Chitinase Production by Conidiobolus lamprauges and Other Conidiobolus Species

A class of yeast variants appears after cultivation of a bottom-fermenting brewing yeast strain, IFO2003. Although IFO2003 fails to grow well above 33°C, the variants can grow up to 34°C. Temperature-resistance and an acquired phenotype of maltose poor-fermentation ability are strictly correlated in the bottom-fermenting brewing yeast, enabling us to develop easy estimation of the fermentation ability of the variants.     

Role of bottom-fermenting brewer's yeast KEX2 in high temperature resistance and poor proliferation at low temperatures

Variants of bottom-fermenting brewer's yeast that grew at high temperatures and showed poor proliferation and fermentation at low temperatures were isolated. Similar variants of laboratory yeast were also isolated and found to be incapable of mating. The KEX2 gene was cloned by complementation. It was shown to be responsible for these traits, because a KEX2 disruptant of Saccharomyces cerevisiae (S. cerevisiae) laboratory yeast grew poorly at low temperatures and was resistant to high temperatures. In addition, a Saccharomyces bayanus (S. bayanus)-type KEX2 (Sb-KEX2) disruptant of bottom-fermenting brewer's yeast grew poorly at low temperatures and was resistant to high temperatures. The KEX2 gene product plays an important role in proliferation of yeast at low temperatures, which is an important trait of bottom-fermenting brewer's yeast. These findings advance our understanding of the proliferation of yeast at low temperatures, especially that of bottom-fermenting brewer's yeast.     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

Antigenicity of Candida tropicalis strain cells cultured at 27 and 37 degrees C

All cells of four Candida tropicalis strains IFO 0199 (Ct-0199), IFO 0587 (Ct-0587), IFO 1400 (Ct-1400), and IFO 1647 (Ct-1647), obtained by cultivation at 27 and 37 degrees C for 48 h in yeast extract-added Sabouraud liquid medium, showed the shapes of typical budding yeast and the same agglutination patterns against factor sera 1, 4, 5 and 6 in the commercially available kit 'Candida Check'. The cells of the C. tropicalis IFO 0589 strain display the same properties at 27 degrees C but formed hyphae at 37 degrees C. The cell wall mannan (Ct-0589-37-M) obtained from the strain cells cultured at 37 degrees C had lost most of its reactivity against factor sera 4, 5 and 6 in an enzyme-linked immunosorbent assay, in contrast to the mannan (Ct-0589-27-M) at 27 degrees C. The 1H-nuclear magnetic resonance patterns of the mannans obtained from the cells of the four C. tropicalis strains IFO 0199, IFO 0587, IFO 1400, and IFO 1647, obtained by cultivation at 37 degrees C, did not change compared to those at 27 degrees C. By contrast, the Ct-0589-37-M had significantly lost the beta-1,2-linked mannopyranose units, corresponding to the serum factors 5 and 6. These results show that the IFO 0589 strain is an unusual strain among the general C. tropicalis strains studied.     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

Breeding of yeast strains able to grow at 42 degrees C

Saccharomyces cerevisiae strain 2-39/10A is able to ferment alcohol at 42 degrees C. The ability of various yeast strains, including 2-39/10A, to grow at high temperatures was compared. The strain 2-39/10A was able to grow at 42 degrees C and the high temperature growth was found to be governed by more than one gene. The yeast strains that can grow at 42 degrees C were bred by crossing the haploid strains, which are inherently unable to grow at high temperatures.     

