酵母菌磷脂酰肌醇（PI）的生物合成及其重要的生理学功能

本文对近年来在酵母菌磷脂酰肌醇（PI）生物合成,PI与酵母菌信号传导的相互关系,PI在酵母菌耐浓度酒精中的作用和PI在酵母菌胞外酶分泌解阻遏中的作用等方面最新研究进展进行了较为全面的讨论。     

酵母菌在疏水性有机污染物去除方面的应用

介绍了酵母菌处理技术在疏水性有机污染物降解中的研究和应用进展,并分析了酵母菌表面特征与其降解活性及在废水处理系统中的稳定性之间的关系。酵母菌技术可以在高含油色拉油废水及含高分于量多环芳烃（PAH）废水的处理中发挥细菌无法替代的作用,酵母菌重要表面特征,特别是其疏水性和乳化能力对于其在疏水性有机污染物处理体系中的稳定性具有重要影响。     

酵母菌在红葡萄酒酒精发酵串罐中稳定性研究

在生产条件下,对酵母菌在红葡萄酒酒精发酵串罐过程中的稳定性进行了研究。结果表明,在近1个月的时间内（相当于酵母菌细胞无性繁殖了200代）,串罐过程中的酵母菌细胞不仅能保持初始酵母菌的发酵活性和优良特性的稳定性,而且由于葡萄汁的选择作用,串罐用的酵母菌细胞的发酵活性比初始酵母菌的活性更强,因而其酒精发酵的启动和速度都更快。     

蜡螟抗白假丝酵母菌自噬机制的初步研究

目的探究白假丝酵母菌感染蜡螟时蜡螟的自噬相关通路蛋白的表达情况。方法用一定量的白假丝酵母菌的活化孢子感染蜡螟,经过12h,解剖蜡螟收集蜡螟细胞并裂解细胞,用真菌活性检测试剂盒检测孢子活性;解剖蜡螟取肠道组织并用PI染死细胞。取不同感染时间段的淋巴细胞,用裂解液裂解,离心取上清,用Western blot法检测上清液中的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平。结果活化的孢子注射至蜡螟体内后,其活性受到抑制;蜡螟的肠道细胞被定位在上面的菌丝损伤并且孢子活性受到抑制。随着蜡螟感染白假丝酵母菌时间的递增,其自身的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平在不断增高且在感染24h最高。结论白假丝酵母菌感染蜡螟后,蜡螟的淋巴细胞和肠道细胞通过升高Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平发挥杀伤孢子的作用。     

拟南芥AtPI基因植物表达载体的构建及其在烟草中的遗传转化

花是被子植物主要的繁殖器官,在繁育后代的过程中,发挥着极其重要的作用,PISTILLATA(PI)基因作为控制花器官发育的B类功能基因中的一员,在花器官发育中起到重要的作用。为探究PI基因在花瓣和雄蕊发育中的功能,本文以拟南芥(Arabidopsis thaliana)中的PI基因作为研究对象,利用PCR技术从拟南芥花序c DNA扩增出At PI基因,构建植物表达载体(p ROKⅡ-At PI)并进行烟草(Nicotiana tobacum)转化。转基因植株的PCR检测结果表明,At PI基因已经整合到了烟草基因组中。在T2代植株中,通过实时定量荧光PCR检测显示,At PI在m RNA水平也均有表达。过量表达PI的转基因烟草在花器官中存在明显表型,与野生型相比主要表现为转基因植株花冠变小,雄蕊缩短,果实畸形且子房基部比野生型长5~10 mm,上述结果表明At PI基因是特异性参与雄蕊和花瓣的发育并起着至关重要的作用。     

