NAD Kinase Activity Research Articles

Stripe rust effector Pst03724 modulates host immunity by inhibiting NAD kinase activation by a calmodulin.

Puccinia striiformis f. sp. tritici (Pst) secretes effector proteins that enter plant cells to manipulate host immune processes. In this report, we present an important Pst effector, Pst03724, whose mRNA expression level increases during Pst infection of wheat (Triticum aestivum). Silencing of Pst03724 reduced the growth and development of Pst. Pst03724 targeted the wheat calmodulin TaCaM3-2B, a positive regulator of wheat immunity. Subsequent investigations revealed that Pst03724 interferes with the TaCaM3-2B-NAD kinase (NADK) TaNADK2 association and thus inhibits the enzyme activity of TaNADK2 activated by TaCaM3-2B. Knocking down TaNADK2 expression by virus-mediated gene silencing significantly increased fungal growth and development, suggesting a decrease in resistance against Pst infection. In conclusion, our findings indicate that Pst effector Pst03724 inhibits the activity of NADK by interfering with the TaCaM3-2B-TaNADK2 association, thereby facilitating Pst infection.

Riboflavin application regulates sugar and energy metabolism in strawberries during cold storage

The commercial and nutritional value of strawberry fruit is drastically reduced due to its susceptibility to decay fungal diseases. In this study, we employed 40 μM riboflavin to delay senescence and decay of strawberry fruit during storage at 4 °C for 12 d. The findings showed that the treatment decreased strawberries mass loss, respiration rate, ROS level, and MDA content and maintained soluble sugars level. Riboflavin treatment maintained high levels of ATP content and energy charge, and activated the activities of succinate dehydrogenase (SDH), cytochrome C oxidase (CCO), H+-ATPase, Ca+-ATPase, glucokinase (GK), fructokinase (FRK), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6-P-GDH) and up-regulated the expression of their encoding genes. In addition, riboflavin-mediated NAD kinase (NADK) activation accelerated the conversion of NAD+ to DADP+ and the accumulation of DADPH. The respiratory pathway switched from the glycolytic (EMP)-tricarboxylic acid cycle (TCA) to the pentose phosphate pathway (PPP), reducing respiratory consumption. As a result, our findings suggest that riboflavin supplementation after harvest maintains a higher level of energy and delays senescence in strawberries.

Uncovering the Conditionally Essential Roles of NAD Kinases in Human Cells

Forward genetic screens across hundreds of human cancer cell lines have been used to identify genetic dependencies of proliferating human cells and how these vary by genotype and cell lineage. However, while it is appreciated that environmental factors influence cell physiology, there has been little investigation into how nutrient availability impacts gene essentiality. Further, most such screens have been performed in vitro using culture media that poorly reflect the metabolic composition of human blood. Previously, we developed Human Plasma‐Like Medium (HPLM), a physiologic medium designed to more closely reflect the biochemical conditions in human blood. To determine how medium composition affects gene essentiality, we recently performed CRISPR‐based screens of K562 leukemia cells in HPLM versus RPMI, the medium historically used to culture human blood cells. Among the strongest scoring HPLM‐essential genes is Nicotinamide Adenine Dinucleotide Kinase (NADK), which encodes one of two human NAD kinases (NADK, cytosolic; NADK2, mitochondrial) that can catalyze the phosphorylation of NAD(H)to NADP(H). While most human cells co‐express both isozymes, only NADK was identified as HPLM‐essential, suggesting that endogenous NADK2 expression is not sufficient to complement NADK knockout in HPLM‐cultured cells. Interestingly, NADK has been annotated as an essential gene in less than 1% of over 1000 genome‐wide CRISPR‐based screens from the Broad Institute Cancer Dependency Map project. To begin to investigate the conditional CRISPR phenotype for NADK, we first engineered NADK‐knockout cells and then found that they showed a 40% growth defect versus control cells in HPLM relative to RPMI using a short‐term growth assay. Next, to test the hypothesis that cytosolic NAD kinase activity is required for the conditionally essential role of NADK, we performed similar short‐term growth assays with NADK‐knockout cells expressing cDNAs for wild‐type and cytosol‐restricted NADK2, as well as kinase‐dead NADK. These experiments revealed that neither kinase‐dead NADK nor NADK2 overexpression could rescue the growth defect of NADK‐knockout cells, but that cytosol‐restricted NADK2 could. Collectively, these results indicate that the conditionally essential role of NADK is linked to its cytosolic NAD kinase activity. Currently, we are applying systematic approaches in media engineering and metabolomics to identify the medium component(s) that contribute to the NADK gene‐nutrient interaction, and to gain insights into the non‐redundant, context‐dependent role of NADK. Lastly, through further conditional essentiality profiling, we identified NADK2as DMEM‐essential versus HPLM. Consistent with two recently published studies, we traced this effect to the differential availability of proline between HPLM and DMEM. Collectively, our results demonstrate the human NADKs fulfill distinct cell‐essential roles and form distinct gene‐nutrient interactions that vary with their subcellular localization.

