The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001

Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l(-1) xylose or chemostat cultures of 20 g l(-1) glucose and 20 g l(-1) xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However, the specific growth rate on xylulose increased from 0.02-0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l(-1) xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase.     

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiae

This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time. The mass isotopomer distributions of free intracellular metabolites in central carbon metabolism were measured by liquid chromatography-mass spectrometry. For four time points during the culture, these distributions were used to obtain the best estimates for the metabolic fluxes. The obtained flux fits suggested that the optimally fitted split ratio for the pentose phosphate pathway changed by almost a factor of 2 up and down around a value of 0.27 during the experiment. Statistical analysis revealed that some of the fitted flux distributions for different time points were significantly different from each other, indicating that cell cycle-dependent variations in cytosolic metabolic fluxes indeed occurred.     

系列过表达非氧化磷酸戊糖途径基因对重组酿酒酵母菌株木糖代谢的影响

【目的】通过系统研究一个、两个及多个非氧化磷酸戊糖(PP)途径基因组合过表达对酿酒酵母木糖代谢的影响,以优化重组菌株的构建过程,构建高效的木糖代谢酿酒酵母菌株。【方法】在酿酒酵母中双拷贝过表达上游代谢途径的关键酶(木糖还原酶XR,木糖醇脱氢酶XDH,木酮糖激酶XKS),在此基础上构建了一系列PP途径基因过表达菌株,并对其木糖发酵性能进行比较研究。【结果】木糖发酵结果显示,不同组合过表达PP途径基因能不同程度改善重组菌株的木糖发酵性能。其中,过表达PP途径全部基因(RKI1,RPE1,TAL1和TKL1)使菌株的发酵性能最优,其乙醇产率和产量较对照菌株分别提高了39.25%和12.57%,同时较其他基因组合过表达菌株也有不同程度的改善。【结论】通过构建PP途径基因不同组合过表达酿酒酵母菌株,首次对PP途径基因对酿酒酵母木糖代谢的影响进行了系统研究,结果表明,不同组合强化PP途径基因对重组菌株木糖代谢的影响存在差异,相对于其他基因过表达组合,同步过表达PP途径全部基因最有利于碳通量流向乙醇。     

Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.     

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by ³¹P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.     

New aspects on phosphate sensing and signalling in Saccharomyces cerevisiae

The mechanism involved in the cellular phosphate response of Saccharomyces cerevisiae forms part of the PHO pathway, which upon expression allows a co-ordinated cellular response and adaptation to changes in availability of external phosphate. Although genetic studies and analyses of the S. cerevisiae genome have produced much information on the components of the PHO pathway, little is known about how cells sense the environmental phosphate level and the mechanistic regulation of phosphate acquisition. Recent studies emphasize different levels in phosphate sensing and signalling in response to external phosphate fluctuations. This review integrates all these findings into a model involving rapid and long-term effects of phosphate sensing and signalling in S. cerevisiae. The model describes in particular how yeast cells are able to adjust phosphate acquisition by integrating the status of the intracellular phosphate pools together with the extracellular phosphate concentration.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

Significance of the non-oxidative route of the pentose phosphate pathway for supplying carbon to the purine-nucleotide pathway in <Emphasis Type="Italic">Corynebacterium ammoniagenes</Emphasis>

To evaluate the strategy of supplying ribose 5-phosphate to the purine-nucleotide pathway exclusively via the nonoxidative route, the glucose 6-phosphate dehydrogenase gene zwf was disrupted in inosine- and 5′-xanthylic acid-producers of Corynebacterium ammoniagenes. In both producers, interruption of the oxidative route caused a decrease in production yields of about 50%. Attempts to increase the capacity of the nonoxidative route through overexpression of the transketolase or transaldolase gene in the zwf mutants led to no discernable effects on production, indicating that, in C. ammoniagenes, the nonoxidative route alone cannot provide sufficient ribose 5-phosphate for high-level production, although nonoxidative synthesis of the precursor is possible. Electronic Publication     

Distributed control of the glycolytic flux in wild-type cells and catabolite repression mutants of Saccharomyces cerevisiae growing in carbon-limited chemostat cultures

The sensitivity of the control of glycolysis was studied in the wild-type (WT) strain CEN.PK122 and in isogenic catabolite-repression mutants growing in carbon-limited, aerobic chemostat cultures at different dilution rates, D. Based on a model of glycolysis in which the glucose transport step was considered reversible and inhibited by glucose 6-phosphate (G6P), the matrix method of metabolic control analysis was applied. In the present work, we report that the control of glycolysis was significantly distributed between the glucose uptake, hexokinase, and phosphofructokinase steps. The flux control properties were sensitive to the glucose gradient through the membrane and the extent of inhibition of the transport by G6P as parameters of the glucose-uptake kinetics in all strains tested. In the WT strain at low and high D, most of the control was exerted by the phosphofructokinase (PFK)-catalyzed step. In the cat1 mutant, the step catalyzed by PFK was the most rate controlling while in the cat3 strain, the control was shared between the PFK, hexokinase (HK), and glucose transport steps. On the other hand, the mig1 mutant exhibited high control by the glucose transporter depending on the glucose gradient across the membrane. The results obtained are discussed in terms of the dependence upon the type of metabolism displayed by yeast and the kinetics of the sugar transport step.     

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26 μM of BCB for 90 min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10 min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10 min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P < 0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P < 0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P < 0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P > 0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P < 0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.     

Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.     

酿酒酵母糖酵解途径中酶量变化对乙醇浓度影响的模拟分析

代谢组学是系统生物学的一个重要组成部分,应用相关方法获得了大量的数据。如何处理这些数据以及如何将这些数据与其他组学数据结合起来的问题不容忽视。在酶的反应动力学方程中引入“酶量倍数因子”能够解决其中的部分问题。如果反应动力学方程中酶的量发生变化,只需要改变相应的酶量倍数因子的数值。为了观察酿酒酵母糖酵解途径中酶量变化对乙醇浓度的影响,设定了高低两个酶量水平进行计算机模拟,对应的酶量倍数因子分别为10和0.1。基于计算机模拟结果,使用聚类分析方法,12种酶被分为两类。属于第一大类的四种酶ADH、HK、PFK和PDC,均催化不可逆反应。第二大类8种酶中的6种,ALD、GAPDH、GlcTrans、lpPEP、PGI和TIM均催化可逆反应。第二大类中另外两种酶lpGlyc和PK催化不可逆反应。按照这种方法,代谢组和蛋白质组数据能较容易地结合起来对系统作出较全面的分析。     

Acceleration of the rate of fermentation by Saccharomyces cerevisiae in the presence of ammonium ion

The rate of fermentation of both d-glucose and maltose in a defined medium by a brewing strain of Saccharomyces cerevisiae was found to be dependent on the availability of NH₄⁺. The glycolytic rate did not correlate with intracellular NH₄⁺and activation by NH₄⁺was blocked by cycloheximide. The ability of several amino acids to activate glycolysis followed the same order as their effectiveness as sole sources of nitrogen. It therefore seems that NH₄⁺does not stimulate fermentation through direct activation of glycolytic enzymes, but through its function as a substrate for protein synthesis.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates

Abstract Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases. The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains. However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol₂₄P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors. It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants. Moreover, a block in the ergosterol branch of the metabolic pathway ( erg 9 ) causes an increase in the de novo synthesis of dolichyl phosphate.