一种增加毕赤酵母生产胰岛素前体的方法

为了提高胰岛素前体(PI)的产量,构建了p PIC9K-PI表达载体并电转化至毕赤酵母菌株GS115中,在浓度为4.0 mg/m L的G418抗性平板上筛选到了1株拷贝数为12的菌株CL012。将SNAREs(可溶性N-乙基马来酰亚胺敏感因子受体蛋白)组分中的SNC2和SNC2-SSO2分别转入菌株CL012中,并在摇瓶和5 L发酵罐水平上检测SNAREs对PI产量的影响。结果表明:摇瓶水平上,甲醇诱导96 h后,菌株CL012的PI产量为1.53 mg/L;表达SNC2和SNC2-SSO2的菌株的PI产量分别为1.89 mg/L和2.21 mg/L,分别比菌株CL012提高了23.53%和44.44%。在5 L发酵罐上进行高密度发酵,甲醇诱导96 h后菌株CL012的PI产量为53 mg/L,是摇瓶水平的34.64倍;表达SNC2和SNC2-SSO2的菌株的PI产量分别达到64 mg/L和78 mg/L,分别比菌株CL012提高了20.75%和47.17%。由此得出结论 SNAREs可以促进胰岛素前体的分泌,从而提高在毕赤酵母中的异源表达。     

纳豆杆菌对白假丝酵母菌的拮抗作用

目的探讨纳豆杆菌对白假丝酵母菌的拮抗作用。方法将纳豆杆菌和白假丝酵母菌混合培养24 h后,应用沙保弱平板培养基分离白假丝酵母菌,计数菌落,计算纳豆杆菌对白假丝酵母菌的拮抗率。结果纳豆杆菌对白假丝酵母菌的拮抗作用明显,拮抗率高达91.91%;纳豆杆菌肉汤培养物的除菌滤液对白假丝酵母菌也有明显的拮抗作用,拮抗率为79.05%。结论纳豆杆菌对白假丝酵母菌具有明显拮抗作用,是白假丝酵母菌的理想拮抗菌株。     

磷脂酰肌醇3激酶通过Akt和ERK抑制盘基网柄菌细胞的趋电性

目的 磷脂酰肌醇3激酶（PI3Ks）通过调控肌动蛋白在细胞定向运动中发挥重要作用。然而,PI3Ks的结构和功能很复杂,人们对PI3Ks在细胞趋电性运动中的作用并不完全清楚。因此,本文以模式生物盘基网柄菌细胞为实验材料,探究其中的PI3K1和PI3K2在细胞趋电性运动中的作用。方法 首先利用CRISPR/Cas9系统介导分别构建PI3K1编码基因pikA基因敲除突变株和PI3K2编码基因pikB基因敲除突变株;随后将2个突变株置于强度为12 V/cm的直流电场中,记录并分析两个突变株的趋电性。结果 数据分析显示,野生型细胞在直流电场中的方向指数为（0.86±0.03）,而pikA-和pikB-突变株在直流电场中的运动方向指数分别为（0.95±0.02）和（0.94±0.03）;此外,野生型细胞在电场中的平均轨迹速度（3.34±0.08）μm/min,而pikA-和pikB-突变株的平均轨迹速度分别为（4.85±0.20）μm/min和（5.48±0.15）μm/min,t检验表明突变株和野生型的方向性指数和运动速度都存在极显著的差异。蛋白质印迹实验结果显示,pikA-和pikB-突变株中...     

黄连解毒汤乙酸乙酯提取物对白假丝酵母菌黏附作用的影响

目的探讨黄连解毒汤乙酸乙酯提取物（ethyl acetateextract of Huanglianjiedu decoction,EAHD）对白假丝酵母菌黏附作用的影响。方法XTT法检测白假丝酵母菌黏附力;平板法计数导管上黏附的白假丝酵母菌CFU;倒置显微镜观察黏附的白假丝酵母菌;qRT—PCR法定量检测黏附相关基因EAP1、HWP1、ALS1、ALS3、MP65的表达。结果312μg/mL EDHA可显著抑制白假丝酵母菌在聚苯乙烯和聚酯导管上的黏附;白假丝酵母菌生长早期在EDHA作用下,菌细胞出芽数均呈现剂量依赖性降低;EDHA作用后,EAP1、HWP1均下调,ALS3、MP65均上调,ALS1几乎未变化。结论EAHD可显著抑制白假丝酵母菌的黏附作用。     