Chimeric types of chromosome X in bottom-fermenting yeasts

Aim:  To determine the structure of the chimeric chromosome X of bottom-fermenting yeasts.
Methods and Results:  Eight cosmid clones carrying DNA from chromosome X of bottom-fermenting yeasts were selected by end-sequencing. Four of the cosmid clones had Saccharomyces cerevisiae (SC)-type and Saccharomyces bayanus (SB)-type chimeric ends, two had SC-type ends and two had SB-type ends. Sequencing revealed that the bottom-fermenting yeast strains in this study had four types of chromosome X: SC–SC, SC–SB, SB–SC and SB–SB. The translocation site in the chimeric chromosome is conserved among bottom-fermenting yeast strains, and was created by homologous recombination within a region of high sequence identity between the SC-type sequence and the SB-type sequence.
Conclusions:  Existing bottom-fermenting yeast strains share a common ancestor in which the chimeric chromosome X was generated.
Significance and Impact of the Study:  The knowledge gained in this study sheds light on the evolution of bottom-fermenting yeasts.     

Enhancing adhesion of yeast brewery strains to chamotte carriers through aminosilane surface modification

The adhesion of cells to solid supports is described as surface-dependent, being largely determined by the properties of the surface. In this study, ceramic surfaces modified using different organosilanes were tested for proadhesive properties using industrial brewery yeast strains in different physiological states. Eight brewing strains were tested: bottom-fermenting Saccharomyces pastorianus and top-fermenting Saccharomyces cerevisiae. To determine adhesion efficiency light microscopy, scanning electron microscopy and the fluorymetric method were used. Modification of chamotte carriers by 3-(3-anino-2-hydroxy-1-propoxy) propyldimethoxysilane and 3-(N, N-dimethyl-N-2-hydroxyethyl) ammonium propyldimethoxysilane groups increased their biomass load significantly.     

Isolation and characterization of brewer's yeast variants with improved fermentation performance under high-gravity conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22 degrees Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11 degrees C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

Growth under alkaline conditions of the salt-tolerant yeast Debaryomyces hansenii IFO10939

A salt-tolerant yeast Debaryomyces hansenii IFO 10939, which is able to grow at pH 10.0, was isolated and characterized. IFO 10939 had the ability of maintaining intracellular pH. The in vivo activation of plasma membrane ATPase was observed in cells grown at pH 6.2 during conditioning in buffer at pH 9.0. Alkalification of growth medium exhibited a significant increase in acetate and propionate production. The results suggested that the regulation of intracellular pH was involved in plasma membrane ATPase pumping protons out of the cells and weak acid formation for the source of the protons in cells growing at high pH. Received: 4 December 2001 / Accepted: 24 January 2002     

Induction of Glutathione Peroxidase by Linoleic Acid Hydroperoxide in Hansenula mrakii

The yeast Hansenula mrakii IFO 0895 could grow in a minimal medium containing 4 mm linoleic acid hydroperoxide, and the hydroperoxide moiety of linoleic acid hydroperoxide was reduced to the alcohol moiety. The activity of glutathione peroxidase was detected from the insoluble fractions of the cell homogenates of H. mrakii IFO 0895 only when the cells were grown in a linoleic acid hydroperoxide-containing medium.     

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.     

Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

酵母菌属间原生质体融合构建高温酵母菌株

酿酒酵母(Saccharomyces cerevisiae) A001和克鲁维酵母(Kluyveromyces sp.) Y034属间原生质体融合构建高温酵母菌株。对制备高再生活性原生质体及融合子细胞形态、生理化特征、同工酶性质、遗传稳定性和高温发酵等方面进行了研究。结果表明,融合子AY023和AY680遗传性能稳定,表达了双亲优良性状,获得了在45℃培养条件下产酒率7.4%的属间融合菌株,是目前已见文献报道的产酒率最高的高温(45℃)酵母菌株。     

