PSK1 coordinates glucose metabolism and utilization and regulates energy-metabolism oscillation in Saccharomyces cerevisiae

Energy-metabolism oscillations (EMO) are ultradian biological rhythms observed in in aerobic chemostat cultures of Saccharomyces cerevisiae. EMO regulates energy metabolism such as glucose, carbohydrate storage, O₂ uptake, and CO₂ production. PSK1 is a nutrient responsive protein kinase involved in regulation of glucose metabolism, sensory response to light, oxygen, and redox state. The aim of this investigation was to assess the function of PSK1 in regulation of EMO. The mRNA levels of PSK1 fluctuated in concert with EMO, and deletion of PSK1 resulted in unstable EMO with disappearance of the fluctuations and reduced amplitude, compared with the wild type. Furthermore, the mutant PSK1Δ showed downregulation of the synthesis and breakdown of glycogen with resultant decrease in glucose concentrations. The redox state represented by NADH also decreased in PSK1Δ compared with the wild type. These data suggest that PSK1 plays an important role in the regulation of energy metabolism and stabilizes ultradian biological rhythms. These results enhance our understanding of the mechanisms of biorhythms in the budding yeast.     

Gluconeogenesis in the liver of rats receiving 1,3-butanediol in their diet

Inclusion of 1,3-butandiol as a synthetic source of nutritional energy into the composition of a low-carbohydrate diet produced a fall in the ration of free [NAD+]:[NADN] and [NADP]:[NADPN] calculated for the cytoplasm and mitochondria of the liver cell in rats according to the concentration of oxidated and reduced metabolites and the equilibrium constant of the lactate-dehydrogenase, glutamate-dehydrogenase and malic-fermentative systems. In these conditions the concentration of metabolites, at whose level the conjugation of the carbohydrates decomposition during glycolysis and their synthesis at the time of gluconeogenesis (phosphoenol-pyruvate, malate, oxaloacetate) is realized, as well as the activity of key gluconeogenesis enzymes (phosphoenol-pyruvate carboxykinase, fructose-1,6-diphosphatase) increase. The NADN generation in the course of oxidative metabolism of 1,3-butandiol gives rise to reducing properties of the free NAD-par cytoplasm and mitochondria pool, which leads to the intensification of gluconeogenesis in the liver, attended by a drop of the phosphate potential level.     

Expression of a cytoplasmic transhydrogenase in Saccharomyces cerevisiae results in formation of 2-oxoglutarate due to depletion of the NADPH pool

The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/NADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can catalyse transfer of reducing equivalents between the two nucleotide systems, occurs in several organisms, but not in yeasts. The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomyces cerevisiae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme, and expressed it under the control of a S. cerevisiae promoter in a strain derived from the industrial model strain S. cerevisiae CBS8066. Anaerobic batch cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein was measured in the extracts from the strain expressing the sth gene from A. vinelandii, while no transhydrogenase activity could be detected in control strains without the gene. Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate. Since NADPH is used to convert 2-oxoglutarate to glutamate while glycerol formation increases when excess NADH is formed, this suggested that transhydrogenase converted NADH and NADP(+) to NAD(+) and NADPH. This was further supported by measurements of the intracellular nucleotide concentrations. Thus, the (NADPH/NADP(+)):(NADH/NAD(+)) ratio was reduced from 35 to 17 by the transhydrogenase. The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate. Also the biomass and ethanol yields were significantly lowered by the transhydrogenase.     

Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions,using alcohol dehydrogenase as sensor reaction

With the current quantitative metabolomics techniques, only whole‐cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH‐catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose‐limited chemostats under steady‐state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose‐limited steady‐state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD(+). The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-Delta1), formation of glycerol was significantly impaired, which resulted in reduction of the maximum specific growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-Delta1 gpd2-Delta1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD(+) by synthesis of glycerol. Also, strain TN23 (gpd1-Delta1 gpd2-Delta1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD(+) pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP(+) to NADPH and NAD(+). Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-Delta1) and TN5 (gpd2-Delta1) without serious effects on the maximum specific growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-Delta1 gpd2-Delta1) resulted in a dramatic reduction in the maximum specific growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in micromax from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP(+) to NADH and NADP(+). Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.     