Change in expression levels of NAD kinase-encoding genes in Flaveria species

Nicotinamide adenine dinucleotides (NAD(H)) and NAD phosphates (NADP(H)) are electron carriers involved in redox reactions and metabolic processes in all organisms. NAD kinase (NADK) is the only enzyme that phosphorylates NAD+ into NADP+, using ATP as a phosphate donor. In NADP-dependent malic enzyme (NADP-ME)-type C4 photosynthesis, NADP(H) are required for dehydrogenation by NADP-dependent malate dehydrogenase (NADP-MDH) in mesophyll cells, and decarboxylation by NADP-ME in bundle sheath cells. In this study, we identified five NADK genes (FbNADK1a, 1b, 2a, 2b, and 3) from the C4 model species Flaveria bidentis. RNA-Seq database analysis revealed higher transcript abundance in one of the chloroplast-type NADK2 genes of C4F. bidentis (FbNADK2a). Comparative analysis of NADK activity in leaves of C3, C3–C4, and C4Flaveria showed that C4Flaveria (F. bidentis and F. trinervia) had higher NADK activity than the other photosynthetic-types of Flaveria. Taken together, our results suggest that chloroplastic NAD kinase appeared to increase in importance as C3 plants evolved into C4 plants in the genus Flaveria.

The Ca2+-CaM Signaling Pathway Mediates Potassium Uptake by Regulating Reactive Oxygen Species Homeostasis in Tobacco Roots Under Low-K+ Stress.

Potassium (K+) deficiency severely threatens crop growth and productivity. Calcium (Ca2+) signaling and its sensors play a central role in the response to low-K+ stress. Calmodulin (CaM) is an important Ca2+ sensor. However, the mechanism by which Ca2+ signaling and CaM mediate the response of roots to low-K+ stress remains unclear. In this study, we found that the K+ concentration significantly decreased in both shoots and roots treated with Ca2+ channel blockers, a Ca2+ chelator, and CaM antagonists. Under low-K+ stress, reactive oxygen species (ROS) accumulated, and the activity of antioxidant enzymes, NAD kinase (NADK), and NADP phosphatase (NADPase) decreased. This indicates that antioxidant enzymes, NADK, and NADPase might be downstream target proteins in the Ca2+-CaM signaling pathway, which facilitates K+ uptake in plant roots by mediating ROS homeostasis under low-K+ stress. Moreover, the expression of NtCNGC3, NtCNGC10, K+ channel genes, and transporter genes was significantly downregulated in blocker-treated, chelator-treated, and antagonist-treated plant roots in the low K+ treatment, suggesting that the Ca2+-CaM signaling pathway may mediate K+ uptake by regulating the expression of these genes. Overall, this study shows that the Ca2+-CaM signaling pathway promotes K+ absorption by regulating ROS homeostasis and the expression of K+ uptake-related genes in plant roots under low-K+ stress.

