类胡萝卜素产生菌深红酵母(Rhodotorula rubra)的培养条件的优化研究

本文研究深红酵母及其产生的类胡萝卜素的培养优化条件,并对其进行了小型发酵试验,结果表明pH值对深红酵母和类胡萝卜素有影响,初始pH值越低,色素的积累量越高,发酵过程中控制pH值能有效地增加色素积累的速度和初始色素积累量,但随着发酵时间的延长,色素积累量逐渐下降。     

糠醛对粘红酵母生长与油脂积累的影响

为了探究纤维素水解液中常见的发酵抑制物糠醛对粘红酵母Rhodotorula glutinis生长与油脂积累的影响,对比了不同的糠醛浓度(0.1、0.4、0.6、1.5 g/L)下粘红酵母的生物量和油脂积累情况,并探究了1.0 g/L的糠醛对粘红酵母不同碳源(葡萄糖和木糖)利用的影响。研究表明,当糠醛浓度达1.5 g/L时,粘红酵母的延迟期延长至96 h,残糖高达17.7 g/L,生物量最高6.6 g/L,仅为正常积累量的47%,油脂含量也减少了约50%;以木糖为碳源时,糠醛对粘红酵母的抑制程度小于葡萄糖为碳源时的情况;在糠醛存在的逆境中,粘红酵母倾向于生成更多的18碳脂肪酸或18碳不饱和脂肪酸。     

Sporobolomyces salmonicolor var. fischerii,a new yeast

A new variety of Sporobolomyces salmonicolor, a basidiomycetous yeast isolated from the cerebrospinal fluid of a human case of meningitis has been described. The variety can be differentiated from the species primarily by its ability to assimilate D-galactose and L-arabinose.     

Considerations on the use of commercially available yeast biomass for the treatment of metal-containing effluents

Summary Three strains ofSaccharomyces cerevisiae and one strain of aCandida sp. obtained from different industrial sources were screened for uptake of silver and copper. Considerable differences in metal uptake capacities were found between the different strains ofS. cerevisiae and betweenS. cerevisiae and theCandida sp. used. Copper uptake capacities ranged from 0.05 mmol g^–1 dry wt to 0.184 mmol g^–1 dry wt while values of 0.034 mmol Ag g^–1 dry wt and 0.193 mmol Ag g^–1 dry wt biomass were observed. Use of ion-selective electrodes (ISEs) enabled the detection of copper complexing agents (possibly proteins and carbohydrates) released by yeasts into the surrounding medium. In contrast, these compounds had no silver complexation abilities. Langmuir and Scatchard transformations of metal adsorption isotherms suggested differences in the mechanisms involved in metal uptake by the various yeasts. The differences between strains ofS. cerevisiae were due possibly to differences in cell wal composition. Different methods of preparation of biomass (fresh, air, oven and freeze-dried) had little effect on metal uptake in comparison with fresh biomass. Storage of fresh waste biomass at 4°C for 20 days had no effect on metal biosorption capacities. It was also observed that individual batches of waste biomass produced from different fermentation runs had consistent metal uptake capacities. The implications of the above results on the use of waste yeast biomass for treatment of metal-containing effluents are discussed.     

Study on the wall-breaking method of carotenoids producing yeast Sporidiobolus pararoseus and the antioxidant effect of four carotenoids on SK-HEP-1 cells

Abstract

The cell wall of carotenoids producing yeast Sporidiobolus pararoseus was broken through five different methods: acid-heating method, dimethyl sulfoxide (DMSO) method, enzymatic method, high-pressure homogenization (HPH) method, and cell autolysis method. HPH method not only brought the optimum breaking effect (wall-breaking extent of 72.3%) and the highest carotenoid extraction rate (67.2%), but also had the advantages of short-time, simple process, safe, and pollution-free. After optimization, the wall-breaking extent and the carotenoid extraction rate were enhanced to 78.3% and 82.5%, respectively. And the optimum conditions of HPH were obtained as homogenization pressure 80?MPa, bacterial liquid concentration 8% and homogenization for three times. Moreover, cell experiments demonstrated that all of the four carotenoids (β-carotene, γ-carotene, torulene, and torularhodin) purified from intracellular products of S. pararoseus. had the effect of resistance to oxidative damage from hydrogen peroxide on SK-HEP-1 cells, and torulene showed the most notable effect among them.     

