Differential killer sensitivity as a tool for fingerprinting wine-yeast strains ofSaccharomyces cerevisiae

The extreme variability of the killer phenomenon in nature, expressed differently in different strains of the same yeast species, embodies an exceptional potential for the discrimination of yeasts at the strain level. Killer-sensitive relationships between a killer reference panel of 24 yeasts belonging to 13 species of six genera, and different industrial wine-starters ofSaccharomyces cerevisiae can be used profitably for a rapid and simple fingerprinting procedure.     

Restoration of respiratory competence in the yeastCandida utilis after somatic fusion withSaccharomyces cerevisiae

After somatic fusion between a mitochondrial mutant ofCandida utilis andSaccharomyces cerevisiae respiratory competent strain, the complex III of the respiratory chain seems to be restored. Fusion products, FP, synthesizing normal apocytochromeb were recovered and showed normal-shaped mitochondria along the cytoplasm as in theCandida utilis original, respiratory-competent strain.     

Biosynthesis of superoxide dismutase and catalase inSaccharomyces cerevisiae: effects of oxygen and cytochromec deficiency

Summary Two strains ofSaccharomyces cerevisiae were used to study the synthesis of superoxide dismutase. One strain (cytochromec-deficient) contained 5–10% of the normal amounts of total cytochromec, while the other strain was a wild type. The cytochromec-deficient mutant had lower specific growth rate, growth yield, and oxygen uptake than the wild type. The superoxide dismutase and catalase activities, in both strains, were significantly lower under anaerobic than under aerobic conditions. Furthermore, under aerobic conditions the mutant contained higher levels of superoxide dismutase than the wild type which may be attributed to the higher intracellular flux of superoxide radicals caused by the cytochromec deficiency. The mutant also showed a lower level of catalase which was due to glucose repression.Paper Number 10007 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, U.S.A. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.     

Killer phenotype of indigenous yeasts isolated from Argentinian wine cellars and their potential starter cultures for winemaking

Of 31 yeasts, from different surfaces of two cellars from the northwest region of Argentina, 11 expressed killer activity against the sensitive strain Saccharomyces cerevisiae P351. Five of these killer yeasts were identified as S. cerevisiae by phenotypic tests and PCR-RFLP analysis. Two S. cerevisiae killer strains, Cf5 and Cf8, were selected based on their excellent kinetic and enological properties as potential autochthonous mixed starter cultures to be used during wine fermentation. They could dominate the natural microbiota in fermentation vats and keep the typical sensorial characteristics of the wine of this region.     

Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion

Conditions were optimized for rapid release and improved regeneration of protoplasts ofSaccharomyces cerevisiae NCIM 3458. Rapid protoplast release was also obtained with representatives of several other yeast genera under the modified conditions of treatment. The application of the procedure in construction of a highly flocculentSaccharomyces cerevisiae with a killer character is described. Fusion was effected between UV-killed protoplasts ofS. cerevisiae NCIM 3578 with a killer character and live protoplasts of the highly flocculentS. cerevisiae NCIM 3528 in the presence of polyethylene glycol (PEG) 6000. Fusants were selected using benomyl resistance as marker, the killer toxin producer rather than the highly flocculent yeast being resistant to the fungicide at a concentration of 100 g ml^–1. Fusants were also characterized by their DNA contents, capacity for ethanolic fermentation of molasses sugar and levels of invertase, alcohol dehydrogenase and pyruvate decarboxylase activities.     

Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

Effect of Brestan on Saccharomyces cerevisiae during continuous cultivation

An increase in Brestan concentration in nutrient media decreased the content of protein, phosphorus, total ribonucleic acid, activity of pyruvate carboxylase and isocitrate lyase in cells ofSaccharomyces cerevisiae parent strain and respiratory deficient (RD) mutant while the trehalose content increased. The respiration quotient value for the RD mutant was higher than for the parent strain. The RD mutant lacked cytochromeaa ₃; cytochromec andb contents were lower than those of the parent strain.     

Industrial yeast strain improvement: construction of strains having the killer character and capable of utilizing starch

Summary A hybrid of Saccharomyces cerevisiae with the ability to utilize starch and to produce the killer toxin was constructed by the protoplast fusion technique. The hybrid was obtained in two steps. In the first, a wild killer strain was fused with a laboratory strain (S. cerevisiae STA2). A fusion product which carried the killer factor and the ability to grow on starch was selected. In the second step, this hybrid was fused with a baker's yeast.     