应用流式细胞术对白假丝酵母菌耐药机制的研究

目的 探讨流式细胞术研究白假丝酵母菌耐药机制的可行性.方法 临床分离对氟康唑敏感的白假丝酵母菌种,经体外诱导产生耐药.应用荧光染料PI(碘化丙啶)和罗丹明123染色,通过流式细胞术检测敏感株、耐药株、回复敏感株的细胞周期和药物外排活性.结果 与敏感株和回复敏感株比较,耐药株增殖活性明显下降,但是药物外排活性显著增强.结论 应用氟康唑能够体外诱导白假丝酵母菌产生耐药.流式细胞术应用于白假丝酵母菌增殖活性和药物外排活性研究是可行的.     

Inositol and phosphatidylinositol mediated mediated glucose derepression, gene expression and invertase secretion in yeasts

Glucose Repression [1,2] Saccharomyces cerevisiae and other yeasts can growwell on different kinds of carbon sources. However,glucose and fructose are the best carbon sources for theirgrowth. When the medium contains glucose or fructose,the biosynthesis of enzyme catalyzing degradation of othercarbon sources will be greatly reduced or stopped. Thisphenomenon is called glucose repression. Although much progress has been made in this field,the exact mechanisms of glucose repression in yeastsa…     

Characterization of polar lipids of oral isolates of Candida, Pichia and Saccharomyces by Fast Atom Bombardment Mass Spectrometry (FAB MS)

AIMS: To characterize fatty acid and phospholipid analogue profiles of oral yeasts. METHODS AND RESULTS: Twenty-seven strains of oral yeasts were cultured on SDA and lipids of freeze-dried cells were extracted and analysed by FAB MS. The most abundant carboxylate anion was m/z 281 (C18 : 1). The most intense phospholipid analogue ions were of PE, PG, PA and PI. Pichia etchellsii contained molecular species of PG and PE, whereas Saccharomyces cerevisiae had PA, PG and PE analogues. Mass spectra revealed that S. cerevisiae and Candida glabrata were distinct from one another and from the other species tested. CONCLUSION: Oral yeasts largely differ with respect to their polar lipids. It is concluded that oral yeast species have distinctive fatty acid and phospholipid analogue anion profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: FAB MS provided novel chemotaxonomic information.     

Roles of Pichia pastoris Uvrag in vacuolar protein sorting and the phosphatidylinositol 3-kinase complex in phagophore elongation in autophagy pathways

Although it has been established that Atg6/Beclin 1, the phosphatidylinositol 3-kinase (PI3K) Vps34, and associated proteins have direct or indirect roles in autophagic pathways in both mammals and yeasts, the elucidation of these roles and the proteins required for them is ongoing. The involvement of the Beclin 1-binding protein, UVRAG, has been a particular source of disagreement. We found that PpAtg6 is required for all autophagic pathways that have been identified in the yeast Pichia pastoris, as well as for the carboxypeptidase Y (PpCPY) vacuolar protein sorting pathway. We localized PpAtg6 to the phagophore assembly site (PAS) and observed its continued presence at that site as the isolation membrane grew from it and matured into a pexophagosome. PpUvrag, however, was required for proper PpCPY sorting, but not for any autophagic pathway. Rather, the defects in all autophagic pathways observed when PpUvrag was overexpressed support its presence in a complex that competes with the PI3K complex required for autophagy.     

Digestion of Histoplasma capsulatum yeasts by human macrophages.