基于k-tuple组合的酵母ncRNA与mRNA的比较研究

ncRNA和mRNA一样,都是重要的功能分子。以k-tuple(k字)含量为特征,对酵母ncRNA成熟序列和mRNA的编码区、上游序列与下游序列进行了分类与比较研究,结果显示:基于ncRNA成熟序列与mRNA编码区的3-tuple的含量,ncRNA和mRNA的交叉有效性分类精度(leave-one out cross-validation,LOOCV)平均值达到93.93%;基于上游序列4-tuple和5-tuple的含量,分类精度分别为92.49%和92.76%;基于下游序列4-tuple和5-tuple的含量,分类精度分别为91.58%和90.60%;利用上游序列和下游序列的4-tuple与5-tuple的含量,其平均分类精度分别为94.68%和94.83%;通过t检验,得到了在ncRNA和mRNA上、下游序列中具有显著统计学差异的k-tuple。上述结果表明,基于ncRNA成熟序列与mRNA编码区的3-tuple含量和基于ncRNA与mRNA上、下游序列的4或5-tuple含量可以有效地区分ncRNA与mRNA。此研究结果不仅有助于准确识别ncRNA与mRNA,还有助于发现ncRNA特异的转录因子结合位点。     

基于遗传算法酵母核小体定位性质预测

在DNA序列上,定位模糊的特殊核小体与定位良好的普通核小体同时存在于染色体区域内,但由于二者的化学性质差异不明显,区分较为困难。本文针对实验核小体在真核基因转录起始位点周围的分布规律和保守性建立了一个核小体分布模型,并在前人所做的预测核小体位置的工作基础上,利用遗传算法寻找模型上不同性质核小体的分布中心,构建核小体定位性质判别准则,最终确定了转录起始位点上、下游定位良好和模糊核小体的位置。     

基于对数线性模型的酵母基因转录调控模体分析

识别真核基因的转录因子结合位点(或称模体)是后基因组时代的一项主要工作,对共表达或共调控的基因同时进行分析可以提高模体识别的准确性.本文基于2×2列联表的对数线性模型,以模体出现的基因条数计数,对酵母核糖体蛋白(RP)基因普遍使用的转录调控模体进行分析,然后用U-检验进一步筛选出相对于背景序列来说过表达的模体.这些模体为酵母RP基因潜在的转录调控元件,与实验获得的转录因子结合位点的符合率达90%.本方法的优点在于用严格的统计标准在一组基因启动子中搜索普遍使用的模体,克服了以往分析中对模体使用普遍性的模糊判断.本文的方法也可以有效地搜索共表达基因族的组合调控模体对.研究中还发现一个现象:2×2列联表中反映属性相关程度的Pearson相关系数与对数线性模型的交互效应之间存在着明显的相关性.这一结果提示,可以用对数线性模型的交互效应来评价两属性的关联情况.     

Growth of a yeast mutant on ring a modified cholesterol derivatives

The ability of cholesterol derivatives without a hydroxyl group or a side-chain, to support the growth of heme and cyclase deficient Saccharomyces cerevisiae mutant GL 7 was tried and found to be in conformity with the results obtained using liposomes. On the other hand, results with other Ring A modified steroids involving saturation or movement of C5-C6 double bond, or isomeric 3-hydroxy-3-methyl cholestane derivatives, indicated that even minor structural variations can cause considerable changes in their growth supporting potential. The consequence of such structural variations need not be obviated by studies using liposomes or vesicles.     

