Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO(3) but Not NH(4) by a Green Alga

Addition of NO₃⁻ to N-limited Selenastrum minutum during photosynthesis resulted in an immediate drop in the NADPH/NADP ratio and a slower increase of the NADH/NAD ratio. These changes were accompanied by a rapid decrease in glucose-6-phosphate and increase in 6-phosphogluconate, indicating activation of glucose-6-phosphate dehydrogenase and a role for the oxidation pentose phosphate pathway during photosynthetic NO₃⁻ assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH₄⁺ were not consistent with a stimulation of the oxidative pentose phosphate pathway.     

Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis

Anion exchangers (AEs) of the Cl^-/HCO3^- exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4’-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400–1000 μM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC. The authors Chih-Yang Huang and Jer-Yuh Lin contributed equally to this article.     

Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO₃⁻ or NH₄⁺ assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH₄⁺ was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO₃⁻ addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO₃⁻ assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO₃⁻ assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH₄⁺ assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO₃⁻ assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.     

Involvement of the ornithine decarboxylase/polyamine system in precondition-induced cardioprotection through an interaction with PKC in rat hearts

Anion exchanger (AE) 2, belonging to the chloride–bicarbonate transporter family, has been reported to involve cell survival for hepatocellular carcinoma (HCC) cells. Our previous findings showed that AE2 gene was highly expressed in a poorly differentiated HCC cell line, HA22T/VGH. Additionally, treatment with 4,4′-diisothiocyanatostilbene-2,20-disulfonic acid (DIDS), an AE-specific inhibitor, significantly inhibited cell proliferation and induced cell apoptosis for the HA22T/VGH. To further investigate the biological functions of AE2 in human HCC, suppression of AE2 expression by the antisense oligonucleotide-AE2 (AS-AE2) was performed, and the cell viability, cell cycle regulation, and cell apoptosis for HCC cell lines were monitored. The results showed that AS-AE2 treatment could efficiently suppress the mRNA expression of AE2 for various differentiated HCC cells, including HA22T/VGH, SK-Hep-1, PLC/PRF/5, Hep3B, and HepG2. Moreover, AS-AE2 treatment significantly reduced cell viability, arrested cell cycle at sub-G1 phase, and induced cell apoptosis for the poorly differentiated HA22T/VGH, but not for other moderately or well-differentiated HCC cell lines. The findings indicated that AE2 may play an important role in the progression of HCC cells, and provide a new strategy for the development of therapeutic treatment against human HCC.     

Novel combination of Celecoxib and proteasome inhibitor MG132 provides synergistic antiproliferative and proapoptotic effects in human liver tumor cells

Molecular targeted therapy has shown promise as a treatment for advanced hepatocellular carcinoma (HCC). Celecoxib (Celebrex®) exhibits antitumor effects in human HCC cells, and its mechanism of action is mediated either by its ability to inhibit cyclooxygenase 2 (COX-2) or by a number of various other COX-2 independent effects. Proteasome inhibitors (PIs) can exert cell growth inhibitory and apoptotic effects in different tumor cell types, including HCC cells. The present study examined the interaction between celecoxib and the PI MG132 in two human liver tumor cell lines HepG2 and HA22T/VGH. Our data showed that each inhibitor reduced proliferation and induced apoptosis in a dose-dependent manner in both cell lines. Moreover, the combination of celecoxib with MG132 synergistically inhibited cell viability and increased apoptosis, as documented by caspase 3 and 7 activation, PARP cleavage, and down-regulation of Bcl-2. Celecoxib and MG132, both alone and synergistically in combination, induced expression of the endoplasmic reticulum (ER) stress genes ATF4, CHOP, TRB3 and promoted the splicing of XBP1 mRNA. Knockdown of TRB3 mRNA expression by small interference RNA significantly decreased combination-induced cell death in HA22T/VGH cells, whereas it increased combination-induced cell death in HepG2 cells, suggesting that activation of the ER stress response might have either a detrimental or a protective role in liver tumor cell survival. In conclusion, our data indicate that combination treatment with celecoxib and MG132 resulted in synergistic antiproliferative and proapoptotic effects against liver cancer cells, providing a rational basis for the clinical use of this combination in the treatment of liver cancer.     

Induction of plasma protein secretion in a newly established human hepatoma cell line.