NADK is activated by oncogenic signaling to sustain pancreatic ductal adenocarcinoma.

Metabolic adaptations and the signaling events that control them promote the survival of pancreatic ductal adenocarcinoma (PDAC) at the fibrotic tumor site, overcoming stresses associated with nutrient and oxygen deprivation. Recently, rewiring of NADPH production has been shown to play a key role in this process. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells. However, de novo NADP+ is synthesized only through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). In this study, we show that oncogenic KRAS promotes protein kinase C (PKC)-mediated NADK phosphorylation, leading to its hyperactivation, thus sustaining both NADP+ and NADPH levels in PDAC cells. Together, our data show that increased NADK activity is an important adaptation driven by oncogenic signaling. Our findings indicate that NADK could serve as a much-needed therapeutic target for PDAC.

CPPU-induced changes in energy status and respiration metabolism of grape young berry development in relation to Berry setting

The energy status, the level of respiration metabolism are improtant index reflecting plant growth state, participates in many growth processes. However, their relationship to berry setting remain unclear. In this study, the grape of ‘kyoho’ of CPPU [CPPU, N-(2-coloro-4-pyridinyl)-N-phenylurea] treated were used to measurement physiological indicators. The results showed that CPPU treatment reduced respiratory rate, reduced Cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO) activity, reduced NAD kinase (NADK) activity, maintained low nicotinamide adenine denucleotie phosphate (NADP) and nicotinamide adenine denucleotie phosphate reduced form (NADPH) levels, reduced nicotinamide adenine denucleotie (NADH) levels, and slowed down the reduction of energy charge. These results provided strong evidence that CPPU treatment promoted grape berry setting due to increased energy status, increased Embden-Meyerhof pathway (EMP) and the tricarboxylic acid (TCA) cycle respiratory metabolic pathways, decreased pentose phosphate pathway (PPP), and decreased CCO and AAO activity of respiratory terminal oxidase.

Protein kinase C activates NAD kinase in human neutrophils

NAD kinase (NADK) is required for the de novo synthesis of NADP+ from NAD+. In neutrophils, NADK plays an essential role by providing sufficient levels of NADPH to support a robust oxidative burst. Activation of NADPH oxidase-2 (NOX-2) in neutrophils by stimulators of protein kinase C (PKC), such as phorbol myristate acetate (PMA), results in the rapid generation of superoxide at the expense of oxidation of NADPH to NADP+. In this study, we measured the levels of pyridine nucleotides following the addition of PMA to neutrophils. PMA elicited a rapid increase in NADP+ in neutrophils, which was not due to oxidation of NADPH, the levels of which also rose. This was mirrored by a rapid reduction in NAD+ levels, suggesting that NADK had been activated. PMA-induced depletion of NAD+ in neutrophils was blocked by PKC inhibitors, but was not dependent on NOX-2, as it was not blocked by the NOX inhibitor, diphenyleneiodonium. PMA also increased NADK activity in neutrophil lysates as well as NADK phosphorylation, as revealed by a monoclonal antibody selective for phospho-NADK. Human recombinant NADK was phosphorylated by PKCδ, resulting in increased immunoreactivity, but unchanged enzyme activity, suggesting that PKC-induced phosphorylation alone is insufficient to increase NADK activity in neutrophils. This leads us to speculate that phosphorylation of NADK promotes the dissociation of an inhibitory molecule from a complex, thereby increasing enzyme activity. Activation of NADK by PKC in phagocytic cells could be critical for the rapid provision of sufficient levels of superoxide for host defence against invading microorganisms.