A Comparative Study of Certain Parameters of Energy Metabolism in Two Strains of Saccharomyces cerevisiae

A comparative study of energy metabolism in two strains Saccharomyces cerevisiae (the initial strain no. 73 and laser-irradiated mutant strain Y-503) was performed. In all growth phases, the rates of oxygen consumption by cells of Y-503 were higher than in the initial strain. The maximum (threefold) increase in the rate of oxygen consumption was observed in the linear phase. The effects of respiratory chain inhibitors rotenone, antimycin A, and cyanide on cellular and mitochondrial respiration were identical. There are two sites of energy coupling in the respiratory chain of mitochondria in S. cerevisiae no. 73 and Y-503, and electron flow is mainly mediated by cytochrome oxidase. The data suggest that the higher respiratory activity ofS. cerevisiaeY-503 cells in comparison with no. 73 is associated with greater amounts of mitochondria and total surface area of coupling mitochondrial membranes, which appears to be a factor contributing to the high physiological and biochemical activity of this strain.     

Combined use of killer biotype and mtDNA-RFLP patterns in a Patagonian wine Saccharomyces cerevisiae diversity study

The aim of this work was to characterize the indigenous wine Saccharomyces cerevisiae diversity within the Argentinean Patagonia. Two cellars with particular enological practices located in different winegrowing areas were selected and 112 indigenous S. cerevisiae isolates were obtained from spontaneous red wine fermentations carried out in them. Thirty-five and 19 patterns were distinguished among the total indigenous isolates using mtDNA-RFLP and killer biotype analysis, respectively. The combination of both typing techniques rendered a higher variability with 42 different patterns, i.e. 42 strains, evidencing a great diversity in S. cerevisiae populations associated with spontaneous red wine fermentations in Northwestern Patagonia. The analysis of the relatedness among strains using Principal Coordinates Analysis from combined data allowed the clustering of the strains into two populations significantly related to their origin fermentations. The combined use of the mtDNA-RFLP analysis together with the killer biotype method proved to be a powerful tool in the fingerprinting of the enological S. cerevisiae strains.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.     

Killer-toxin-resistantkre12 mutants ofSaccharomyces cerevisiae: genetic and biochemical evidence for a secondary K1 membrane receptor

TheSaccharomyces cerevisiae killer toxin K1 is a secreted α/β-heterodimeric protein toxin that kills sensitive yeast cells in a receptor-mediated two-stage process. The first step involves toxin binding to β-1,6-d-glucan-components of the outer yeast cell surface; this step is blocked in yeast mutants bearing nuclear mutations in any of theKRE genes whose products are involved in synthesis and/or assembly of cell wall β-d-glucans. After binding to the yeast cell wall, the killer toxin is transferred to the cytoplasmic membrane, subsequently leading to cell death by forming lethal ion channels. In an attempt to identify a secondary K1 toxin receptor at the plasma membrane level, we mutagenized sensitive yeast strains and isolated killer-resistant (kre) mutants that were resistant as spheroplasts. Classical yeast genetics and successive back-crossings to sensitive wild-type strain indicated that this toxin resistance is due to mutation(s) in a single chromosomal yeast gene (KRE12), renderingkrel2 mutants incapable of binding significant amounts of toxin to the membrane. Sincekrel2 mutants showed normal toxin binding to the cell wall, but markedly reduced membrane binding, we isolated and purified cytoplasmic membranes from akrel2 mutant and from an isogenicKre12+ strain and analyzed the membrane protein patterns by 2D-electrophoresis using a combination of isoelectric focusing and SDS-PAGE. Using this technique, three different proteins (or subunits of a single multimeric protein) were identified that were present in much lower amounts in thekre12 mutant. A model for K1 killer toxin action is presented in which the gene product ofKRE12 functions in vivo as a K1 docking protein, facilitating toxin binding to the membrane and subsequent ion channel formation.     

Phenotypic character of the lipid hydroperoxide-resistant mutant: Positive relationship between flocculation and resistance against lipid hydroperoxide inSaccharomyces cerevisiae

A lipid hydroperoxide-resistant mutant was isolated from a strain ofSaccharomyces cerevisiae. The mutant was resistant to 1.5mm tert-butylhydroperoxide and 1.0mm linoleic acid hydroperoxide. It flocculated in a Ca²⁺-dependent manner and the resistance against lipid hydroperoxide was suppressed by mannose, which also inhibited flocculation. A positive relationship between the acquirement of, the flocculent phenotype and resistance against lipid hydroperoxide is suggested. A protein with a molecular weight of 33 kDa was found on the surface of the mutant cell.     

Isolation and identification of killer yeasts from Agave sap (aguamiel) and pulque

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic beverages such as beer and wine; therefore, killer or killer-resistant strains have been sought for use in alcoholic fermentations. In the current paper a total of 16 strains belonging to six species were isolated. From two samples of Agave sap (aguamiel) the following yeast strains were isolated: Candida lusitaneae (1), Kluyveromyces marxianus var. bulgaricus (2), and Saccharomyces cerevisiae (capensis) (1). Additionally, in seven samples of pulque (the fermented product), the species C. valida (six strains), S. cerevisiae (chevalieri) (4), S. cerevisiae (capensis) (1), and K. marxianus var. lactis (1) were found. The killer strains were C. valida and K. marxianus var. lactis from pulque and K. marxianus var. bulgaricus from aguamiel. One strain of S. cerevisiae (chevalieri) isolated from pulque which did not show killer activity was, on the other hand, resistant to other killer strains and it had a remarkable ethanol tolerance, suggesting that this strain could be used for alcohol production.     

Induction of alkane-oxidizing andα-olefin-epoxidizing enzymes by a non-hydrocarbon in aPseudomonas

A strain ofPseudomonas aeruginosa could be induced to oxidizen-paraffins and to epoxidize-olefins by treating peptone-grown cells with 1,6-hexanediol or by growing them on this substrate. Of some related alcohols and acids investigated, only a few showed weak inducing capacities.Shell Research N.V.     

The use of killer biotyping in an ecological survey of yeast in an old patagonian winery

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been made in the same winery on two consecutive years (1993 and 1994) with Merlot type musts, and with Malbec type must on a third year (1998). Saccharomyces cerevisiae strains associated with winery surfaces were also analysed. Differential killer sensitivity patterns related to a killer reference panel of 10 killer yeasts belonging to nine species of four genera were used as a quick and simple procedure to discriminate between indigenous S. cerevisiae isolates at the strain level. Although a great diversity of wild strains was observed, two main indigenous S. cerevisiae strains, designated as S. cerevisiae 9 and S. cerevisiae 13, took over the Merlot type fermentation in both years. These strains also appeared in Malbec must fermentation during the year 1998 and they were again found on the winery surface the next year. These results show that some few and stable indigenous S. cerevisiae strains remained in the environmental winery over the considered period of time (1993–1999) and they represent an additional evidence of the taking over of musts by local strains of S. cerevisiae.     

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.     

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.     

Patagonian wines: the selection of an indigenous yeast starter

The use of selected yeasts for winemaking has clear advantages over the traditional spontaneous fermentation. The aim of this study was to select an indigenous Saccharomyces cerevisiae yeast isolate in order to develop a regional North Patagonian red wine starter culture. A two-step selection protocol developed according to physiological, technological and ecological criteria based on killer interactions was used. Following this methodology, S. cerevisiae isolate MMf9 was selected among 32 indigenous yeasts previously characterized as belonging to different strains according to molecular patterns and killer biotype. This isolate showed interesting technological and qualitative features including high fermentative power and low volatile acidity production, low foam and low sulphide production, as well as relevant ecological characteristics such as resistance to all indigenous and commercial S. cerevisiae killer strains assayed. Red wines with differential volatile profiles and interesting enological features were obtained at laboratory scale by using this selected indigenous strain.     

Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae

An efficient yeast gene expression system with GAL10 promoter that does not require galactose as an inducer was developed using Δgal80 mutant strain of Saccharomyces cerevisiae. We constructed several combinations of gal mutations (Δgal1, Δgal80, Δmig1, Δmig2, and Δgal6) of S. cerevisiae and tested for their effect on efficiency of recombinant protein production by GAL10 promoter using a lipase, Candida antarctica lipase B (CalB), as a reporter. While the use of Δgal1 mutant strain required the addition of a certain amount of galactose to the medium, Δgal80 mutant strain did not require galactose. Furthermore, it was found that the recombinant CalB could be produced more efficiently (1.6-fold at 5 L-scale fermentation) in Δgal80 mutant strain than in the Δgal1 mutant. The Δgal80 mutant strain showed glucose repressible mode of expression of GAL10 promoter. Using Δgal80 mutant strain of S. cerevisiae, CalB was efficiently produced in a glucose-only fermentation at volumes up to 500 L.