propionibacterium freudenreichii CCTCC M207015 have been investigated. Potassium ferricyanide and sodium borohydride were determined as ORP control agents through serum bottle experiment. In batch fermentation, cell growth, propionic acid and by-products distribution were changed with ORP levels in the range of 0–160 mV. Based on these analysis results, an ORP-shift control strategy was proposed: at first 156 h, ORP was controlled at 120 mV to obtain higher cell growth rate and propionic acid formation rate, and then it was shifted to 80 mV after 156 h to maintain the higher propionic acid formation rate. By applying this strategy, the optimal parameters were obtained as follows: the propionic acid concentration 45.99 g L?1, productivity 0.192 g L?1 h?1, the proportion of propionic acid to total organic acids 92.26 % (w/w) and glycerol conversion efficiency 76.65 %. The mechanism of ORP regulation was discussed by the ratio of NADH/NAD+, ATP levels, and metabolic flux analysis. The results suggest that it is possible to redistribute energy and metabolic fluxes by the ORP-shift control strategy, and the strategy could provide a simple and efficient tool to realize high purity propionic acid production with glycerol as carbon source. 相似文献
13.
Klebsiella oxytoca ZS fermented glucose to ethanol and lactic, formic, and acetic acids, but, in contrast to many strains, accumulates pyruvic and acetic acids as the principal end products in aerobic growth conditions. This strain was grown in sulfate-limited chemostat at a fixed low dilution rate (D=0.033 h–1) with glucose present in excess. When oxygen was supplied at a high level, pyruvate and acetate were produced, and the ratio NADH/NAD+ was low (0.04) while the internal pyruvate concentration increased to 100 mol (g dry wt)–1. A shortage of oxygen supply was accompanied by lactate production, an increase of the ratio NADH/NAD+ (0.53), and an undetectable level in internal pyruvate concentration. The observed changes in LDH activity found in vitro in extracts of the cells are not strictly related to those found in vivo. In fact, the specific activity of LDH was essentially stable at 30% of dissolved oxygen tension (d.o.t.) and decreased slightly at 60% of d.o.t., whereas specific lactic acid production decreased rapidly. The in vitro LDH activity was strongly affected by the NADH/NAD+ ratio. 相似文献
14.
Luisa Schwarzmann Rainer Ullrich Pliquett Andreas Simm Babett Bartling 《Bioscience reports》2021,41(1)
Nicotinamide adenine dinucleotide (NAD) is a coenzyme in metabolic reactions and cosubstrate in signaling pathways of cells. While the intracellular function of NAD is well described, much less is known about its importance as an extracellular molecule. Moreover, there is only little information about the concentration of extracellular NAD and the ratio between its oxidized (NAD+) and reduced (NADH) form in humans. Therefore, our study aimed at the analysis of total NAD and NAD+/NADH ratio in human plasma depending on sex and age. First, an enzymatic assay was established for detecting NAD+ and NADH in human plasma samples. Then, plasma NAD was analyzed in 205 probands without severe diseases (91 men, 114 women) being 18–83 years old. The total plasma NAD concentration was determined with median 1.34 µM (0.44–2.88 µM) without difference between men and women. Although the amounts of NAD+ and NADH were nearly balanced, women had higher plasma NAD+/NADH ratios than men (median 1.33 vs. 1.09, P<0.001). The sex-related difference in the plasma NAD+/NADH ratio reduces with increasing age, an effect that was more obvious for two parameters of the biological age (skin autofluorescence, brachial-femoral pulse wave velocity (PWV)) than for the chronological age. However, plasma values for total NAD and NAD+/NADH ratio did not generally alter with increasing age. In conclusion, human plasma contains low micromolar concentrations of total NAD with higher NAD+/NADH redox ratios in adult but not older women compared with same-aged men. 相似文献
15.
In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD+ ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD+ ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3. 相似文献
16.
Vijay Tejwani Franz-Josef Schmitt Svea Wilkening Ingo Zebger Marius Horch Oliver Lenz Thomas Friedrich 《BBA》2017,1858(1):86-94
Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 oxidation with the reduction of NAD+ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD+ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD+] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD+] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD+] ratios represents a novel and sensitive tool to determine the redox state of cells. 相似文献
17.
Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD+ and NADH:NADP+ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD+ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD+ system was approximately five times more active than the NADH:NADP+ system. The NADH:NADP+ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD+ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD+ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD+ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD+ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD+ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD+ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system. 相似文献
18.
Jung-Chin Chang Simei Go Eduardo H. Gilglioni Suzanne Duijst Daan M. Panneman Richard J. Rodenburg Hang Lam Li Hsu-Li Huang Lonny R. Levin Jochen Buck Arthur J. Verhoeven Ronald P.J. Oude Elferink 《BBA》2021,1862(4):148367
The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level. 相似文献
19.
Motoyuki Shimizu 《Bioscience, biotechnology, and biochemistry》2018,82(2):216-224
Filamentous fungi are used to produce fermented foods, organic acids, beneficial secondary metabolites and various enzymes. During such processes, these fungi balance cellular NAD+:NADH ratios to adapt to environmental redox stimuli. Cellular NAD(H) status in fungal cells is a trigger of changes in metabolic pathways including those of glycolysis, fermentation, and the production of organic acids, amino acids and secondary metabolites. Under hypoxic conditions, high NADH:NAD+ ratios lead to the inactivation of various dehydrogenases, and the metabolic flow involving NAD+ is down-regulated compared with normoxic conditions. This review provides an overview of the metabolic mechanisms of filamentous fungi under hypoxic conditions that alter the cellular NADH:NAD+ balance. We also discuss the relationship between the intracellular redox balance (NAD/NADH ratio) and the production of beneficial secondary metabolites that arise from repressing the HDAC activity of sirtuin A via Nudix hydrolase A (NdxA)-dependent NAD+ degradation. 相似文献
20.
In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD+ as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD+ is exhausted. Here we show that NAD+ regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD+-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD+. When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD+ enhanced the activity of ALD4. More interestingly, the growth rates of all NAD+-regenerating strains were prolonged in comparison with the control, indicating that NAD+ regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD+ availability but also provides a method for regulating the dha regulon. 相似文献