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。     

Stress-related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae

Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.     

The role of xylulokinase in Saccharomyces cerevisiae xylulose catabolism

Many yeast species have growth rates on D-xylulose of 25-130% of those on glucose, but for Saccharomyces cerevisiae this ratio is only about 6%. The xylulokinase reaction has been proposed to be the rate-limiting step in the D-xylulose fermentation with S. cerevisiae. Over-expression of xylulokinase encoding XKS1 stimulated growth on D-xylulose in a S. cerevisiae strain to about 20% of the growth rate on glucose and deletion of the gene prevented growth on D-xylulose and D-xylulose metabolism. We have partially purified the xylulokinase and characterised its kinetic properties. It is reversible and will also accept D-ribulose as a substrate.     

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.     

Genetic improvement of Saccharomyces cerevisiae for xylose fermentation

There is considerable interest in recent years in the bioconversion of forestry and agricultural residues into ethanol and value-added chemicals. High ethanol yields from lignocellulosic residues are dependent on efficient use of all the available sugars including glucose and xylose. The well-known fermentative yeast Saccharomyces cerevisiae is the preferred microorganism for ethanol production, but unfortunately, this yeast is unable to ferment xylose. Over the last 15 years, this yeast has been the subject of various research efforts aimed at improving its ability to utilize xylose and ferment it to ethanol. This review examines the research on S. cerevisiae strains that have been genetically modified or adapted to ferment xylose to ethanol. The current state of these efforts and areas where further research is required are identified and discussed.     

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.     

The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).     

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     

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.     

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.     

Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4-19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwfl Delta) and TMB3261 (PGK1-XYL1, zwf1 Delta), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.     

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.     

不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。     

Disorders in NADPH generation via pentose phosphate pathway influence the reproductive potential of the Saccharomyces cerevisiae yeast due to changes in redox status

Intermediary metabolites have a crucial impact on basic cell functions. There is a relationship between cellular metabolism and redox balance. To maintain redox homoeostasis, the cooperation of both glutathione and nicotine adenine dinucleotides is necessary. Availability of nicotinamide adenine dinucleotide phosphate (NADPH) as a major electron donor is critical for many intracellular redox reactions. The activity of glucose-6-phosphate dehydrogenase (Zwf1p) and 6-phosphogluconate dehydrogenase (Gnd1p and Gnd2p) is responsible for NADPH formation in a pentose phosphate (PP) pathway. In this study, we examine the impact of redox homoeostasis on cellular physiology and proliferation. We have noted that the Δzwf1 mutant lacking the rate-limiting enzyme of the PP pathway shows changes in the cellular redox status caused by disorders in NADPH generation. This leads to a decrease in reproductive potential but without affecting the total lifespan of the cell. The results presented in this paper show that nicotine adenine dinucleotides play a central role in cellular physiology.     

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization.We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C(J) (transp), for the xylose transport was calculated from the response and elasticity coefficients. The value of C(J) (transp) for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L(-1). However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L(-1), while C(J) (transp) stayed below 0.2 for strain TMB3001 irrespective of xylose concentration.     

Production of ethanol from L-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway

The fungal pathway for L-arabinose catabolism converts L-arabinose to D-xylulose 5-phosphate in five steps. The intermediates are, in this order: L-arabinitol, L-xylulose, xylitol and D-xylulose. Only some of the genes for the corresponding enzymes were known. We have recently identified the two missing genes for L-arabinitol 4-dehydrogenase and L-xylulose reductase and shown that overexpression of all the genes of the pathway in Saccharomyces cerevisiae enables growth on L-arabinose. Under anaerobic conditions ethanol is produced from L-arabinose, but at a very low rate. The reasons for the low rate of L-arabinose fermentation are discussed.     

Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation

Xylose isomerase (XI) is a key enzyme in the conversion of d ‐xylose, which is a major component of lignocellulosic biomass, to d ‐xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI‐related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell‐surface display system, and the xylose assimilation and fermentation properties of this XI‐displaying yeast were examined. XI‐displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013