利用定点突变改变树干毕赤酵母(Pichia stipitis)木糖醇脱氢酶辅酶特性的研究

在酿酒酵母中同时表达木糖还原酶基因(xyl1)和木糖醇脱氢酶基因(xyl2)可使酿酒酵母利用木糖发酵生成乙醇.但由于两种酶所依赖的辅酶不同导致酿酒酵母细胞内氧化还原失衡,致使中间产物大量积累,降低了乙醇产率.本研究从树干毕赤酵母中克隆了木糖醇脱氢酶基因,通过与银叶粉虱山梨醇脱氢酶[其活性依赖NADP+(H)]序列进行对...     

Oxygen-dependent xylitol metabolism in Pichia stipitis

Pichia stipitis CBS 6054 was cultivated in chemostat cultures under aerobic and oxygen-limited conditions with xylitol alone, a mixture of xylitol and glucose and a mixture of xylitol and xylose. Xylitol metabolism was strictly respiratory and no ethanol was formed. Simultaneous feeding of xylitol and glucose and xylitol and xylose to oxygen-limited xylitol-pregrown cells resulted in ethanol formation. In vitro both pyruvate decarboxylase activity and alcohol dehydrogenase activity were present in cells metabolising xylitol under oxygen-limited conditions; however, this did not result in ethanol formation. Glucose, xylose and xylitol utilisation, respectively, were compared under anaerobic conditions with regard to growth rate, carbon source and oxygenation level during pre-cultivation. Irrespective of pre-growth conditions, xylitol was not metabolised under anaerobic conditions, whereas ethanol was formed from both xylose and glucose. Anaerobic xylose utilisation required induction of a xylose-utilising metabolic pathway during pre-cultivation. Received: 23 February 1999 / Received last revision: 20 July 1999 / Accepted: 1 August 1999     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Influence of magnesium concentration on growth,ethanol and xylitol production by Pichia stipitis NRRL Y-7124

Summary The effect of Mg⁺² on Pichia stipitis growth and ethanol production was studied under condition of constant oxygen uptake rate (OUR) . Biomass/xylose and biomass/Mg⁺² yields increased with Mg⁺² concentration with a maximum value at Mg⁺² 4mM, ethanol being the main product obtained. At low Mg⁺² levels (ImM) 49 % of carbon flux to ethanol was redirected to xylitol production, accomplished through NADH intracellular accumulation.     

Heterologous expression of D-xylulokinase from Pichia stipitis enables high levels of xylitol production by engineered Escherichia coli growing on xylose

Deletion of the Escherichia coli xylulokinase gene (xylB) is essential for achieving high xylitol titers from xylitol-producing E. coli strains growing on glucose in the presence of xylose. Our study suggests that this is due to XylB-catalyzed toxic synthesis of xylitol-phosphate. This activity prohibits the use of xylose as the sole carbon source during xylitol production by E. coli. To overcome this limitation we turned to the yeast Pichia stipitis, which naturally produces xylitol, as a source of xylulokinase (Xyl3). We examined the effects of plasmid-based expression of Xyl3 versus XylB on growth and xylitol production by engineered E. coli strains. Xylulokinase activity assays show similar levels of functional expression of both enzymes (determined as activity on xylulose), and reveal significantly more activity on xylitol by XylB compared to Xyl3. (31)P NMR confirms the production of xylitol-phosphate from in vitro reactions with XylB. Lastly, the replacement of xylB with XYL3 results in drastically enhanced xylitol titers from E. coli strains co-expressing xylose reductase during growth on xylose.     

Structural and mechanistic insight into stem‐loop RNA processing by yeast Pichia stipitis Dicer

Dicer is a member of the ribonuclease III enzyme family and processes double‐stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non‐canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double‐stranded RNA‐binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA‐binding surface. The second dsRNA binding domain at C‐terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem‐loop structure of the RNA substrate, suggesting the possibility that stem‐loop RNA‐guided gene silencing pathway exists in budding yeast.     

木糖发酵生产乙醇的研究

选育出一株优良的木糖发酵菌株树干毕赤酵母菌7124,并利用纯木糖优化了木糖发酵条件,利用海藻酸钠固定化树干毕赤酵母菌增殖细胞,不仅能较好满足限氧发酵条件,而且能耐较高糖浓度,使乙醇发酵浓度提高到20g/L。利用半纤维素水解液进行了乙醇发酵的初步研究,基本达到了纯木糖发酵的效果。     

Xylitol production by a Pichia stipitis D-xylulokinase mutant

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g xylitol/g xylose). However, xylose consumption increased five-fold under aerobic compared to oxygen-limited conditions. This suggests that the efficiency of the alternative route of xylose assimilation is affected by respiration. As a result, the FPL-YS30 strain produced 26 g/l of xylitol, and exhibited a higher volumetric productivity (0.22 g xylitol l^–1 h^–1) under aerobic conditions.     

