酵母海藻糖酶缺失突变株的构建及其耐性

[目的]构建酵母海藻糖酶缺失突变株,并进行耐性分析,进一步研究海藻糖与酵母耐性之间的关系,为商业生产打下一定的基础.[方法]利用同源重组的方法,敲除了编码酸性海藻糖酶的ATH1基因和中性海藻糖酶的NTH1基因,构建了酸性海藻糖酶缺失突变株(△ath1)、中性海藻糖酶缺失突变株(△nth1)和双缺失突变株(△ath1△nth1),并进行了耐性分析.[结果]结合PCR和Southernblot的结果,验证了突变株构建的正确.所有突变株的海藻糖积累量和细胞密度均高于亲本,冷冻、高温、高糖和酒精耐性提高了.[结论]说明海藻糖含量与酵母耐性有一定的相关性.突变株耐性的改善,表明它们在酿造和烘焙产业中具有潜在的商业价值.     

不同渗透压调节剂对Candida krusei生理代谢的影响

比较了氯化钠、氯化钾、甘露醇存在的高渗环境下克鲁氏假丝酵母(Candida kru-sei)的生理代谢。3种渗透压调节剂对C.krusei生理代谢影响有显著差异。与甘露醇相比,氯化钠和氯化钾对细胞生长的影响更为显著,而氯化钾对细胞的毒性则又小于氯化钠。细胞对糖的消耗速率依次为甘露醇>氯化钾>氯化钠。甘油和海藻糖是C.krusei在高渗环境下的主要相容性溶质。氯化钠和氯化钾对甘油合成的促进作用明显高于甘露醇。在0.6mol/L氯化钠、氯化钾、甘露醇存在时,细胞甘油浓度较对照提高了74%、63%、57%;胞内甘油最大含量也分别达到对照的3.1,2.4和1.8倍。高渗环境下胞内海藻糖含量在发酵前期均有所降低,但发酵后期在0.6mol/L氯化钾和甘露醇存在时海藻糖迅速积累,其含量分别达对照的1.6和1.4倍。     

啤酒酵母AS2.1416海藻糖合成酶基因tps1的cDNA克隆及序列分析

海藻糖 (Ｔｒｅｈａｌｏｓｅ,α ｇｌｕｃｏｐｙｒａｎｏｓｙｌ α 1,1 Ｄ ｇｌｕｃｏｐｙｒａ ｎｏｓｅ)是一种非还原性二糖 ,广泛存在于藻类、细菌、昆虫、无脊椎动物及酵母等许多生物体内。海藻糖除了作为一种储存性碳源外 ,业已被证明在许多逆境 ,诸如高温、高盐、干旱、重金属离子污染、冷冻、辐射等情况下 ,可以有效地保护生物的细胞膜、蛋白质及核酸[1～ 6] 。海藻糖合成酶为一多酶体系。在酵母细胞中 ,其合成分为两步进行。第一步 ,在 6 磷酸海藻糖合成酶 (Ｔｐｓ1)的作用下 ,由ＵＤＰ 葡萄糖和葡萄糖 6 磷酸合成海藻糖 6 磷酸 …     

鲁氏接合酵母海藻糖耦合发酵过程的转录组差异分析

[目的]为探究鲁氏接合酵母(Zygosaccharomyces rouxii)在海藻糖耦合发酵过程中适应高温胁迫的基因表达差异及内在代谢调节机制。[方法]采用GC色谱仪和Illumina HiSeq^TM高通量测序技术对海藻糖耦合发酵过程(无调控阶段、补料响应阶段、补料与中温刺激耦合响应阶段、补料与高温刺激耦合响应阶段)的生物量、胞内海藻糖含量、基因转录序列等指标进行检测。[结果]转录组测序结果显示，与无调控阶段对比，三个调控响应阶段分别筛选得到1717、522、2038个差异表达基因。海藻糖耦合发酵过程中的鲁氏接合酵母细胞差异表达基因主要富集到次级代谢产物的生物合成、核糖体、氨基酸的生物合成等代谢途径上，高温胁迫条件下(海藻糖高速合成过程)分别涉及158、97、58个差异表达基因，次级代谢主要涉及赖氨酸等氨基酸和嘌呤/嘧啶核苷酸，氨基酸的生物合成主要涉及赖氨酸代谢、半胱氨酸和谷氨酸代谢。[结论]通过转录组分析揭示了鲁氏接合酵母主要通过调控次级代谢产物的生物合成与氨基酸代谢来适应高温热激胁迫。     