Saccharomyces cerevisiae QNS1 codes for NAD(+) synthetase that is functionally conserved in mammals

NAD(+), an essential molecule involved in a variety of cellular processes, is synthesized through de novo and salvage pathways. NAD(+) synthetase catalyses the final step in both pathways. Here we show that this enzyme is encoded by the QNS1 gene in Saccharomyces cerevisiae. Expression of Escherichia coli or Bacillus subtilis NAD(+) synthetases was able to suppress the lethality of a qns1 deletion, while a B. subtilis NAD(+) synthetase mutant with lowered catalytic activity was not. Overexpression of QNS1 tagged with HA led to elevated levels of NAD(+) synthetase activity in yeast extracts, and this activity can be recovered by immunoprecipitation using anti-HA antibody. An allele of QNS1 was constructed that carries a point mutation predicted to reduce the catalytic activity. Overexpression of this allele, qns1(G521E), failed to elevate NAD(+) synthetase levels and qns1(G521E) could not rescue the lethality caused by the depletion of Qns1p. These results demonstrate that NAD(+) synthetase activity is essential for cell viability. A GFP-tagged version of Qns1p displayed a diffuse localization in both the nucleus and the cytosol. Finally, the rat homologue of QNS1 was cloned and shown to functionally replace yeast QNS1, indicating that NAD(+) synthetase is functionally conserved from bacteria to yeast and mammals.     

SHAM-sensitive alternative respiration in the xylose-metabolizing yeast Pichia stipitis

SHAM-sensitive (STO) alternative respiration is present in the xylose-metabolizing, Crabtree-negative yeast, Pichia stipitis, but its pathway components and physiological roles during xylose metabolism are poorly understood. We cloned PsSTO1, which encodes the SHAM-sensitive terminal oxidase (PsSto1p), by genome walking from wild-type CBS 6054 and subsequently deleted PsSTO1 by targeted gene disruption. The resulting sto1-delta deletion mutant, FPL-Shi31, did not contain other isoforms of Sto protein that were detectable by Western blot analysis using an alternative oxidase monoclonal antibody raised against the Sto protein from Sauromatum guttatum. Levels of cytochromes b, c, c(1) and a.a(3) did not change in the sto1-delta mutant, which indicated that deleting PsSto1p did not alter the cytochrome pool. Interestingly, the sto1-delta deletion mutant stopped growing earlier than the parent and produced 20% more ethanol from xylose. Heterologous expression of PsSTO1 in Saccharomyces cerevisiae increased its total oxygen consumption rate and imparted cyanide-resistant oxygen uptake but did not enable growth on ethanol, indicating that PsSto1p is not coupled to ATP synthesis. We present evidence that the mitochondrial NADH dehydrogenase complex (Complex I) was present in wild-type CBS 6054 but was bypassed in the cells during xylose metabolism. Unexpectedly, deleting PsSto1p led to the use of Complex I in the mutant cells when xylose was the carbon source. We propose that the non-proton-translocating NAD(P)H dehydrogenases are linked to PsSto1p in xylose-metabolizing cells and that this non-ATP-generating route serves a regulatory function in the complex redox network of P. stipitis.     

Low ergosterol content in yeast adh1 mutant enhances chitin maldistribution and sensitivity to paraquat-induced oxidative stress

Alcohol dehydrogenases catalyse the reversible oxidation of alcohols to aldehydes or ketones, with concomitant reduction of NAD(+) or NADP(+) . Adh1p is responsible for the reduction of acetaldehyde to ethanol, while Adh2p catalyses the reverse reaction, the oxidation of ethanol to acetaldehyde. Lack of Adh1p shifts the cellular redox balance towards excess NADH/NADPH and acetaldehyde, while absence of Adh2p does the opposite. Yeast mutant adh1Δ had a slow growth rate, whereas adh2Δ grew like the isogenic wild-type (WT) during prediauxic shift fermentative metabolism. After 48 h WT and mutants reached the same number of viable cells. When exponentially growing (LOG) cells were exposed to calcofluor white, only mutant adh1Δ displayed an irregular deposition of chitin. Quantitative analyses of both LOG and stationary-phase cells showed that adh1Δ mutant contained significantly less ergosterol than cells of WT and adh2Δ mutant, whereas the erg3Δ mutant contained extremely low ergosterol pools. Both adh1Δ and adh2Δ mutants showed higher-than-WT resistance to heat shock and to H(2) O(2) but had WT resistance when exposed to ultraviolet (UV) light and the DNA cross-linking agent diepoxyoctane, indicating normal DNA repair capacity. Mutant adh1Δ was specifically sensitive to acetaldehyde and to membrane peroxidizing paraquat. Our results link the pleiotropic phenotype of adh1Δ mutants to low pools of ergosterol and to reductive stress, and introduce the two new phenotypes, resistance to heat shock and to H(2) O(2) , for the adh2Δ mutant, most probably related to increased ROS production in mitochondria, which leads to the induction of oxidative stress protection.     

