Antifungal cellobiose lipid secreted by the epiphytic yeast <Emphasis Type="Italic">Pseudozyma graminicola</Emphasis>

The yeast Pseudozyma graminicola isolated from plants inhibited growth of almost all ascomycetes and basidiomycetes tested (over 270 species of ca. 100 genera) including pathogenic species. This yeast secreted a fungicidal agent, which was identified as a glycolipid composed of cellobiose residue with two O-substituents (acetyl and 3-hydroxycaproic acid) and 2,15,16-trihydroxypalmitic acid. The release of ATP from the glycolipid-treated cells indicated that this glycolipid impaired the permeability of the cytoplasmic membrane. Basidiomycetes were more sensitive to the cellobiose lipid than ascomycetes.     

Fungicidal activity of cellobiose lipids from cultural fluid of yeast Cryptococcus humicola and Pseudozyma fusiformata

Cellobiose lipids of yeast fungi Cryptococcus humicola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

Production of Antifungal Cellobiose Lipids by <Emphasis Type="Italic">Trichosporon porosum</Emphasis>

The yeast Trichosporon porosum suppresses growth of ascomycetes and basidiomycetes belonging to 52 genera. It is due to secretion of a thermostable fungicidal agent. The suppression was maximal at pH 3.5–4.0. Fungicidal preparation obtained from the culture broth was shown to be a mixture of cellobiosides of dihydrodecane acid with different degree of acetylation of cellobiose residue. The preparation caused the death of Candida albicans and Filobasidiella neoformans cells in the concentrations of 0.2 and 0.03 mM, respectively.     

Candida queiroziae sp. nov., a cellobiose-fermenting yeast species isolated from rotting wood in Atlantic Rain Forest

Eight strains of a novel yeast species were isolated from rotting wood and wood-boring insects in Atlantic Rain Forest ecosystems in Brazil. Sequences of the D1/D2 domains of the large subunit of the rRNA gene showed that the yeast belongs to the Scheffersomyces clade and that it is related to Candida lignicola and Candida coipomoensis. The new species was isolated from rotting wood of three different localities and a wood-boring insect suggesting that these substrates are its ecological niche. This new yeast species is able to assimilate cellobiose and other compounds related to rotting wood. Strong fermentation of cellobiose in Durham tubes was observed for the strains of this new yeast. The new species produced an intracellular β-glucosidase responsible for cellobiose hydrolysis. The novel species, Candida queiroziae sp. nov., is proposed to accommodate these isolates. The type strain of C. queiroziae is UFMG-CLM 5.1(T) (=CBS 11853(T) = NRRL Y-48722(T)).     

Rapid differentiation of phenotypically similar yeast species by single-strand conformation polymorphism analysis of ribosomal DNA

Single-strand conformation polymorphism (SSCP) analysis of ribosomal DNA (rDNA) was investigated for rapid differentiation of phenotypically similar yeast species. Sensitive tests indicated that some yeast strains with one, most strains with two, and all strains with three or more nucleotide differences in the internal transcribed spacer 1 (ITS1) or ITS2 region could be distinguished by PCR SSCP analysis. The discriminative power of SSCP in yeast species differentiation was demonstrated by comparative studies of representative groups of yeast species from ascomycetes and basidiomycetes, including Saccharomyces species, medically important Candida species, and phylloplane basidiomycetous yeast species. Though the species within each group selected are closely related and have relatively similar rDNA sequences, they were clearly differentiated by PCR-SSCP analysis of the ITS1 region, given the amplified fragments were less than 350 bp in sizes. By using SSCP analysis for rapid screening of yeast strains with different rDNA sequences, species diversity existing in a large collection of yeast strains from natural sources was effectively and thoroughly investigated with substantially reduced time and cost in subsequent DNA sequencing.     

Yeast Diversity Associated with Invasive Dendroctonus valens Killing Pinus tabuliformis in China Using Culturing and Molecular Methods