酶法合成海藻糖研究的新进展

海藻糖（bondooe）是由两个葡萄糖残基经a－l,l键接的非还原二糖,广泛存在于细菌、酵母、真菌、藻类、昆虫中］。海藻糖具有独特而令人感兴趣的生物学功能。例如可以保护蛋白质、生物膜及敏感细胞的细胞壁免受干旱、冷冻、渗透压的变化等造成的伤害,用作不稳定药品、食品和化妆品的稳定剂,多种食品和药品的甜味剂,种子的包衣,冷冻干燥菌株的保护剂l’］等,最近报道海藻糖可保护DNA防止放射线引起的损伤［’］。海藻糖来源目前主要从酵母中提取,但收率较低,成本高,限制了其应用。以往的研究表明,海藻糖在真菌和细菌中的合成主要…     

海藻糖代谢途径相关基因及生物工程

海藻糖(Trehalose)是一种由两个葡萄糖分子通过α,α-1,l糖苷键连接的非还原性双糖。最早的记录是在19世纪初期作为黑麦的麦角菌的一种成分而被描述,后来发现海藻糖广泛存在于微生物、动物和植物体内,特别是在那些能抗脱水作用的生物中起着重要作用。这些特殊生物具有在脱水条件下存活多年的性质,包括所谓的“复苏植物”(Selaginella lepidophylla)、某些咸水虾、线虫及面包酵母等。当它们体内99％的水分被去掉之后,仍保持着能在获水后迅速复活的能力^[1]。研究表明,海藻糖对于生物抗逆具有重要的保护作用。海藻糖的应用研究因此得到了人们的广泛关注和重视,目前海藻糖已被用作酶、其它蛋白、生物制品甚至移植器官的保护剂。海藻糖作为生物体对抗环境胁迫的重要应激保护物质,在不同生物中存在多种合成和分解代谢途径,相关基因已相继被克隆和分析。海藻糖合成、分解及其调控是生物抗逆的重要机制,其相关基因的研究也是海藻糖生物工程的重要基础。     

乙醇胁迫抑制毕赤酵母表达外源蛋白的转录组学分析

在5 L发酵罐中进行毕赤酵母发酵表达猪?干扰素的实验,发现甘油培养末期乙醇的积累会抑制外源蛋白的表达。从转录组学角度系统分析不同浓度乙醇胁迫条件下,毕赤酵母甘油培养期和甲醇诱导期细胞的生理状态变化。研究结果表明,在甘油培养期,乙醇胁迫使得毕赤酵母细胞中的545个基因发生了显著差异表达(265个基因表达上调,280个基因表达下调),这些差异表达基因的功能主要涉及蛋白质合成、能量代谢、细胞周期和过氧化物酶代谢。乙醇胁迫增加了蛋白质错误折叠的情况,降低了核糖体和线粒体的结构完整性,使得甘油培养末期无法得到大量具有健全功能的酵母细胞。在甲醇诱导期,与甲醇代谢、蛋白质加工合成、氨基酸代谢等途径相关的294个基因发生了显著差异表达(171个基因表达上调,123个基因表达下调),导致内质网胁迫不能被及时解除,破坏了细胞内的氨基酸正常代谢。     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

胰岛素蛋白质工程: [B16Ala]胰岛素和[B26Ala]胰岛素——两个新型的速效人胰岛素

用大动物(猪)降血糖实验证明[B16Ala]胰岛素和[B26Ala]胰岛素是快速降血糖胰岛素. 它们的前体[B16Ala]PIP和[B26Ala]PIP, 在甲醇酵母体系中的分泌表达量分别为650和130 mg/L. 由于它们是新型的分别保留全部和几乎全部胰岛素体内生物活力的速效胰岛素, 且在甲醇酵母表达体系中得到比较高的表达量, 所以具有很好的临床应用前景.     

利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.     