To study the expression and the regulation of hepatocyte markers, we have undertaken to establish human hepatoma cell lines of various phenotypes. We now report the establishment of a new human hepatoma cell line, HA22T/VGH. This cell line has many of the properties of human hepatocellular carcinoma. Only 5 of 15 plasma proteins investigated were detected in the medium of a 10-day-old HA22T/VGH culture. However, when the HA22T/VGH cells and a clonal derivative, C5, were cultured in an aggregated form, all 15 plasma proteins were found in the culture medium. These results indicate that hepatoma cell lines with different phenotypes can be established, and they provide a good experimental framework to investigate differentiation of human hepatocytes.     

Absence of cytokeratin in human hepatoma cell lines

The immunofluorescence study revealed that both our established human hepatoma cell lines, HA22T/VGH and HA47T/VGH, were absent of cytokeratin. This observation was further confirmed by a western blot study. However, they as well as the other human hepatoma cells, Hep G2, Hep 3B, and SK-Hep-1 expressed vimentin.     

Antitumor effects of curcumin and structurally beta-diketone modified analogs on multidrug resistant cancer cells

Using concepts of bioisostery a series of curcumin analogs were synthesized: the diketonic system of the compound was elaborated into enaminones, oximes, and the isoxazole heterocycle. The cell growth inhibitory and apoptosis inducing effects of the new analogs were evaluated by in vitro assays in the hepatocellular carcinoma HA22T/VGH cells, as well as in the MCF-7 breast cancer cell line and in its multidrug resistant (MDR) variant MCF-7R. Increased antitumor activity on all cell lines was found with the isoxazole analog and especially with the benzyl oxime derivative; in the HA22T/VGH cell model, the latter compound inhibited constitutive NF-kappaB activation.     

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite

An NADPH-dependent NO₂⁻-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP⁺ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO₂⁻ reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO₂⁻ reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP⁺. NADP⁺ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO₂⁻ reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO₂⁻ reduction and (b) consume NADP⁺, releasing FNR from NADP⁺ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO₂⁻ reduction observed in vitro is capable of accounting for the observed rates of dark NO₃⁻ assimilation by C. reinhardtii.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly regulated by the ratio of NADPH/NADP⁺, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP⁺ ratio. With a ratio of NADPH/NADP⁺ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited.This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP⁺ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions.Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP⁺ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP⁺.It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP⁺ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolite unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP⁺ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film modified electrode for the simultaneous determination of ascorbic acid, dopamine, uric acid and nitrite

A novel biosensor has been constructed by the electrodeposition of Au-nanoclusters (nano-Au) on poly(3-amino-5-mercapto-1,2,4-triazole) (p-TA) film modified glassy carbon electrode (GCE) and employed for the simultaneous determination of dopamine (DA), ascorbic acid (AA), uric acid (UA) and nitrite (NO₂⁻). NH₂ and SH groups exposed to the p-TA layer are helpful for the electrodeposition of nano-Au. The combination of nano-Au and p-TA endow the biosensor with large surface area, good biological compatibility, electricity and stability, high selectivity and sensitivity and flexible and controllable electrodeposition process. In the fourfold co-existence system, the linear calibration plots for AA, DA, UA and NO₂⁻ were obtained over the range of 2.1–50.1 μM, 0.6–340.0 μM, 1.6–110.0 μM and 15.9–277.0 μM with detection limits of 1.1 × 10⁻⁶ M, 5.0 × 10⁻⁸ M, 8.0 × 10⁻⁸ M and 8.9 × 10⁻⁷ M, respectively. In addition, the modified biosensor was applied to the determination of AA, DA, UA and NO₂⁻ in urine and serum samples by using standard adding method with satisfactory results.     