Bark beetle-associated yeasts are much less studied than filamentous fungi, yet they are also considered to play important roles in beetle nutrition, detoxification, and chemical communication. The red turpentine beetle, Dendroctonus valens, an invasive bark beetle introduced from North America, became one of the most destructive pests in China, having killed more than 10 million Pinus tabuliformis as well as other pine species. No investigation of yeasts associated with this bark beetle in its invaded ranges has been conducted so far. The aim of this study was to assess the diversity of yeast communities in different microhabitats and during different developmental stages of Den. valens in China using culturing and denaturing gradient gel electrophoresis (DGGE) approaches and to compare the yeast flora between China and the USA. The yeast identity was confirmed by sequencing the D1/D2 domain of LSU ribosomal DNA (rDNA). In total, 21 species (13 ascomycetes and eight basidiomycetes) were detected by culturing method, and 12 species (11 ascomycetes and one basidiomycetes) were detected by molecular methods from China. The most frequent five species in China were Candida piceae (Ogataea clade), Cyberlindnera americana, Candida oregonensis (Metschnikowia clade), Candida nitratophila (Ogataea clade) and an undescribed Saccharomycopsis sp., detected by both methods. Seven species were exclusively detected by DGGE. Ca. oregonensis (Metschnikowia clade) was the most frequently detected species by DGGE method. Eight species (all were ascomycetes) from the USA were isolated; seven of those were also found in China. We found significant differences in yeast total abundance as well as community composition between different developmental stages and significant differences between the surface and the gut. The frass yeast community was more similar to that of Den. valens surface or larvae than to the community of the gut or adults. Possible functions of the yeast associates are discussed.     

Production of β-glucosidase,exo-β-l,4-glucanase and endo-³-l,4-glucanase by selected microscopic fungi

Production of beta-glucosidase, exo-beta-1,4-glucanase and endo-beta-1,4-glucanase was screened in 58 species of imperfect fungi, mucoral fungi and some ascomycetes. beta-Glucosidase activity was found in all of the tested microorganisms, exo-beta-1,4-glucanase activity in 23, and endo-beta-1,4=glucanase activity in 38 microorganisms. Growth on cellobiose was found in all tested microorganisms, growth on carboxymethylcellulose in in 38 tested strains.     

The fungicidal activity of an extracellular glycolipid from Sympodiomycopsis paphiopedili sugiyama et al

The yeast Sympodiomycopsis paphiopedili (Ustilaginomycetes) produces an extracellular glycolipid, which possesses the maximum antifungal activity at the pH of the medium equal to 4.0-4.5. Among the approximately 300 tested species of yeastlike and mycelial fungi, more than 80% (including species pathogenic for plants, animals, and humans) were found to be susceptible to this glycolipid.     

Fermentation and aerobic metabolism of cellodextrins by yeasts

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.     

Fermentation and aerobic metabolism of cellodextrins by yeasts.

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.     

Pichia norvegensis sp. nov.

A new yeast species Pichia norvegensis Leask et Yarrow is described as the perfect state of Candida norvegensis (Dietrichson) van Uden et Farinha ex van Uden et Buckley. Strains of this species were isolated on 3 occasions from human vaginas. This species differs from other Pichia species that assimilate glucose but not galactose, sucrose, maltose, lactose, D-mannitol and D-glucitol by assimilating cellobiose.     

Purification and characterization of mycoferritin from Aspergillus parasiticus (255)

As intracellular iron storage molecules, only hydroxymate type siderophores have been reported in ascomycetes and basidiomycetes. This is the first report documenting the presence of mycoferritin in ascomycetes. The fungus, Aspergillus parasiticus (255), is capable of producing mycoferritin only upon induction with iron in yeast extract sucrose (YES) medium. The same has been purified from Aspergillus sps by application of conventional biochemical techniques. The molecular mass, yield, iron and carbohydrate contents of the HPLC purified protein were 460kDa, 0.012mg/g of wet mycelia, 1.6% and 6.0%, respectively. The iron content was much lower than Mortierella alpina mycoferritin (17%). Native PAGE revealed the presence of trimeric and monomeric forms of ferritin. Subunit analysis by SDS-PAGE showed a single protein subunit of approximately 20kDa suggesting structural simplicity of the apoferritin shell. Variation in amino acid composition was noted upon comparison with ferritins of other species. Interestingly, no phenylalanine could be detected in the mycoferritin of Aspergillus sps. The acidic amino acid content was 1.5-1.6 fold higher than mammalian and fish ferritins. The spectral characteristics (UV/VIS and fluorescence) of mycoferritin were akin to equine spleen ferritin. However, circular dichroic spectra revealed a lower degree of helicity.     

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.     

Regulation of hydroxydocosanoic acid sophoroside production in Candida bogoriensis by the levels of glucose and yeast extract in the growth medium.