高糖胁迫下蜂蜜接合酵母碳代谢流的研究

研究蜂蜜接合酵母LGL-1在高糖胁迫下的代谢特性及代谢流变化。采用高效液相色谱等方法测定蜂蜜接合酵母LGL-1在葡萄糖质量浓度100 g/L和400 g/L条件下积累的代谢产物(酒精、海藻糖、甘油、苹果酸、α-酮戊二酸、柠檬酸及乳酸),通过构建代谢矩阵方程组,采用Matlab软件和归一法分析关键节点的代谢流分布情况。结果表明,蜂蜜接合酵母LGL-1高糖胁迫时的碳流量偏向流入海藻糖、甘油、乙醇和TCA循环的代谢支路,与常规培养相比,海藻糖、甘油和TCA循环的通量分别增加了50.00%,17.73%和4.79%,高糖培养与常规培养的乙醇代谢通量接近。在高糖胁迫条件下蜂蜜接合酵母LGL-1积累的海藻糖、甘油以及TCA循环的通量变化也反映海藻糖、甘油以及TCA循环对酵母细胞的重要保护功能。     

乙醇发酵中酿酒酵母辅酶NAD+及NADH测定方法

乙醇发酵过程中酿酒酵母细胞辅酶NAD+和NADH质量分数及其比例反映了胞内的氧化还原状态和细胞代谢活性,具有重要的生理意义。作者主要研究了加热萃取、珠磨破碎、反复冻融破碎3种方法对乙醇发酵过程酿酒酵母细胞辅酶NAD+和NADH提取和测定的影响,提出基于珠磨破碎、辅以加酸或碱并加热的萃取模式,以及合适的酶循环反应体系,从而建立了高效的胞内NAD+和NADH检测方法。     

冰温贮藏对猪肉宰后品质及糖酵解途径相关酶活性的影响

为研究冰温贮藏对猪肉感官品质和宰后糖酵解途径中乳酸代谢的影响,以猪里脊肉为对象,分别采用冰温(-1℃)和冷鲜(4℃)贮藏方式,测定其宰后0.5、2、6、12、24、72、120、168h时包括汁液损失率、色泽、pH值在内的品质指标以及糖酵解及相关代谢途径主要酶的活力.结果表明:冰温贮藏可有效降低猪肉品质劣变,减缓糖酵解...     

Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ

Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae. To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase δ is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase δ, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1, which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.     

翅果油对高脂饮食小鼠的降脂减肥作用

目的:研究翅果油干预对高脂饮食小鼠降脂减肥作用。方法:将40只C57BL/6小鼠随机分为5组(n=8):正常组、高脂饮食组、翅果油低、中、高剂量组,灌胃量分别为1.5、3.0和6.0 g/kg·d。正常组给予基础饲料,其余各组给予高脂饲料,干预8周。实验结束后记录小鼠体重、摄食量等基础指标,测定血清中总胆固醇(Total cholesterol,TC)、甘油三酯(Triglyceride,TG)、高密度胆固醇(High density lipoprotein cholesterol,HDL-C)及肝脏TC、TG的含量,并计算血清中低密度脂蛋白(Low density lipoprotein cholesterol,LDL-C)含量及动脉粥样硬化指数(Atherosclerosis index,AI),苏木精伊红染色(Hematoxylin and eosin,HE)染色观察脂肪组织细胞数量及大小,并进行相关性分析。结果:与高脂饮食组相比,翅果油干预组显著降低小鼠体重、脂肪重、肝重、肝脏TG含量及血清TC、TG、LDL-C含量(P<0.05),其中翅果油中剂量组干预效果最好;翅果油干预能增加血清HDL-C含量,降低AI值,但不具有显著性差异(P>0.05);HE染色结果显示,高脂饮食小鼠脂肪细胞变大且排列不均匀,翅果油干预能够抑制高脂饮食引起的脂肪细胞增大,且中剂量效果较好;相关性分析结果显示,脂肪细胞面积与肝脏TG、TC含量呈显著正相关,与血清HDL-C呈显著负相关,与AI值呈显著正相关(P<0.05),说明翅果油可以通过降低肝脏脂质含量,调节机体脂质代谢,从而抑制脂肪细胞增大,达到降脂减肥的作用。结论:翅果油干预可以在一定程度上有效预防肥胖及与肥胖相关的代谢疾病的发生。     