树干毕赤酵母NLP31发酵产乙醇的研究

研究了树干毕赤酵母NLP31在木糖质量浓度为45 g/L的3种发酵培养基Ⅰ、Ⅱ和Ⅲ上发酵3轮的发酵性能以及在45 g/L木糖或混合糖(葡萄糖30 g/L,木糖15 g/L)的发酵培养基Ⅲ上的代谢历程。结果表明:树干毕赤酵母NLP31在发酵培养基Ⅲ上,乙醇浓度和乙醇得率均达到最高,分别为(17.29±0.15)g/L和(84.65±0.58)%。在45 g/L木糖或混合糖(葡萄糖30 g/L,木糖15 g/L)的发酵培养基Ⅲ上的代谢历程表明:混合糖发酵达到最大乙醇得率的时间仅为12 h,要比单一木糖发酵缩短了8 h。树干毕赤酵母NLP31在以廉价的无机N源为发酵培养基上的乙醇发酵性能高,能够降低燃料乙醇的生产成本。     

Effect of Oxygenation on Xylose Fermentation by Pichia stipitis

The effect of oxygen limitation on xylose fermentation by Pichia stipitis (CBS 6054) was investigated in continuous culture. The maximum specific ethanol productivity (0.20 g of ethanol g dry weight⁻¹ h⁻¹) and ethanol yield (0.48 g/g) was reached at an oxygen transfer rate below 1 mmol/liter per h. In the studied range of oxygenation, the xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were constant as well as the ratio between the NADPH and NADH activities of xylose reductase. No xylitol production was found. The pyruvate decarboxylase (EC 4.1.1.1) activity increased and the malate dehydrogenase (EC 1.1.1.37) activity decreased with decreasing oxygenation. With decreasing oxygenation, the intracellular intermediary metabolites sedoheptulose 7-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, and malate accumulated slightly while pyruvate decreased. The ratio of the xylose uptake rate under aerobic conditions, in contrast to that under anaerobic assay conditions, increased with increasing oxygenation in the culture. The results are discussed in relation to the energy level in the cell, the redox balance, and the mitochondrial function.     

Xylose metabolism by Pichia stipitis: the effect of ethanol

Summary Pichia stipitis Y7124 was grown anaerobically on d-xylose in the presence of an initial ethanol concentration (E₀) varying from 0 to 40 g/l. When E₀ increased, the yield of xylitol increased linearly, reaching a value of 0.20 mol xylitol/mol xylose at E₀=40 g/l. When a hydrogen acceptor (acetoin) was added to the cultures, the cylitol yield decreased with the contaminant stoichiometric reduction of acetoin to 2,3-butanediol. Furthermore, it was demonstrated that xylitol dehydrogenase and acetoin reductase activities from cell-free extracts of P. stipitis Y7124 were NAD⁺ and NADH₂-linked, respectively. A hypothesis is put forward explaining that the xylitol yield is dependent on the ethanol concentration. It is suggested that ethanol may cause a disturbed NAD⁺/NADH₂ balance during anaerobic xylose metabolism by P. stipitis. Metabolic mechanisms are proposed and their validity is discussed. Offprint requests to: J. P. Delgenes     

Xylan-hydrolysing enzymes of the yeast Pichia stipitis

Summary Two xylanolytic enzymes, xylanase and -xylosidase from the yeast Pichia stipitis were purified to homogeneity and characterized. Both enzymes are secreted into the culture medium upon growth on xylan. The xylanase is a glycoprotein with an approximate molecular mass of 43 kDa. The N-linked carbohydrate content was estimated to be 26% by endoglycosidase H digestion. The -xylosidase protein has a molecular mass of 37 kDa as determined by sodium dodecyl sulphate gel electrophoresis. Synthesis of xylanase was found to be inducible by xylan and repressible by xylose and glucose. By contrast, -xylosidase is synthesized constitutively to a considerable degree. The purified -xylosidase is able to hydrolyse aryl--D-glucosides with an even higher rate than -xylosides. Thus, this enzyme may not be a specific component of the xylan-degrading system of P. stipitis. Offprint requests to: M. Ciriary     

Efficient production of L-lactic acid from xylose by Pichia stipitis

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to approximately 15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis

Ethanol production was evaluated from wheat straw (WS) hemicellulose acid hydrolysate using an adapted and parent strain of Pichia stipitis. NRRL Y-7124. The treatment by boiling and overliming with Ca(OH)(2) significantly improved the fermentability of the hydrolysate. Ethanol yield (Yp/s) and productivity (Qp av) were increased 2.4+/-0.10 and 5.7+/-0.24 folds, respectively, compared to neutralized hydrolysate. Adaptation of the yeast to the hydrolysate resulted further improvement in yield and productivity. The maximum yield was 0.41+/-0.01 g(p) g(s)(-1), equivalent to 80.4+/-0.55% theoretical conversion efficiency. Acetic acid, furfurals and lignins present in the hydrolysate were inhibitory to microbial growth and ethanol production. The addition of these inhibitory components individually or in various combinations at a concentrations similar to that found in hydrolysate to simulated medium resulted a reduction in ethanol yield (Yp/s) and productivity (Qp av). The hydrolysate used had the following composition (expressed in g x l(-1)): xylose 12.8+/-0.25; glucose 1.7+/-0.3; arabinose 2.6+/-0.21 and acetic acid 2.7+/-0.33.     