Cells of Candida bogoriensis produce as a major extracellular lipid 13-[(2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]docosanoic acid 6',6'-diacetate (Ac2Glc2HDA), the diacetylated sophoroside of 13-hydroxydocosanoic acid (HDA), along with mono- and unacetylated derivatives. The HDA glycolipid production is greater than 2 g/liter when cells are grown on a "standard" medium of 3% glucose and 0.15% yeast extract. Either lowering the glucose concentration (0.5 to 2.0% glucose, at 0.2% yeast extract) or raising the yeast extract concentration (2 to 4% yeast extract at 3% glucose) greatly decreased the yield of this glycolipid, as well as its rate of synthesis measured by [14C]acetate incorporation. Total HDA production was also depressed on the low glucose medium, as was the activity of UDP-glucose:HDA glucosyltransferase, the first enzyme involved in the synthesis of Ac2Glc2HDA from HDA. Levels of acetyl-CoA:Glc2HDA acetyltransferase were not decreased by growth on a low glucose medium, however, even under conditions in which glycolipid production was less than 4% of that found in the standard medium. Low levels of the HDA glycolipids were monitored by high pressure liquid chromatography of their p-bromophenacyl esters, formed by the action of alpha,beta-dibromoacetophenone on the sodium salt of the lipid in the presence of a crown reagent catalyst. This regulation of extracellular Ac2Glc2HDA production by the nutrient composition of the growth medium may represent an important property in the adaptation of C. bogoriensis to its natural environment, the phyllosphere.     

The fungicidal activity of an extracellular glycolipid from <Emphasis Type="Italic">Sympodiomycopsis paphiopedili</Emphasis> sugiyama <Emphasis Type="Italic">et al.</Emphasis>

The yeast Sympodiomycopsis paphiopedili (Ustilaginomycetes) produces an extracellular glycolipid, which possesses the maximum antifungal activity at a pH of the medium equal to 4.0–4.5. Among the approximately 300 tested species of yeastlike and mycelial fungi, more than 80% (including species pathogenic for plants, animals, and humans) were found to be sensitive to this glycolipid.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 841–845.Original Russian Text Copyright © 2004 by W. Golubev, T. Kulakovskaya, E. Kulakovskaya, N. Golubev.     

Characterisation of the diversity and physiology of cellobiose-fermenting yeasts isolated from rotting wood in Brazilian ecosystems

We investigated the yeast species associated with rotting wood samples obtained from Brazilian ecosystems, with a special focus on cellobiose-fermenting species. About 647 yeast strains were isolated from rotting wood samples collected from the areas of Atlantic rainforest, Cerrado, and Amazonian forest. Eighty-six known species and 47 novel species of yeasts were isolated. Candida boidinii, Cyberlindnera subsufficiens, Meyerozyma guilliermondii, Schwanniomyces polymorphus, Candida natalensis, and Debaryomyces hansenii were the most frequently isolated species. Among the cellobiose-fermenting yeasts, 14 known and three novel yeast species were identified. Scheffersomyces queiroziae, Sc. amazonensis, Yamadazyma sp.1, Hanseniaspora opuntiae, C. jaroonii, and Candida tammaniensis were the main ethanol-producing yeasts. These species also produced an intracellular β-glucosidase responsible for cellobiose hydrolysis. In fermentation assays using a culture medium containing 50 g L^?1 cellobiose, ethanol production was observed in all cases; Sc. queiroziae and Sc. amazonensis showed the highest yield, efficiency, and productivity. Candida jaroonii and Yamadazyma sp.1 strains also showed high efficiency in cellobiose fermentation, while C. tammaniensis and H. opuntiae strains produced low amounts of ethanol. This study shows the potential of rotting wood samples from Brazilian ecosystems as a source of yeasts, including new species as well as those with promising biotechnological properties.     

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae. These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.     

Glycolipids of the smut fungus Ustilago maydis from cultivation on renewable resources

When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l⁻¹ sunflower oil fatty acids (technical grade) a yield of 30 g l⁻¹ glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques. Received: 22 June 1998 / Received revision: 11 September 1998 / Accepted: 13 September 1998     

Fermentation of Cellodextrins by Different Yeast Strains

The fermentation of cellodextrins by eight yeast species capable of fermenting cellobiose was monitored. Only two of these species, Torulopsis molischiana and T. wickerhamii, were able to ferment β-glucosides with a degree of polymerization between one and six. These two species showed exocellular β-glucosidase activity. Four other species were able to ferment cellotriose, and the last two species only fermented cellobiose. These latter six species produced a β-glucosidase capable of attacking cellodextrins, but this enzyme was endocellular.