Influence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast Rhodotorula aurantiaca A19, isolated from Antarctic ice, that the inability to grow at temperatures close to 20 degrees C is associated with profound alterations in cell morphology and integrity. High performance liquid chromatography analysis of the intracellular acyl-CoA esters revealed an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated close to the nonpermissive temperature. Its concentration (500 microm) was found to be 28-fold higher than in cells cultivated at 0 degrees C. If one considers its ability to disrupt membrane bilayers and to inhibit many cellular enzymes and functions, intracellular myristoyl-CoA accumulation in the psychrophile R. aurantiaca represents one of the principal causes of growth arrest at moderate temperatures. Intracellular acyl-CoA concentrations are believed to be regulated by thioesterase activity. Thus in an attempt to explore the mechanism by which temperature disrupts myristoyl-CoA metabolism, we isolated and characterized a long chain acyl-CoA thioesterase. The monomeric 80-kDa thioesterase from the psychrophilic yeast shows a very strong specificity for myristoyl-CoA. The affinity for substrate and the catalytic efficiency of the thioesterase are optimal below 5 degrees C (temperatures habitually experienced by the strain) and dramatically decrease with increasing temperature. The loss of affinity for substrate is related to the intracellular increase of myristoyl-CoA concentration. Our observations reveal one of the probable mechanisms by which temperature fixes the limit of growth for this psychrophilic yeast.     

Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.

Saccharomyces cerevisiae strains with a disrupted RAS1 gene and with an intact RAS2 gene (ras1- RAS2 strains) grew well on both fermentable and nonfermentable carbon sources. By constructing isogenic mutants having a disrupted RAS1 locus and a randomly mutagenized chromosomal RAS2 gene, we obtained yeast strains with specific growth defects. The strain TS1 was unable to grow on nonfermentable carbon sources and galactose at 37 degrees C, while it could grow on glucose at the same temperature. The mutated RAS2 gene in TS1 cells encoded a protein with the glycines at positions 82 and 84 replaced by serine and arginine respectively. Both mutations were necessary for temperature sensitivity. We also isolated a mutant yeast that was unable to grow on nonfermentable carbon sources both at 30 and 37 degrees C, while growing on glucose at both temperatures. This phenotype was caused by a single chromosomal mutation, leading to the replacement of aspartic acid 40 of the RAS2 protein by asparagine. A ras1- yeast strain with a chromosomal RAS2 gene harbouring the three mutations together did not grow at any temperature using non-fermentable carbon sources, but it was able to grow on glucose at 30 degrees C, and not at 37 degrees C. The mutated proteins were much less effective than the wild-type RAS2 protein in the stimulation of adenylate cyclase, but were efficiently expressed in vivo. The possible roles of residues 40, 82 and 84 of the RAS2 protein in the regulation of adenylate cyclase are discussed.     

Construction and analysis of recombinant glucanolytic brewer's yeast strains

Summary Four different types of endo--1,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the egl1 gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The egl1 gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or ADH1 locus of the yeast. Integration into the PGK1 or ADH1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the egl1 gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of -glucans in worts. Offprint requests to: M.-L. Suihko     

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: the flocculation as a separation process

In this work, a brewer's yeast strain was used to remove heavy metals from a synthetic effluent. The solid-liquid separation process was carried out using the flocculation ability of the strain. The yeast strain was able to sediment in the presence of Cu2+, Ni2+, Zn2+, Cd2+ and Cr3+, which evidences that the flocculation can be used as a cheap and natural separation process for an enlarged range of industrial effluents. For a biomass concentration higher than 0.5 g/l, more than 95% of the cells were settled after 5 min; this fact shows that the auto-aggregation of yeast biomass is a rapid and efficient separation process. Cells inactivated at 45 degrees C maintain the sedimentation characteristics, while cells inactivated at 80 degrees C lose partially (40%) the flocculation. The passage of metal-loaded effluent through a series of sequential batches allowed, after the second batch, the reduction of the Ni2+ concentration in solution for values below the legal limit of discharge of wastewater in natural waters (2mg/l); this procedure corresponds to a removal of 91%. A subsequent batch had a marginal effect on Ni2+ removal (96%). Together, the results obtained suggest that the use of brewing flocculent biomass looks a promising alternative in the bioremediation of metal-loaded industrial effluents since the removal of the heavy metals and cell separation are simultaneously achieved.     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.