Osmoregulation in Saccharomyces cerevisiae. Studies on the osmotic induction of glycerol production and glycerol-3-phosphate dehydrogenase (NAD+)

Production of glycerol and a key enzyme in glycerol production, glycerol 3-phosphate dehydrogenase (NAD+) (GPD), was studied in Saccharomyces cerevisiae cultured in basal media or media of high salinity, with glucose, raffinose or ethanol as the sole carbon source. At high salinity, glycerol production was stimulated with all carbon sources and glycerol was accumulated to high intracellular concentration in cells grown on glucose and raffinose. Cells grown on ethanol accumulated glycerol to a lower level but showed an increased content of trehalose at high salinity. However, the trehalose concentration corresponded only to about 20% of the glycerol level, and did not compensate for the shortfall in intracellular osmolyte content. Immunoblot analysis demonstrated an increased production of GPD at high salinity. This increase was osmotically mediated but was lower when glycerol was substituted for NaCl or sorbitol as the stress-solute. The enzyme also appeared to be subject to glucose repression; the specific activity of GPD was significantly lower in cells grown on glucose, than on raffinose or ethanol.     

Solute stresses affect growth patterns, endogenous water potentials and accumulation of sugars and sugar alcohols in cells of the biocontrol yeast Candida sake

AIM: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake. METHODS AND RESULTS: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c). CONCLUSIONS AND SIGNIFICANCE: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Osmoregulation in the Halotolerant Alga Asteromonas gracilis

Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol.     

Improving water stress tolerance of the biocontrol yeast Candida sake grown in molasses-based media by physiological manipulation

The biocontrol agent Candida sake was cultured on either an unmodified molasses-based medium (water activity, a(w) 0.996) or on water stressed media produced by the addition of glycerol, glucose, NaCl, sorbitol, or proline to 0.98, and 0.96 a(w) for 24, 48, and 72 h, to study their impact on subsequent cell viability, and on concentrations of endogenous sugars (trehalose and glucose) and polyols (glycerol, erythritol, arabitol, and mannitol). The viability of cells of different ages cultured on these media was evaluated on NYDA medium with freely available water (a(w) 0.995), and on medium modified with polyethylene glycol to a(w) 0.95. Regardless of solute used, viable counts of cells grown on molasses-based medium (a(w) 0.98) were equal to or higher than those obtained from the medium with water freely available. The amino acid proline stimulated growth at 10% concentration. In contrast, water stress induced by addition of NaCl, glucose, or sorbitol at a(w) 0.96 caused a significant reduction in viable counts. Older cultures were more resistant to water stress. Glycerol and arabitol were the main solutes accumulated by C. sake cells in response to lowered a(w). Intracellular concentration of these polyols depended more on the solute used to adjust the a(w) than on the a(w) itself. Candida sake was more resistant to water stress with higher intracellular concentration of glycerol and erythritol.     

ADAPTATION OF THE UNICELLULAR ALGA DUNALIELLA PARVA TO A SALINE ENVIRONMENT1

The holophilic alga Dunaliella parva produces glycerol as a major product of photosynthetic ¹⁴CO₂ incorporation and accumulates very large amounts of intracellular glycerol. A method was adopted for the determination of the cell water space based on the distribution of ¹⁴C sorbitol and ³H₂O between the cells and the medium. Using these measurements the internal concentration of glycerol was found to be isoomotic with that of the medium over a broad range of 0.6 to 2.1 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular water content immediately varied so as to keep an osmotic equilibrium between the cells and the medium. During the following 90 min under metabolic conditions, glycerol content changed until a new level was reached. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. Determination of the glycerol sensitivity of enzymic and photosynthetic reactions of cell-free preparations from D. parva showed a broad range of tolerance to high concentrations of glycerol. These results indicate that osmoregulation in Dunaliella depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. A proposed scheme of glycerol synthesis in Dunaliella is suggested.     

The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva

Dunaliella parva, a green halophilic alga, was found to accumulate very large amounts of intracellular glycerol. Through measurements of the intracellular volume the internal concentration of glycerol was calculated and found to be around 2.1 m in cells cultured in 1.5 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular glycerol varied accordingly, reaching its new osmotic equilibrium after about 90 minutes. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. The above results indicate the existence of a new type of algal osmoregulation, in which the osmotic balance depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration.