Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

Expression of the Ruminococcus flavefaciens cellodextrinase gene in Saccharomyces cerevisiae

Summary A recombinant strain of Saccharomyces cerevisiae secreting bacterial cellodextrinase was constructed. The Ruminococcus flavefaciens cellodextrinase gene (celA) was inserted between a yeast expression-secretion cassette and yeast gene terminator, and cloned into a yeast-centromeric shuttle vector. Enzyme assays revealed growth-associated production of biologically active cellodextrinase by S. cerevisiae transformants.     

Spheroplast fusion of a brewing yeast strain with Saccharomyces diastaticus

Summary One haploid and one diploid strain of Saccharomyces diastaticus carrying genes responsible for glucoamylase synthesis were fused with a brewing polyploid Saccharomyces uvarum lager strain. With the spheroplast fusion technique, the ability to use dextrin and starch was introduced in the brewing yeast. Spheroplasts of the strains to be used were obtained by enzymatic digestion of the cell walls. Fusion took place in polyethylene glycol; complete cells were then regenerated in hypertonic medium containing 3% agar at 37°C. In the first fusion experiment melibiose was used as carbon source; in the second fusion experiment glycerol was employed as carbon source, for the parental Saccharomyces diastaticus diploid strain was a petite mutant. Fusion products were capable of utilizing melibiose and dextrin as carbon sources.     

Construction of flocculent industrial yeast by the yeast flocculation gene <Emphasis Type="Italic">FLO1</Emphasis>

The structure gene FLO1 from Saccharomyces cerevisiae W303-1A encoding a flocculation protein and the G418 resistance gene kanMX from plasmid pUG6 were amplified by PCR method. The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Angel yeast. The transformant Angel yeast F6 was obtained and showed strong and stable flocculation ability during 20 batches inoculation. And the flocculation ability of the transformant Angel yeast F6 showed no difference in the medium with the initial pH ranging from 3.5 to 6.0. Noteworthily, the flocculation onset of the transformant strain was in the early stationary growth phase, not coincident with the glucose depletion in the cultural medium. And in the experiment the ethanol yield and other properties of the transformant Angel yeast F6 were similar to those of the wild-type strain, although its fermentation time was a little slower comparing with the wild-type strain. Those would be potential application for yeast cells to separate and recycle in the fuel ethanol industry.     

Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation. Revisions requested 14 November 2005; Revisions received 17 January 2006     

Construction of a xylose-metabolizing yeast by genome integration of xylose isomerase gene and investigation of the effect of xylitol on fermentation

A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.     

Selection of yeast hybrids by the sulfonylurea herbicide chlorsulfuron

Summary A new selection method based on the use of chlorsulfuron (CS) resistance as the selection marker for protoplast fusion in industrial yeast has been introduced using the system of protoplast fusion. A petite mutant of a spontaneously CS-resistant distiller's Saccharomyces cerevisiae strain and a wild-type CS-sensitive strain of the osmotolerant yeast Zygosaccharomyces mellis were fused in order to obtain a distiller's yeast suitable for fermentations on concentrated molasses. Fusion products were isolated as large colonies on minimal glycerol agar with 0.5 mg ml^–1 of the herbicide Glean (75% CS). Following prolonged cultivation on molasses, stable hybrid subxlones were obtained. Offprint requests to: F. Cvrková     

Production of Hormoconis resinae glucoamylase P by a stable industrial strain of Saccharomyces cerevisiae

A stable strain of Saccharomyces cerevisiae secreting glucoamylase (EC 3.2.1.3) with high debranching activity was constructed using recombinant DNA technology. An expression cassette without bacterial sequences, containing Hormoconis resinae glucoamylase P cDNA and the dominant selection marker MEL1 was integrated into the yeast chromosome using ARS1 homology. The glucoamylase expression level of the integrant yeast strain was increased by chemical mutagenesis. The yeast strains secreting glucoamylase were able to grow on soluble starch (5%, w/v) and ferment it to ethanol.Correspondence to: A. Vainio     

Amine-catalyzed Racemization of the Imidazoline Derivatives

Protoplast fusion was carried out between a saké brewer’s yeast strain, Saccharomyces cerevisiae Kyokai 7, and a lactose utilizing yeast strain, Kluyveromyces lactis T396. A stable hybrid, PN 13, which was selected from the many resultant fusants, showed physiologically complemented traits with respect to sugar utilization, vitamin requirements and so on. Biochemical investigations also revealed that fusant PN 13 was an intermediate hybrid between the parental strains. In glucose and lactose media, moreover, the fusant grew and produced ethanol at higher rates than K. lactis T396.     

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.     

Amplification and loss of repeat units of the human minisatellite MS1 integrated in chromosome III of a haploid yeast strain

Minisatellites comprise arrays of tandemly repeated short DNA sequences which show extensive variation in repeat unit number. The mechanisms that underlie this length variation are not understood. In order to study processes influencing length changes of minisatellites, we integrated the human minisatellite MS1 into a haploid strain of the yeast Saccharomyces cerevisiae. Frequent spontaneous generation of MS1 alleles with new lengths were observed in this yeast strain. Hence it is concluded that recombination between members of a pair of homologous chromosomes is not a prerequisite for the generation of length changes in MS1 in yeast.     

Immunostimulatory activity of potential probiotic yeast strains in the dorsal air pouch system and the gut mucosa

Aims: To determine the immunostimulatory activity of 15 presumptive probiotic yeast strains in the dorsal air pouch system in comparison with their activity in the gut mucosa. Methods and Results: Presumptive probiotic yeast strains previously isolated from human gastrointestinal tract and Feta cheese were further characterized genotypically and biochemically. The Saccharomyces cerevisiae 982, Saccharomyces boulardii KK1 and Kluyveromyces lactis 630 strains exhibited in the air pouch increased polymorphonuclear cell influx and phagocytic activity as well as cytokine production with similar potency as the probiotics Ultra levure S. boulardii and Lactobacillus acidophilus NCFB 1748. Oral administration of these strains in mice results in differential activation of small intestine immune responses concerning IgA and cytokine production as well as Toll‐like receptor expression. Conclusion: Besides the Saccharomyces strains 982 and KK1, the K. lactis 630 strain could also be considered as a candidate probiotic. Significance and Impact of the Study: The air pouch model may be used as an alternative and rapid method for the discrimination and selection of potential probiotic yeast strains.     

Contribution of the fermenting yeast strain to ethyl carbamate generation in stone fruit spirits

Fermented fruit and beverages frequently contain ethyl carbamate (EC), a potentially carcinogenic compound that can be formed by the reaction of urea with ethanol. Both are produced by the yeast Saccharomyces cerevisiae with ethanol as the major end product of hexose fermentation and urea as a by-product in arginine catabolism. In spirit production, EC can also be derived from cyanide introduced by stone fruit. To determine the relative contribution of yeast metabolism to EC production, we genetically engineered a diploid laboratory strain to reduce the arginase activity, thus blocking the pathway to urea production. For this purpose, strains with either a heterozygous CAR1/car1 deletion or a homozygous defect (car1/car1) were constructed. These strains were compared to the parental wild type and to an industrial yeast strain in cherry mash fermentations and spirit production. The strain with the homozygous car1 deletion showed a significant reduction of EC in the final spirits in comparison to the non-engineered controls. Nevertheless, using this strain for fermentation of stoneless cherry mashes did not completely impede EC formation. This indicates another, as yet unidentified, source for this compound.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Cloning and sequencing of the ura3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement

The ura3 gene of Hansenula polymorpha was cloned, sequenced and used to generate a ura3 mutant from the wild-type strain of this yeast via integrative mutagenesis. The Tn5 neomycin-resistance marker (neo) under control of the ADH1 promoter from Saccharomyces cerevisiae served as a transformation marker. The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration. Correspondence to: C. P. Hollenberg     

Retion of human apolipoprotein A-II by a mutant of Saccharomyces cerevisiæ

Summary A modified cDNA of mature human apolipoprotein A-II (apoA-II) was expressed by a sterol-uptake yeast strain (erg 10) of Saccharomyces cerevisiæ. ApoA-II cDNA was fused with a modified yeast alpha factor leader peptide coding sequence under phosphoglycerate kinase promoter control in a 2 micron-based plasmid construction. The use of cholesterol allowed apoA-II secretion in the culture medium. The mono and dimeric forms of apoA-II (approximately 3 micrograms per ml) were detected.     

Construction and evaluation of self‐cloning bottom‐fermenting yeast with high SSU1 expression

Aim: To construct a self‐cloning brewer’s yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds. Methods and Results: DNA fragments of a high‐expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae‐type and Saccharomyces bayanus‐type SSU1 genes of bottom‐fermenting brewer’s yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3‐methyl‐2‐buten‐1‐thiol (MBT) and 2‐mercapto‐3‐methyl‐1‐butanol (2M3MB) were lower when compared with the parent strain. Conclusion: We successfully constructed a self‐cloning brewer’s yeast with high SSU1 expression that enhanced the sulfite‐excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB. Significance and Impact of the Study: The self‐cloning brewer’s yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB.     

Studies on yeast host strains for heterologous P450 expression

The Saccharomyces cerevisiae strain AH22 was capable of human P4501A1 expression without detectable background of yeast P450, unlike ATCC44773. Repeated backcrossing to AH22 produced a strain allowing transformation by vectors carrying various common selectable markers. Background yeast xenobiotic metabolism was observed only with growth on complex medium.     

Effect of cultivation conditions on invertase production by hyperproducing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolates

Invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) finds major uses in confectionery and in the production of invert syrup. In the present study, we report on invertase production by wild cultures of Saccharomyces cerevisiae. The yeast strains were isolated from dates available in a local market. Five hyperproducing yeast strains (>100- fold higher invertase activity) were kinetically analysed for invertase production. Saccharomyces cerevisiae strain GCA-II was found to be a better invertase-yielding strain than all the other isolates. The values of Q_p and Y_p/s for GCA-II were economical as compared to other Saccharomyces cultures. The effect of sucrose concentration, rate of invertase synthesis, initial pH of fermentation medium and different organic nitrogen sources on the production of invertase under submerged culture conditions was investigated. Optimum concentrations of sucrose, urea and pH were 3, 0.2 (w/v), and 6 respectively. The increase in the enzyme yield obtained after optimization of the cultural conditions was 47.7%.     

酿酒酵母表达和纯化功能性的铁载体合成蛋白PchE

[目的] 利用酿酒酵母表达系统,通过乙醇脱氢酶启动子异源表达细菌源的铁载体合成蛋白PchE,并与来源于枯草芽孢杆菌的泛酰化酶Sfp同宿主共表达,探索真核表达体系表达具有生化活性的细菌源蛋白。[方法] 从大肠杆菌BAP 1染色体上扩增sfp基因,将pchE基因及串联的pchE与sfp基因分别构建到酵母-大肠杆菌穿梭质粒pXW55中,各自转化酿酒酵母BJ5464-npgA表达,经过亲和层析和离子交换层析纯化蛋白,利用HPLC检测细菌源与酵母源表达的PchE在体外重构生化反应中的催化活性。[结果] 利用酿酒酵母表达系统可以获得高纯度的原核蛋白PchE。真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰PchE,合成中间产物HPT-Cys。[结论] 在酿酒酵母Saccharomyces cerevisiae BJ5464-npgA表达系统中,首次证明真菌源的泛酰化基因NpgA和细菌源的Sfp,均可泛酰化修饰细菌源的非核糖体肽合酶。比较酵母和细菌宿主的目标蛋白表达,证明酵母表达的巨大蛋白PchE的纯度更高,非特异性条带减少,推测酵母宿主可能更适合表达纯化功能性的巨型蛋白质。