Increase of Bax/ Bcl-XL ratio and arrest of cell cycle by luteolin in immortalized human hepatoma cell line

Luteolin is a common constituent of many kinds of fruits and vegetables. It possesses the anti-neoplastic activities against several human cancers, but its activity against hepatocellular carcinoma (HCC) is seldom mentioned. To evaluate the activity against HCC and to provide information about the mechanism, we tested luteolin against five human hepatoma cell lines, namely HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH, with XTT assay and flow cytometry. The results showed that luteolin inhibited PLC/PRF/5, Hep3B and HA22T/VGH at a concentration of 1 microg/ml, but it needed 5 microg/ml to inhibit HepG2 and 10 microg/ml for SK-Hep1 (P <0.05). The inhibitive concentrations of 50% (IC50) of luteolin were between 7.29 microg/ml and 32.59 microg/ml, which were comparable with those of 5-FU (15.35 microg/ml to 32.84 microg/ml). The least effective cell line as affected by luteolin (SK-Hep1) was the most effective one when treating with 5-FU. The least effective cell line as affected by 5-FU (HA22T/VGH) was effectively affected by luteolin. It seemed that luteolin had some complementary activity to 5-FU against these HCC cell lines. The luteolin-treated PLC/PRF/5 cells exhibited typical changes of apoptosis with a characteristic DNA laddering pattern on gel electrophoresis. Luteolin also activated casepase-3, increased Bax protein with a concomitant decrease in Bcl-XL level. Increase in Bax/ Bcl-XL ratio and activation of caspase-3 supported the apoptotic finding on gel electrophoresis. Luteolin also induced cell cycle arrest at G0/G1 phase. We suggested that luteolin might exhibit anti-HCC activity as efficient as 5-FU by the mechanism of not only cell cycle arrest but also apoptosis.     

Effects of nitrate contamination and seasonal variation on the denitrification and greenhouse gas production in La Rocina Stream (Doñana National Park, SW Spain)

Climatic influence (global warming and decreased rainfall) could lead to an increase in the ecological and toxicological effects of the pollution in aquatic ecosystems, especially contamination from agricultural nitrate (NO₃⁻) fertilizers. Physicochemical properties of the surface waters and sediments of four selected sites varying in NO₃⁻ concentration along La Rocina Stream, which feeds Marisma del Rocio in Doñana National Park (South West, Spain), were studied. Electrical conductivity, pH, content in macro and microelements, total organic carbon and nitrogen, and dissolved carbon and nitrogen were affected by each sampling site and sampling time. Contaminant NO₃⁻ in surface water at the site with the highest NO₃⁻ concentration (ranged in 61.6-106.6 mg L⁻¹) was of inorganic origin, most probably from chemical fertilizers, as determined chemically (90% of the total dissolved nitrogen from NO₃⁻) and by isotopic analysis of δ¹⁵N-NO₃⁻. Changes in seasonal weather conditions and hydrological effects at the sampling sites were also responsible for variations in some biological activities (dehydrogenase, β-glucosidase, arylsulphatase, acid phosphatase and urease) in sediments, as well as in the production of the greenhouse gases CO₂, CH₄ and N₂O. Both organic matter and NO₃⁻ contents influenced rates of gas production. Increased NO₃⁻ concentration also resulted in enhanced levels of potential denitrification measured as N₂O production. The denitrification process was affected by NO₃⁻ contamination and the rainfall regimen, increasing the greenhouse gases emissions (CO₂, CH₄ and especially N₂O) during the driest season in all sampling sites studied.     

Effect of phosphon-D on photosynthetic light reactions and on reactions of the oxidative and reductive pentose phosphate cycle in a reconstituted spinach (Spinacia oleracea L.) chloroplast system

Phosphon-D (tributyl-2, 4-dichlorobenzylphosphonium chloride), known as an inhibitor of gibberellin biosynthesis, enhances photosynthetic electron transport by up to 200%, with Fe(CN) ₆ ^3- and NADP⁺ being the electron acceptors. Maximum stimulation is reached at phosphon-D concentrations around 2–5 M. At the same time photosynthetic ATP formation is gradually inhibited. Phosphon-D concentrations over 0.1 mM inhibit electron transport. The uncoupling activity of phosphon-D is manifested by inhibition of noncyclic ATP synthesis and by stimulation of light-induced electron flow. The inhibition of ATP synthesis drastically decreases photosynthetic carbon assimilation in a reconstituted spinach chloroplast system. The two ATP-dependent kinase reactions of the reductive pentose phosphate cycle become the rate-limiting steps. On the other hand a stimulated photoelectron transport increases the NADPH/NADP⁺ ratio, resulting in a drastic inhibition of chloroplast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), the key enzyme of the oxidative pentose phosphate cycle. When light-induced electron flow is inhibited by high phosphon-D concentrations and the NADPH/NADP⁺ ratio is low, the light-dependent inhibition of glucose-6-phosphate dehydrogenase is gradually abolished.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - B-Nine N-dimethylaminosuccinamic acid - CCC (2-chloroethyl)-trimethylammonium chloride - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCPIP dichlorophenolindophenol - G-6-PDH glucose-6-phosphate dehydrogenase - FBP fructose bisphosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglyceric acid - Posphon-D tributyl-2,4-dichlorobenzylphosphonium chloride - PMP pentose monophosphates - PPC pentose phosphate cycle - RuBP ribulose bisphosphate - Ru-5-P ribulose-5-phosphate Dedicated to Prof. Dr. Drs.h.c. Adolf Butenandt on the occasion of his 75. birthday     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

