拉格啤酒酵母菌物种和株系的快速分子鉴定

近年来的基因组学研究结果已证实拉格啤酒酵母Saccharomyces pastorianus是一个由艾尔啤酒酵母S. cerevisiae和真贝氏酿酒酵母S. eubayanus杂交而成的杂交种,并可根据地域传承和染色体倍性分为两个株系,即I型/Saaz系和II型/Frohberg系。前者主要为异源3倍体,后者则主要为异源4倍体。为了探讨中国啤酒酿造酵母菌的物种和菌系归属,我们根据拉格啤酒酵母及其两个菌系的基因组特性,制定了一套基于IntFR片段种特异性扩增和ITS-RFLP分析的精确但简便易行的拉格啤酒酵母菌物种和株系鉴定新方法,并以酿酒酵母属内相关种的模式或权威菌株和部分酒精及面包酵母为参照,对保藏于中国普通微生物菌种保藏中心（CGMCC）的41株啤酒酿造酵母菌进行了重新鉴定和分型。这些菌株除1株原定名为贝氏酿酒酵母S. bayanus外,其余菌株的原定名均为S. cerevisiae。研究结果确认了S. bayanus菌株鉴定的正确性,但在其余的40株啤酒酵母菌株中,21株属于S. cerevisiae,1株属于葡萄汁酿酒酵母S. uvarum,18株属于S. pastorianus。菌系鉴定和流式细胞测定结果显示在确认的S. pastorianus菌株中,1株为I型/Saaz系,3倍体;17株为II型/Frohberg系,其中9株为4倍体,两株为3倍体,5株介于3倍至4倍体之间。啤酒酵母物种和株系的确认对优化发酵工艺和菌种选育及遗传改造等具有重要意义。     

《微生物学报》编委白逢彦在啤酒酵母菌的起源与驯化研究方面取得新进展

<正>发表时间:2014.05.22啤酒根据发酵工艺和所用菌种的不同可分为两大类,即爱尔(ale)啤酒和拉格(lager)啤酒。前者使用酿酒酵母菌(Saccharomyces cerevisiae)在相对较高的温度下发酵,酵母菌往往浮在上层,故又称上层发酵啤酒。后者在低温(10°C左右)条件下发酵,酵母菌往往沉于底层,故又称下层发酵啤酒。人类最开始酿造的是爱尔啤酒,已有数千年的历史。拉格啤酒一般认为最早于15世纪出现在德国巴伐利亚,但很快流行开来,逐步取代爱尔啤酒成为全球生产和消费量最大的酒精饮料。我国生产的啤酒基本上都属于拉格啤酒。     

走出中国：酿酒酵母的起源、驯养与演化

酿酒酵母(Saccharomyces cerevisiae)被广泛应用于酒类酿造和食品发酵等行业,其被人类利用的历史已有近万年。酿酒酵母也是遗传学、分子生物学、基因组学和合成生物学等研究中常用的模式生物。近年来研究者对其自然和驯养种群进行了全球范围的生态学、群体遗传学和群体基因组学等方面的研究,更新了对其生态分布、遗传多样性、自然进化和驯养史以及进化动力等方面的认知。发现酿酒酵母在原始森林等自然环境中普遍存在,并可能偏好阔叶树树干、腐木和周围土壤等生境。中国酿酒酵母的遗传多样性显著高于世界其他地区,该物种最古老的谱系也仅发现于中国,说明中国可能是该物种的起源地。生态适应是塑造该物种群体结构的主要力量,导致其野生和驯养群体之间的明显分化。驯养群体又分化为固态发酵和液态发酵两大类群,每个类群内又形成不同的驯养谱系。该物种野生群体的遗传多样性远高于其驯养群体,而野生群体遗传多样性的形成主要由中性突变引起。中国野生和驯养群体在麦芽糖利用能力、基因组杂合性、子囊孢子形成率和孢子活力等方面表现出显著差异,表明这2个群体采取不同的生活策略来适应其不同的生活环境。驯养群体通过群体或谱系特异性基因拷贝数...     

高SO2产量啤酒酵母工业菌株的构建

二氧化硫在啤酒中具有抗氧化的重要功能,而在其形成过程中APS激酶(MET14编码)起着非常重要的作用。以二氧化硫产量较高的青岛啤酒酵母(Saccharomyces cerevisiae)YSF-5的总DNA为模板,用PCR方法克隆得到MET14基因。为使目的基因在酿酒酵母中表达,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以PGK1强启动子为调控元件,构建了重组表达质粒pPM,并转化酿酒酵母YS58。转化子在YNB添加亮氨酸、组氨酸和色氨酸的选择性培养基上筛选鉴定,盐酸副玫瑰苯胺法测得转化子的SO2产量是受体菌的2倍左右。在重组表达质粒pPM的基础上添加铜抗性标记基因构建了重组表达质粒pCPM,并转化青岛啤酒工业酵母菌株YSF-38,转化子在YEPD 4mmol/L CuSO4的选择性培养基上筛选鉴定,实验室条件下培养后,测得转化子YSF-38(pCPM)的SO2产量是受体菌的3.2倍。用该转化子在青岛啤酒厂进行小型发酵实验,结果表明在发酵结束时,YSF-38(pCPM)转化子的SO2产量是受体菌的1.4倍。因此,MET14基因的有效表达可以提高啤酒工业酵母的SO2产量。     

异柠檬酸脱氢酶基因调控对拉格酵母抗自溶性能的影响

啤酒酵母的自溶会影响啤酒的风味和品质,对酵母自溶的调控是工业啤酒生产的迫切需求。前期在啤酒酵母自溶的研究中发现柠檬酸循环相关基因对酵母自溶有较大影响。为探究柠檬酸循环中异柠檬酸脱氢酶基因调控对自溶的影响,在典型拉格啤酒酵母(Saccharomyces pastorianus H.)Pilsner中对IDP1和IDP2基因进行了破坏和过表达。结果发现IDP1基因的破坏能提高酵母的抗自溶能力,自溶96 h抗自溶指数为8.40,比原始菌提高了1.5倍。IDP1基因的破坏提高还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate,NADPH)的供应,NADPH/NADP^(+)的比值为1.94,发酵结束时胞内ATP水平比原始菌提高1.8倍,活性氧(reactive oxygen species,ROS)相比原始菌减少10%。IDP2基因的缺失造成酵母快速自溶和NADPH供应的减少,自溶96 h抗自溶指数为4.03,NADPH/NADP^(+)的比值为0.89。发酵结束时胞内ATP水平相比原始菌减少8%,ROS是原始菌的1.3倍。本研究进一步阐释了柠檬酸循环相关基因对酵母自溶的调控机理,为选育自溶性能可控的优良酵母提供了理论基础。     

酿酒酵母单倍体与双倍体原生质体的融合*

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

酿酒酵母单倍体与双倍体原生质体的融合

本文报道用酿酒酵母(Saccharomyces cerevisiae)原生质体融合,得到营养互补的融合子为三倍体,其生长速度、发酵速率均较亲株提高1—2倍。部分融合子酒精的产量高于亲株,同时高于目前使用的酒精发酵生产菌株。     

一株分离于工业污水池的耐碱酵母

目的:从新疆温泉县一个碱性工业污水处理池中分离并鉴定耐碱酵母菌。方法:用稀释平板法分离菌种,通过形态学观察、生理生化特征及26S rDNA D1/D2区基因序列分析鉴定菌种。结果:从水样中分离得到一株耐碱酵母菌,它们能在pH3.5~11.0,12%NaCl,4~45℃生长,经形态观察及生理生化特征鉴定为酵母属,对其26S rDNA 5’端D1/D2区基因序列进行了PCR扩增并测序,GenBank注册号为DQ132884,同源序列分析结果表明该序列与酿酒酵母(Saccharomyces cerevisiae)Sb4有99.8%的同源性,因此将其命名为酿酒酵母(Saccharomyces cerevisiae)XJU-2,该菌种已保藏于中国微生物菌种保藏委员会普通微生物中心(CGM-CC),保藏号为CGMCC No.2.3095。结论:XJU-2的最高耐碱值可达pH 11.0,而且酸碱耐受范围很大,性能明显优于国内外已报道的酿酒酵母菌种。     

调控酿酒酵母类异戊二烯合成途径强化芳樟醇合成

芳樟醇是一种重要单萜,广泛应用于食品、医药、日化等工业领域.然而芳樟醇在植物中含量低且难提取,限制了其大规模生产.目前通常以酿酒酵母Saccharomyces cerevisiae作为单萜生物合成宿主,其内源类异戊二烯合成途径提供合成单萜物质的前体——香叶基二磷酸(GPP).由于该途径代谢通量较低,导致GPP供应不足,极大地降低了异源单萜的合成效率.为了调节该途径的代谢通量,构建酿酒酵母整合表达载体pRS305-tHMG1和游离表达载体pYLIS-IDI1,并分别转入酿酒酵母CEN.PK2-lC中,获得酿酒酵母工程菌LS01和LS02.同时将载体pYLIS-IDIl转入酿酒酵母工程菌LS01中,构建酿酒酵母工程菌LS03.GC-MS检测结果显示,通过提高异戊二烯二磷酸异构酶(IDIl)和羟甲基戊二酸单酰辅酶A(HMG-CoA)还原酶活性区域(tHMGl)的表达水平,最终使芳樟醇产量提高1.3倍至(127.71±7.68) tg/L.结果表明,通过调控类异戊二烯合成途径,强化GPP合成前体供给,可以显著提高酿酒酵母中芳樟醇的产量.     

表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*

透明颤菌血红蛋白基因vgb在多种研究和工业发酵菌中异源表达很好的解决了高密度发酵中的溶氧率问题。酿酒酵母是经典的真核模型,且在发酵工业中具有重要的应用价值,但vgb在酿酒酵母中异源表达对细胞生长的影响并不清楚。以ADH1为启动子构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb的异源表达质粒YEplac195-ADH1pr-vgb,并转化至酿酒酵母BY4741。通过生长敏感性实验,发现在发酵碳源和非发酵碳源中,vgb的异源表达均抑制了菌株生长。接着,通过2',7'-二氯荧光黄双乙酸盐和PI染色和脂质过氧化产物检测分析,发现过表达vgb的酿酒酵母细胞中活性氧(ROS)的积累、细胞膜通透性改变以及脂质过氧化。结果表明,酿酒酵母中过表达vgb改变细胞的氧化状态促进活性氧的累积,氧化应激导致菌株的生长抑制。     

酿酒酵母SRP40基因对细胞耐受性影响的研究

【目的】本论文研究酿酒酵母srp40³⁹突变基因对酵母细胞异丁醇耐受性的影响。【方法】首先,以酿酒酵母野生型W303-1A和突变株EMS39染色体DNA为模板克隆野生型SRP40基因和srp40³⁹突变基因;然后,将野生型SRP40基因和srp40³⁹突变基因分别连接到质粒YCplac22上,构建质粒YCplac22-SRP40和YCplac22-srp40³⁹。将质粒YCplac22-SRP40、YCplac22-srp40³⁹以及YCplac22空质粒分别转化入野生型酿酒酵母W303-1A中,分别得到W303-1A-SRP40工程菌、W303-1A-srp40³⁹工程菌和W303-1A-control工程菌。将3株工程菌分别置于含1.0%异丁醇、1.3%异丁醇、8.0%乙醇和0.5%异戊醇的CM培养基中进行发酵,测定细胞密度(OD₆₀₀)和生长情况,并计算2–10 h的比生长速率（μ）。将3株工程菌于55°C热激4 min后做稀释...     

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.     

Effects of mutations in the N terminal region of the yeast G protein α-subunit Gpa1p on signaling by pheromone receptors

The sites and modes of interaction between G protein-coupled receptors and their cognate heterotrimeric G proteins remain poorly defined. The C-terminus of the G subunit is the best established site of contact of G proteins with receptors, but structural analyses and crosslinking studies suggest the possibility of interactions at the N-terminus of G as well. We screened for mutations in the N-terminal region of the G subunit encoded by the yeast GPA1 gene that specifically affect the ability of the G protein to be activated by the yeast -mating factor receptor. The screen led to identification of substitutions of glutamine or proline for Leu18 of Gpa1p that reduce the response to the pheromones -factor and a-factor without affecting cellular levels of the subunit or its ability to interact with and subunits. The mutations do not appear to affect the intrinsic ability of the G protein to be converted to the activated state. The low yield of different mutations with this phenotype indicates either that the N-terminal segment of the yeast G subunit does not undergo extensive interactions with the -factor receptor, or that this region can not be altered without detrimental effects upon the formation of G protein trimers.Communicated by D. Y. Thomas     

Assessing the viability of a clumpy <Emphasis Type="Italic">mnn9</Emphasis> strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> used in the manufacture of recombinant pharmaceutical proteins

Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.     

Prevalence and susceptibility of Saccharomyces cerevisiae causing vaginitis in Greek women

Saccharomyces cerevisiae is an ascomycetous yeast, that is traditionally used in wine bread and beer production. Vaginitis caused by S. cerevisiae is rare.The aim of this study was to evaluate the frequency of S. cerevisiae isolation from the vagina in two groups of women and determined the in vitro susceptibility of this fungus.

Subjects and methods

Vaginal samples were collected from a total of262 (asymptomaticandsymptomatic) women with vaginitis attending the centre of family planning of General hospital ofPiraeus. All blastomycetes that isolated from the vaginal samples were examined for microscopic morphological tests and identified by conventional methods: By API 20 C AUX and ID 32 C (Biomerieux). Antifungal susceptility testing for amphotericin B,fluconazole itraconazole,voriconazole, posaconazole and caspofungin was performed by E -test (Ab BIODIKS SWEDEN) against S. cerevisiae.

Results

A total of 16 isolates of S. cerevisiae derived from vaginal sample of the referred women, average 6.10%. Susceptibility of 16 isolates of S. cerevisiae to a variety of antimycotic agents were obtained. So all isolates of S. cerevisiae were resistant to fluconazole, posaconazole and intraconazole, but they were sensitive to voriconazole caspofungin and Amphotericin B which were found sensitive (except 1/16 strains). None of the 16 patients had a history of occupational domestic use of baker’s yeast.

Conclusions

Vaginitis caused by S. cerevisiae occur, is rising and cannot be ignored. Treatment of Saccharomyces vaginitis constitutes a major challenge and may require selected and often prolonged therapy.     

Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.     

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His–Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.     

酒醅来源酵母菌合成异戊醇能力与途径解析

【背景】异戊醇是酵母菌在白酒发酵过程中通过氨基酸合成代谢途径和氨基酸分解代谢途径合成的主要高级醇,其含量影响白酒饮用的舒适度。【目的】分析和比较分离自浓香型白酒酒醅中的酵母菌合成异戊醇的能力,揭示酵母菌合成异戊醇的途径。【方法】从酒醅中分离具有异戊醇合成能力的酵母菌株,比较不同生长时期酵母菌合成异戊醇的能力,通过前体物代谢分析它们合成异戊醇的途径。【结果】分离自酒醅的5株酵母的异戊醇合成能力从强到弱依次为Naumovozyma castellii JP3-1、Saccharomyces cerevisiae JP3、Pichia fermentans JP22、Pichia kudriavzevii JP1和Naumovozyma dairenensis CBS421。这些酵母合成异戊醇的时期主要在对数生长期,N. castellii JP3-1、P. fermentans JP22和N. dairenensis CBS421在稳定生长期也合成异戊醇。S. cerevisiae JP3、N. castellii JP3-1和N. dairenensis CBS421在整个生长时期主要通过Harris途径合成异戊醇;P. kudriavzevii JP1在整个时期主要通过Ehrlich途径合成异戊醇;P. fermentans JP22在对数生长期通过Harris途径和Ehrlich途径合成异戊醇的能力接近,在稳定生长期主要通过Harris途径合成异戊醇。【结论】本研究揭示了酒醅来源5个属种酵母合成异戊醇的途径、能力与其生长时期的关系,研究结果可为解析浓香型白酒发酵过程异戊醇合成、积累机制及实施白酒发酵过程异戊醇合成的精准调控提供理论依据。