Changes in wine yeast storage carbohydrate levels during preadaptation,rehydration and low temperature fermentations

The metabolism of glycogen and trehalose was analysed in a wine yeast strain fermenting at 25 and 13 degrees C. Trehalose and glycogen degradation were completed during the lag phase of fermentation. Ammonia was taken up rapidly and once it had been reduced to negligible amounts, the synthesis of trehalose started. Glycogen followed a similar pattern. If trehalose synthesis was taken as a stress indicator, the fermentation at 13 degrees C could not be considered stressful because the maximum concentrations are similar at both temperatures. In industrial fermentations, and after a preadaptation in grape must for several hours at 18 degrees C, the lag phase was reduced significantly, and this may be why trehalose and glycogen were completely depleted at the beginning of the low temperature fermentation. Various preadaptation conditions were tested so that their influence on trehalose and glycogen degradation could be determined. The presence of fermentable carbon sources, such as glucose or fructose, triggered the mobilisation and use of trehalose. However, just increasing the osmotic pressure did not reduce the trehalose content. No such differences were observed in glycogen metabolism.     

RESERVE CARBOHYDRATE METABOLISM AND CELL SURVIVAL IN AEROBICALLY STARVING BAKER'S YEAST

Changes in the level of reserve carbohydrates, cell number, viability and medium pH values were observed in aerobically starving baker's yeast. The starvation was carried out at 35°C in the physiological saline solution using two different initial pH values (4·4 or 5·5) of the medium. In addition, the formation of ethanol and acetate as well as actual and endogenous respirations were measured at a pH of 4·4. The results revealed that the initial pH value of the medium affected the pattern of glycogen and trehalose degradation and consequently, also caused the loss of viability and cell lysis. Changes in the quantity of metabolic products and respiration activity are discussed in connection with the metabolism of the starving cells and the effect of environmental conditions.     

Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris

Pichia pastoris has been used to produce various recombinant proteins under high oxygen demand conditions. To improve the heterologous production of β-galactosidase, the vgb gene encoding Vitreoscilla hemoglobin (VHb) was co-expressed in the P. pastoris cytoplasm under the control of the methanol-inducible promoter. Co-expression of VHb under different aeration conditions improved cell performance in terms of growth, viability, respiratory rate, and β-galactosidase production. Under limiting aeration conditions, the VHb(+) strain produced 28.2% more biomass but 31.2% less total β-galactosidase activity than the VHb(-) strain. Under non-limiting aeration conditions, the VHb(+) strain showed 20.3% higher cell growth and 9.9% more total β-galactosidase activity than the VHb(-) strain. Moreover, under these conditions, the VHb(+) strain was 7.7% more viable and had a 28.2% higher oxygen uptake rate (OUR) than the VHb(-) strain. Evidently, VHb can enhance the OUR and promote methanol metabolism, thereby improving cell performance and β-galactosidase production.     

温度调节对克鲁氏假丝酵母海藻糖代谢的影响

研究了恒温条件和热冲击条件对克鲁氏假丝酵母海藻糖代谢的影响。结果表明,将指数生长期的克鲁氏假丝酵母细胞置于恒定的45℃和25℃,均不利于细胞生长及海藻糖的合成,而适宜的热冲击能够迅速促进细胞海藻糖的合成。在正常的生长状态下,处于指数生长期的克鲁氏假丝酵母细胞内只积累少量的海藻糖,但此时当细胞受到外界热冲击时海藻糖会大量积累。在热冲击结束后,海藻糖的含量又会恢复至对照水平。周期性热冲击实验表明,随着指数生长期的延续,这种应激性反应逐渐减弱,在3个冲击周期结束时细胞内海藻糖的含量分别是对照的3·9,3及1·6倍。热冲击的温度和持续时间对细胞生长和海藻糖的积累都有影响,过高的温度及过长时间的热冲击均会使细胞生长受到抑制。最佳的热冲击温度为45℃,最佳热冲击持续时间为1h。