The role of the pentose phosphate shunt in glucose-induced insulin release: In vitro studies with 6-aminonicotinamide, methylene blue, NAD, NADH, NADP, NADPH and nicotinamide on isolated pancreatic rat islets

The possible role of the pentose phosphate shunt in insulin release was investigated in vitro with collagenase isolated pancreatic islets of rats. Parameters measured were insulin released into the medium and measured by an immunoassay and formation of ¹⁴CO₂ from glucose labeled either in the C-1 or C-6 position. The in vitro effect of the following substances was studied:

1. 1. 6-Aminonicotinamide, an antimetabolite in the synthesis of pyridine nucleotides. In islets of animals pretreated with 6-amino nicotinamide 6 h previously and in the presence of 3 mg/ml glucose in the incubation medium, 6-aminonicotinamide markedly reduced oxidation of [1-¹⁴C]glucose but did not affect that of glucose labeled in C-6. Concomitantly there was a marked decrease in insulin release. This action of 6-aminonicotinamide did not take place when it was added only to the incubation medium. Pretreatment with 6-aminonicotinamide did not change the insulin concentration of the islets, making it unlikely that it interfered with insulin synthesis. The effect of 6-aminonicotinamide is consistent with partial inhibition of the pentose shunt.

2. 2. Methylene blue: this agent was selected because it is known from studies with red blood cells that it will oxidize NADPH and thus stimulate activity of the pentose shunt. In concentrations of 0.5 and 2 μg/ml, methylene blue markedly stimulated oxidation of [1-¹⁴C]glucose but not that of C-6. Simultaneously there was a dose related decrease of insulin released.

3. 3. Pyridine nucleotides: in the absence of glucose only NADPH exhibited a significant effect of insulin release. If glucose (3 mg/ml) was present 1 or 10 mM of NAD⁺ or NADH exhibited a significant effect, NADP⁺ or NADPH were less effective. If the pentose shunt was blocked by pretreatment with 6-aminonicotinamide, all 4 pyridine nucleotides stimulated insulin release. Similarly there was an increase in oxidation of [1-¹⁴C]glucose, consitent with restimulation of the pentose shunt.

4. 4. Nicotinamide by itself exhibited a small effect; however, it was much less than the one produced by equimolar concentrations of the pyridine nucleotides.

Conclusion: Restricted availability of NADPH either less production or by fast removal leads to a decrease in glucose-induced insulin release. Pyridine nucleotides will restimulate 6-aminonicotinamide blockade insulin release and glucose oxidation by the pentose shunt. Recently it has been proposed by others that the polyol pathway may play a key role in insulin release, our data are consistent with such a hypothesis. Furthermore they do support a major role of the pentose shunt in insulin release.     

Nitrate induces enzymes of the mannitol cycle and suppresses versicolorin synthesis inAspergillus parasiticus

Addition of sodium nitrate to growing cultures ofAspergillus parasiticus (ATCC 36537) induces the synthesis of enzymes involved in nitrate assimilation (NO ₃ ^– reductase), of enzymes in the pentose pathway (glucose-6-phosphate dehydrogenase), and of enzymes in the mannitol cycle (mannitol- and mannitol-1-phosphate dehydrogenases). Addition of NO ₃ ^– also causes a dose-dependent suppression of synthesis of the polyketide secondary metabolite, versicolorin A. We suggest that in the presence of NO ₃ ^– plus peptone, the cytoplasmic NADPH/NADP ratio may be elevated, resulting in increased conversion of malonyl coenzyme A to fatty acid rather than to polyketide.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.