A comparison of sexual and asexual replication strategies in a simplified model based on the yeast life cycle

This paper develops simplified mathematical models describing the mutation-selection balance for the asexual and sexual replication pathways in Saccharomyces cerevisiae, or Baker’s yeast. The simplified models are based on the single-fitness-peak approximation in quasispecies theory. We assume diploid genomes consisting of two chromosomes, and we assume that each chromosome is functional if and only if its base sequence is identical to some master sequence. The growth and replication of the yeast cells is modeled as a first-order process, with first-order growth rate constants that are determined by whether a given genome consists of zero, one, or two functional chromosomes. In the asexual pathway, we assume that a given diploid cell divides into two diploids. For the sake of generality, our model allows for mitotic recombination and asymmetric chromosome segregation. In the sexual pathway, we assume that a given diploid cell divides into two diploids, each of which then divide into two haploids. The resulting four haploids enter a haploid pool, where they grow and replicate until they meet another haploid with which to fuse. In the sexual pathway, we consider two mating strategies: (1) a selective strategy, where only haploids with functional chromosomes can fuse with one another; (2) a random strategy, where haploids randomly fuse with one another. When the cost for sex is low, we find that the selective mating strategy leads to the highest mean fitness of the population, when compared to all of the other strategies. When the cost for sex is low, sexual replication with random mating also has a higher mean fitness than asexual replication without mitotic recombination or asymmetric chromosome segregation. We also show that, at low replication fidelities, sexual replication with random mating has a higher mean fitness than asexual replication, as long as the cost for sex is low. If the fitness penalty for having a defective chromosome is sufficiently high and the cost for sex sufficiently low, then at low replication fidelities the random mating strategy has a mean fitness that is a factor of larger than the asexual mean fitness. We argue that for yeast, the selective mating strategy is the one that is closer to reality, which if true suggests that sex may provide a selective advantage under considerably more relaxed conditions than previous research has indicated. The results of this paper also suggest that S. cerevisiae switches from asexual to sexual replication when stressed, because stressful growth conditions provide an opportunity for the yeast to clear out deleterious mutations from their genomes. That being said, our model does not contradict theories for the evolution of sex that argue that sex evolved because it allows a population to more easily adapt to changing conditions.     

New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison

Yeasts combine the ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. Yeasts thus provide attractive platforms for the production of recombinant proteins. Here, four important species are presented and compared: the methylotrophic Hansenula polymorpha and Pichia pastoris, distinguished by an increasingly large track record as industrial platforms, and the dimorphic species Arxula adeninivorans and Yarrrowia lipolytica, not yet established as industrial platforms, but demonstrating promising technological potential, as discussed in this article.     

The effects of paromomycin on the fidelity of translation in a yeast cell-free system

The effects of the aminoglycoside antibiotic paromomycin on the fidelity of translation of the synthetic template poly(U), and two natural mRNAs (rabbit globin mRNA and Brome Mosaic virus RNA), were examined in an mRNA-dependent cell-free system from the yeast Saccharomyces cerevisiae. At antibiotic concentrations that did not inhibit translation (100 μM) optimal mistranslation of all three templates was observed, with the effects declining at higher antibiotic concentrations. Synthesis of the opal termination read-through protein of rabbit β-globin mRNA was induced by paromomycin, but only in lysates prepared from a [psi⁺] strain of yeast. The antibiotic did not induce detectable levels of either ochre or amber read-through, but did induce general misreading of Brome Mosaic virus RNA to the same degree in both [psi⁺] and [psi^?] lysates. This misreading was enhanced by addition of the polyamine spermidine.     

Five new combinations in the yeast genus Zygofabospora Kudriavzev emend. G. Naumov (pro parte Kluyveromyces) based on genetic data

The rDNA sequencing data obtained during the last 5 years in several laboratories clearly demonstrate that within the heterogeneous genus Kluyveromyces there is a group of highly related species, which we refer to as the genus Zygofabospora Kudriavzev 1960 emend. G. Naumov 2002. This genus includes four hybridizing species, Zf. marxiana, Zf. dobzhanskii, Zf. lactis, Zf. wickerhamii (Zygofabospora sensu stricto), and two taxonomically related species, Zf. aestuarii, Zf. nonfermentans (Nagahama et al.) G. Naumov comb. nov. (Zygofabospora sensu lato). We studied the relationships of the yeasts composing the Zf. lactis complex. Genetic hybridization analysis and molecular karyotyping revealed partial genetic-isolation varieties, Zf. lactis var. drosophilarum (Shehata et al.) G. Naumov comb. nov. and Zf. lactis var. phaseolospora (Shehata et al.) G. Naumov comb. nov. from North America, and Zf. lactis var. krassilnikovii (Kudriavzev) G. Naumov comb. nov. from Europe. The dairy yeast Zf. lactis var. lactis G. Naumov comb. nov. yields highly fertile hybrids with its wild ancestor Zf. lactis var. krassilnikovii and semi-sterile hybrids with North American taxa. Besides, Zf. lactis var. lactis and Zf. lactis var. krassilnikovii formed fertile hybrids with the South African yeast Zf. lactis var. vanudenii (van der Walt et Nel) G. Naumov comb. nov. The reinstatement of the latter yeast at the variety level has been done taking into account the results of the present study and the literature data on its geographic isolation, high divergence of the karyotype and mitochondrial DNA.     