Ethanol production from eucalyptus wood hemicellulose hydrolysate by Pichia stipitis

Ethanol production was evaluated from eucalyptus wood hemicellulose acid hydrolysate using Pichia stipitis NRRL Y-7124. An initial lag phase characterized by flocculation and viability loss of the yeast inoculated was observed. Subsequently, cell regrowth occurred with sequential consumption of sugars and production of ethanol. Polyol formation was detected. Acetic acid present in the hydrolysate was an important inhibitor of the fermentation, reducing the rate and the yield. Its toxic effect was due essentially to its undissociated form. The fermentation was more effective at an oxygen transfer rate between 1.2 and 2.4 mmol/L h and an initial pH of 6.5. The hydrolysate used in the experiences had the following composition (expressed in grams per liter): xylose 30, arabinose 2.8, glucose 1.5, galactose 3.7, mannose 1.0, cellobiose 0.5, acetic acid 10, glucuronic acid 1.5, and galacturonic acid 1.0. The best values obtained were maximum ethanol concentration 12.6 g/L, fermentation time 75 h, fermentable sugar consumption 99% ethanol yield 0.35 g/g sugars consumed, and volumetric ethanol productivity 4 g/L day. (c) 1992 John Wiley & Sons, Inc.     

Fermentation of sunflower seed hull hydrolysate to ethanol by Pichia stipitis

Ethanol production from sunflower seed hull hydrolysate was evaluated using Pichia stipitis NRRL Y-7124. The hydrolysate prepared with 0.7 M H2SO4 at 90 degrees C was fermented as substrate in shaking bath experiments at 30 degrees C. In a group of experiments, the influence of various detoxification methods on the fermentability of hydrolysate was investigated at pH 6. Even though the ability of all employed pretreatments to enhance fermentation performance was close, the sequential application of overliming with sodium sulfite addition was the best detoxification method. Additional experiments were performed with detoxified hydrolysate to investigate the effect of shaking rate (70-130 rpm) and initial pH (5.5-7) on the fermentation. The highest ethanol level 11 gL(-1) was achieved at initial pH of 6 and 100 rpm shaking rate from a hydrolysate containing 48 gL(-1) total reducing sugar. The corresponding alcohol yield and volumetric productivity were 0.32 gg(-1) and 0.065 gL(-1)h(-1).     

树干毕赤酵母酒精发酵碳元素代谢流向分析

实验对树干毕赤酵母（Pichia stipitis）进行了4个阶段共400 h连续恒化培养,在不同阶段以30.0 g/L葡萄糖作为基本碳源,添加30.0或15.0 g/L的木糖,通过控制温度（35±1）℃,进气量100～150mL/min,搅拌转速250～300 r/min。4个阶段共建立4个连续培养的"稳态"。对碳元素进行物料衡算发现,四个阶段碳元素回收率分别为118.0 %、105.6 %、113.5 %和94.7 %。对4个近似"稳态"的碳元素的代谢流向进行分析发现：将近50.0 %左右碳元素流向产物酒精,其次是CO2和酵母细胞;木糖醇浓度与流入底物中木糖浓度有直接关系,在相同发酵条件下流入的木糖浓度越大代谢生成木糖醇浓度也越高;实验所采用的通气条件更适合底物为30.0 g/L葡萄糖和30.0 g/L木糖混合液的连续发酵。     

Characterization of Xylose Uptake in the Yeasts Pichia heedii and Pichia stipitis

The kinetics of xylose uptake were investigated in the efficient xylose fermenter Pichia stipitis and in the more readily genetically manipulated, strictly respiratory yeast Pichia heedii. Both yeasts demonstrated more than one xylose uptake system, differing in substrate affinity. The K_m of high-affinity xylose uptake in both organisms was similar to that of the efficient high-affinity glucose uptake system of Saccharomyces cerevisiae. In P. heedii, low-affinity xylose uptake was enhanced with growth on 2% but not 0.05% xylose and high-affinity uptake was reduced. In contrast to glucose uptake, xylose uptake in P. heedii was inhibited by dinitrophenol. Dinitrophenol inhibited both glucose and xylose uptake by P. stipitis. Glucose uptake was not inhibited by a 100-fold molar excess of xylose in P. heedii. It is suggested that xylose uptake in P. heedii is via a carrier system(s) distinct from those for glucose uptake.