The strategies used by Histoplasma capsulatum yeasts to survive and multiply within human macrophages (M phi) are unknown. To better understand these strategies we studied the intracellular fate of viable vs heat-killed (HK) yeasts in human monocyte-derived M phi. Initial studies demonstrated that phagolysosome fusion was present in M phi ingesting either viable or HK yeasts. Viable yeasts multiplied within M phi phagolysosomes, whereas M phi completely digested intracellular FITC-labeled HK yeasts within 24 h after ingestion. This observation was confirmed by electron microscopy. M phi that had ingested colloidal gold-labeled HK yeasts contained gold particles but no visible yeasts at 24 h. Digestion of HK yeasts was evident as early as 4 h after phagocytosis, and was complete by 24 h. M phi digestion of HK yeasts was blocked completely when M phi were cultured for 24 h in the presence of chloroquine. In M phi simultaneously ingesting both viable and HK yeasts, viable yeasts multiplied, but HK yeasts were digested within the same cell. M phi that had ingested viable yeasts digested them completely when M phi were cultured for 24 h in the presence of cycloheximide or amphotericin B. Coculture of infected M phi with nystatin or ketoconazole resulted in inhibition of growth, but the yeasts were not digested. These data indicate that: 1), HK Hc yeasts are easily digested by preformed M phi lysosomal hydrolases; 2), viable Hc yeasts survive and multiply within M phi phagolysosomes, but the yeasts do not secrete a factor(s) that affects the ability of other phagolysosomes within the same M phi to digest killed yeasts; and 3), inhibition of yeast protein synthesis or cell wall biosynthesis is sufficient to render viable yeasts susceptible to digestion by human M phi.     

工业酵母菌的遗传修饰研究进展及其应用前景

简要概述工业酵母菌的遗传修饰研究进展,主要介绍适用于工业酵母菌遗传修饰的转化系统;敲除工业酵母基因工程菌细胞内不需要的基因的反选择技术;外源基因在工业酵母菌中克隆和表达的非自身克隆技术;工业酵母菌自身已有基因的克隆和表达的自身克隆技术等。此外,对遗传修饰的工业酵母菌的工业化应用前景作了简要展望 。     

Construction of sterile ime1Delta-transgenic Saccharomyces cerevisiae wine yeasts unable to disseminate in nature

The use of new transgenic yeasts in industry carries a potential environmental risk because their dispersal, introducing new artificial genetic combinations into nature, could have unpredictable consequences. This risk could be avoided by using sterile transgenic yeasts that are unable to sporulate and mate with wild yeasts. These sterile yeasts would not survive the annual cyclic harvesting periods, being condemned to disappear in the wineries and vineyards in less than a year. We have constructed new ime1Delta wine yeasts that are unable to sporulate and mate, bear easy-to-detect genetic markers, and quickly disappear in grape must fermentation immediately after sporulation of the yeast population. These sterile yeasts maintained the same biotechnological properties as their parent yeasts without any detectable deleterious effect of the ime1Delta mutation. These yeasts are therefore interesting biotechnologically for food industry applications and for genetically modified microorganism environmental monitoring studies.     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Cyanide-resistant respiration is frequent, but confined to yeasts incapable of aerobic fermentation

In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h. Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts. Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts. We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts.     

Intraspecies diversity of the industrial yeast strains Saccharomyces cerevisiae and Saccharomyces pastorianus based on analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 region of 26S rDNA

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.     

Multiple mechanisms may contribute to the adherence ofCandida yeasts to living cells

The adherence ofCandida yeasts to monolayers of human intestinal epithelium was studied in order to determine the specific and nonspecific mechanisms that might contribute to yeast adherence. Multiple factors were shown to significantly affect the adherence of yeasts to intestinal cells. It was demonstrated that hydrophobic yeasts adhered two times greater than normal yeasts, and positively charged yeasts adhered ten times greater than normal yeasts to monolayers of intestinal epithelium. The binding of yeasts to the intestinal cells was saturable and was most effectively blocked by mucin, which caused an 83% reduction in adherence, whereas the addition ofd-glucose caused a 41% reduction in adherence. Aggregation or coadherence of yeasts occurred as the yeast inocula were increased.Candida appears to possess the ability to adhere to living tissue by several mechanisms, such as adhesin-receptor interactions, nonspecific hydrophobic and ionic bonding, and aggregation or coadherence. This is the first demonstration of multiple forces that may act simultaneously in the process of adherence of yeasts to living cells.