A wide-range integrative yeast expression vector system based on <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>-derived elements

An Arxula adeninivorans integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1 promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level.     

The ribosomal l1 protuberance in yeast is methylated on a lysine residue catalyzed by a seven-beta-strand methyltransferase

Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting (3)H-N-ε-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ε-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38-53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.     

Identification of a ligand for IgG-Fc derived from a soluble peptide library based on fusion proteins secreted by S. cerevisiae

Biological libraries are important tools in the development of new peptide-based compounds. Here, we describe the use of a soluble peptide library system as a complementary tool in the field of ligand development. Random peptides were expressed in S. cerevisiae as carboxy-terminal extensions of the eukaryotic initiation factor 5a (eIF5a) and secreted into the culture supernatant. Expression and screening of this library were performed in a microwell format. As an example of this versatile approach, we describe the identification of a ligand for the human IgG-Fc fragment. Ligands binding IgG-Fc show great potential in a wide variety of applications including development of therapeutics, streamlining the large-scale purification of antibodies, and applications in diagnostic tests. We demonstrated the utility of this system. After screening only 6160 clones, we identified a ligand with the peptide sequence of TRRRTCSPPTWPRARARSTPSGCSSTGPSANRG. An affinity constant of 3.9 x 10(5) M(-1) was determined by a biosensor method. Handling and maintenance of this library is conceptually simple and highly applicable for automated high-throughput systems.     

Application of Mu in vitro transposition for high-precision mapping of protein–protein interfaces on a yeast two-hybrid platform

High-precision mapping of regions involved in protein–protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Transposon-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge.     

Development of an enrichment medium to detect Dekkera/Brettanomyces bruxellensis, a spoilage wine yeast, on the surface of grape berries

Brettanomyces bruxellensis spoilage is a serious problem for the wine industry. Mainly, by producing 4-ethylphenol and 4-ethylguaiacol, it confers off-odors to the wine and changes its aromatic quality. The presence of B. bruxellensis cells on the berry was speculated but it had never been clearly demonstrated. On grape berries, the microbial ecosystem is highly diverse and the population of B. bruxellensis can be very small. The aim of our study was to reveal and confirm the presence of B. bruxellensis on the surface of grape berries. We developed an enrichment medium for B. bruxellensis in order to overcome the detection limit of the molecular methods (species-specific PCR, ITS-RFLP PCR, PCR-DGGE). This medium, named EBB medium, made it possible to detect B. bruxellensis after 10 days of culture. For the first time, the presence of B. bruxellensis has been clearly established in several vineyards and at different stages of the grape development after the veraison. This work led to the conclusion that the grape berry is the primary source of B. bruxellensis. Grape growers and winemakers should take these results into account when deciding on the treatment to apply in the vineyards and the must. With the information provided here, B. bruxellensis prevention could start in the vineyard.     

Effect of acriflavine on the hexokinase isozyme pattern of a yeast, Hansenula jadinii

Dried cells of a yeast, Hansenula jadinii, that had been cultured aerobically with acriflavine, contained three hexokinase isozymes and metabolized glucose at 0.6 M to produce ATP to phosphorylate nucleotides in the presence of a high concentration of phosphate. Dried cells cultured aerobically without acriflavine contained two hexokinase isozymes and could not metabolize glucose under the same conditions. Two of the isozymes of the yeast cultured with acriflavine were similar to isozymes of the yeast cultured without acriflavine. However, the third isozyme was resistant to a high phosphate concentration and caused regeneration of ATP through glycolysis and phosphorylation of nucleotides.