Exogenous phytosulfokine α (PSKα) applying delays senescence and relief decay in strawberry fruits during cold storage by sufficient intracellular ATP and NADPH availability

Herein, we employed exogenous phytosulfokine α (PSKα) for delaying senescence and lessening decay in strawberry fruits during storage at 4 °C for 18 days. Our results showed that the strawberry fruits treated with 150 nM PSKα exhibited lower expression of poly-ADP-ribose polymerase 1 (PARP1) gene, leading to a higher intracellular NAD+ availability, beneficial for a sufficient provision of intracellular NADP+ with the activity of NAD kinase (NADK). Moreover, higher activities of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and methylenetetrahydrofolate dehydrogenase (MTHFD) may be the reason for the sufficient intracellular availability of NADPH in strawberry fruits treated with 150 nM PSKα. In addition, strawberry fruits treated with 150 nM PSKα exhibited a sufficient availability of ATP resulted from higher activities of succinate dehydrogenase (SDH) and cytochrome c oxidase (CCO). Therefore, our results indicate that exogenous PSKα could be beneficial for delaying senescence and reducing decay in strawberry fruits during cold storage.

NADK is Activated by Oncogenic KRAS Signaling to Sustain Pancreatic Ductal Adenocarcinoma

Metabolic adaptations and the signaling events that control them have been implicated in the adaptation of pancreatic ductal adenocarcinoma (PDAC) to survive stresses associated with nutrient and oxygen deprivation at the tumor site. Recently, rewiring of NADPH production has been shown to play a key role in this process. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells. However, de novo NADP+ is only synthesized through one known enzymatic reaction, catalyzed by NAD+ kinase (NADK). In this study, we show that oncogenic KRAS promotes PKC-mediated NADK phosphorylation and consequently leads to its hyperactivation, a phenomenon that is essential for sustaining NADPH levels and redox balance in PDAC cells. Together, our data show that increased NADK activity is an essential adaptation driven by oncogenic KRAS signaling. Our finding indicates that NADK could serve as a new and much-needed therapeutic target for PDAC.

Metabolic adaptation and NADPH homeostasis evoked by a sulfur-deficient environment in Pseudomonas fluorescens.

Sulfur is essential for all living organisms due to its ability to mediate a variety of enzymatic reactions, signalling networks, and redox processes. The interplay between sulfhydryl group (SH) and disulfide bond (S-S) is central to the maintenance of intracellular oxidative balance. Although most aerobic organisms succumb to sulfur starvation, the nutritionally versatile soil microbe Pseudomonas fluorescens elaborates an intricate metabolic reprogramming in order to adapt to this challenge. When cultured in a sulfur-deficient medium with glutamine as the sole carbon and nitrogen source, the microbe reconfigures its metabolism aimed at the enhanced synthesis of NADPH, an antioxidant and the limited production of NADH, a pro-oxidant. While oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle, metabolic modules known to generate reactive oxygen species are impeded, the activities NADPH-producing enzymes such as malic enzyme, and glutamate dehydrogenase (GDH) NADP-dependent are increased. The α-ketoglutarate (KG) generated from glutamine rapidly enters the TCA cycle via α-ketoglutarate dehydrogenase (KGDH), an enzyme that was prominent in the control cultures. In the S-deficient media, the severely impeded KGDH coupled with the increased activity of the reversible isocitrate dehydrogenase (ICDH) that fixes KG into isocitrate in the presence of NADH and HCO3- ensures a constant supply of this critical tricarboxylic acid. The up-regulation of ICDH-NADP dependent in the soluble fraction of the cells obtained from the S-deficient media results in enhanced NADPH synthesis, a reaction aided by the concomitant increase in NAD kinase activity. The latter converts NAD into NADP in the presence of ATP. Taken together, the data point to a metabolic network involving isocitrate, α-KG, and ICDH that converts NADH into NADPH in P. fluorescens subjected to a S-deprived environment.

Enzymatic Characteristics of a Polyphosphate/ATP-NAD Kinase, PanK, from Myxococcus xanthus.

NAD kinase is a crucial enzyme for production of NADP+. Myxococcus xanthus is a gram-negative soil bacterium that forms fruiting bodies and spores under starvation, and it accumulates polyphosphate (poly(P)) during early development. We found that M. xanthus NAD kinase (PanK) utilized both ATP and poly(P) as phosphoryl donors; therefore, PanK was designated as a poly(P)/ATP-NAD kinase. Unlike other poly(P)/ATP-NAD kinases, PanK hardly exhibited NADH kinase activity. The NAD kinase activity of PanK was inhibited by NADPH, but not NADH. Replacement of Thr-90 in the GGDGT motif of PanK with Asn decreased both ATP- and poly(P)-dependent NAD kinase activities; however, poly(P)-dependent NAD kinase activity was further decreased by approximately 6- to 10-fold compared with ATP-dependent NAD kinase activity, suggesting that Thr-90 in the GGDGT motif of PanK may be important for poly(P) utilization. PanK preferred ATP and short-chain poly(P) as phosphoryl donors. The Km of PanK for ATP, poly(P)4, and poly(P)10-15 was 0.66mM, 0.08mM, and 0.71mM, respectively, and the catalytic efficiency (kcat/Km) for poly(P)4 was 2.4-fold higher than that for ATP, suggesting that M. xanthus under starvation conditions may be able to efficiently generate NADP+ using PanK, ATP, and poly(P).

Changes in Activities of Key Enzymes in Sugarcane Stem at Different Growing Stages

Sugarcane is the most important sugar crop in China, which mainly focuses on the upper stem of the harvested land. The proper regulation of the proportion of internode elongation is the key to determine the yield and sugar. Therefore, it is of great significance to study the mechanism of the dynamic change of elongation between cane joints in order to improve the yield of sugar cane and sucrose. To investigate the biochemical mechanism of stem elongation in sugarcane, stem samples were collected at the pre-elongation stage (9-10 leaves) (Ls1), early elongation stage (12-13 leaves) (Ls2) and rapid elongation stage (15-16 leaves) (Ls3). The change trends in the activities of NAD kinase (NADK), calcium-dependent protein kinase (CDPKs), α-mannosidase, α-galactosidase, β-glucosidase, cellulase, xyloglucan endo-transglycosylase/hydrolase (XTH) and catalase (CAT) were completely consistent, showing rapid elongation stage > early elongation stage > pre-elongation stage, while the activities of β-glucosidase, peroxidase (POD) and α-glucosidase were in opposite, and the activities of calmodulin and β-mannosidase showed the same single-peak trend, and the peak was at early elongation stage. The enzyme activities of β-galactosidase and pectinase did not show significant difference at different stages. The results indicate that the elongation of internodes was closely related to the complex physiological metabolism of sugarcane, and the key enzymes play roles at different time but β-galactosidase and pectinase have little effect on internodes elongation in sugarcane.

The effector AvrRxo1 phosphorylates NAD in planta.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3’-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3’-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3’-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

Energy status regulates disease development and respiratory metabolism of Lasiodiplodia theobromae (Pat.) Griff. & Maubl.-infected longan fruit

Lasiodiplodia theobromae (Pat.) Griff. & Maubl. is a major pathogen causing decay of harvested longan fruit. The roles of energy status regulated by 2,4-dinitrophenol (DNP) and adenosine triphosphate (ATP) in disease development regarding respiratory metabolism of L. theobromae-inoculated “Fuyan” longan fruit were studied. Compared with L. theobromae-inoculated longans, DNP treatment could promote the index of fruit disease, accelerate the decrease in energy charge, increase respiration rate and the activities of respiratory terminal oxidases like CCO, AAO and PPO, elevate contents of NAD and NADH, but decrease NAD kinase activity, as well as contents of NADP and NADPH; however, exogenous ATP supply acted contrarily. Above results suggested the different energy status caused by DNP and ATP treatments accelerated or delayed the disease development of L. theobromae-inoculated longans via regulating Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP) and activities of respiratory terminal oxidases.

Enhanced production of poly-3-hydroxybutyrate by recombinant Escherichia coli containing NAD kinase and phbCAB operon

With the help of Tn5 transposon technique, gene yfjB encoding NAD kinase in Escherichia coli ( E . coli ) was inserted into chromosome of recombinant E . coli polyhydroxybutyrate (PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfjB gene copy into genome, the selected recombinant named E . coli PHBTY4 showed stronger NAD kinase activity than that of E . coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E . coli PHBTY4 compared with that of the control. E . coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E . coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions.

Hydrogen Peroxide Induced Changes in Energy Status and Respiration Metabolism of Harvested Longan Fruit in Relation to Pericarp Browning.

Energy status and respiration metabolism of "Fuyan" longan fruit treated by hydrogen peroxide (H2O2) and their relationship to pericarp browning were studied. The results displayed that H2O2 significantly increased the respiration rate, increased activities of respiratory terminal oxidases like cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO), decreased NAD kinase activity, maintained lower contents of NADP and NADPH as well as higher amounts of NAD and NADH, and accelerated the decrease of energy charge. These results gave convincing evidence that the treatment of H2O2 for accelerating longan pericarp browning was due to an increase of energy deficiency, an increase of respiratory metabolic pathways of Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA) cycle, a decrease of pentose phosphate pathway (PPP) of respiratory pathway, and an increase of activities of respiratory terminal oxidases like CCO and AAO.

Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP+. Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme.

Overexpression of ZWF1 and POS5 improves carotenoid biosynthesis in recombinant Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae expressing exogenous carotenogenic genes can synthesize carotenoids. NADPH is a key cofactor for carotenoid biosynthesis, while glucose-6-phosphate dehydrogenase (Zwf1) and an NADH kinase (Pos5) are the two main NADPH-supplying sources in S.cerevisiae. Here, the effect of ZWF1 and POS5 overexpression on carotenoid yield in recombinant S.cerevisiae was explored. The initial carotenogenic strain Sc-EYBIH+I which expressed crtE, crtYB, crtI, cHMG1 and another copy of crtI could synthesize 1·35±0·13mgl(-1) of lycopene and 0·32±0·02mgl(-1) of β-carotene. When ZWF1 was overexpressed (Sc-EYBIZH+I), glucose-6-phosphate dehydrogenase activity increased by 103-fold, the transcription level of crtE and crtI increased significantly, the lycopene and β-carotene yield increased to 2·29±0·06 and 0·38±0·02mgl(-1) respectively. When POS5 was overexpressed (Sc-EYBIPH+I), NAD kinase activity increased by 5·5-fold, the transcription level of crtE, crtYB and crtI increased obviously, the lycopene and β-carotene yield increased to 2·50±0·11 and 0·53±0·03mgl(-1) respectively. Therefore, improvement of NADPH supply contributed to carotenoids biosynthesis in S.cerevisiae and overexpression of POS5 was more effective than overexpression of ZWF1. This study provides a new strategy for enhancing carotenoid biosynthesis. NADPH is a key cofactor for carotenoid biosynthesis. Glucose-6-phosphate dehydrogenase (Zwf1) and an NADH kinase (Pos5) are effective NADPH-supplying sources in Saccharomyces cerevisiae. When ZWF1 and POS5 were overexpressed in a carotenoid-producing S.cerevisiae strain individually, the total yield of lycopene and β-carotene increased by 59·9% and 81·4%, respectively, and the final product β-carotene yield increased by 18·8% and 65·6% respectively. This suggests the improvement of NADPH supply as a useful strategy for carotenoids biosynthesis.

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy.

NAD(+) kinase (NADK) is the only known cytosolic enzyme that converts NAD(+) to NADP(+), which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein, and fatty acid synthesis found in proliferating cells as well as for neutralizing high levels of reactive oxygen species (ROS). We determined whether inhibition of NADK activity is a valid anticancer strategy alone and in combination with chemotherapeutic drugs known to induce ROS. In vitro and in vivo inhibition of NADK with either small-hairpin RNA or thionicotinamide inhibited proliferation. Thionicotinamide enhanced the ROS produced by several chemotherapeutic drugs and produced synergistic cell kill. NADK inhibitors alone or in combination with drugs that increase ROS-mediated stress may represent an efficacious antitumor combination and should be explored further.