Showing papers on "Yeast published in 2004"

Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass.

Abstract: An overview of the different inhibitors formed by pre-treatment of lignocellulosic materials and their inhibition of ethanol production in yeast and bacteria is given. Different high temperature physical pre-treatment methods are available to render the carbohydrates in lignocellulose accessible for ethanol fermentation. The resulting hydrolyzsates contain substances inhibitory to fermentation—depending on both the raw material (biomass) and the pre-treatment applied. An overview of the inhibitory effect on ethanol production by yeast and bacteria is presented. Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed. The furans and phenols generally inhibited growth and ethanol production rate (QEtOH) but not the ethanol yields (YEtOH) in Saccharomyces cerevisiae. Within the same phenol functional group (aldehyde, ketone, and acid) the inhibition of volumetric ethanol productivity was found to depend on the amount of methoxyl substituents and hence hydrophobicity (log P). Many pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but detoxification by overliming is needed. Thermoanaerobacter mathranii A3M3 can grow on pentoses and produce ethanol in hydrolysate without any need for detoxification.

Chronological aging leads to apoptosis in yeast

Abstract: During the past years, yeast has been successfully established as a model to study mechanisms of apoptotic regulation. However, the beneficial effects of such a cell suicide program for a unicellular organism remained obscure. Here, we demonstrate that chronologically aged yeast cultures die exhibiting typical markers of apoptosis, accumulate oxygen radicals, and show caspase activation. Age-induced cell death is strongly delayed by overexpressing YAP1, a key transcriptional regulator in oxygen stress response. Disruption of apoptosis through deletion of yeast caspase YCA1 initially results in better survival of aged cultures. However, surviving cells lose the ability of regrowth, indicating that predamaged cells accumulate in the absence of apoptotic cell removal. Moreover, wild-type cells outlast yca1 disruptants in direct competition assays during long-term aging. We suggest that apoptosis in yeast confers a selective advantage for this unicellular organism, and demonstrate that old yeast cells release substances into the medium that stimulate survival of the clone.

Metabolic engineering for improved fermentation of pentoses by yeasts

Abstract: The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g−1 sugar consumed, so commercialization seems feasible for some applications.

Protein-only transmission of three yeast prion strains

Abstract: Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein1,2,3. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae4,5. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as ‘seeds’, elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle

Abstract: When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h−1. After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h−1, respectively. The anaerobic ethanol yield was 0.42 g ethanol · g xylose−1 and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.

An AIF orthologue regulates apoptosis in yeast

Abstract: Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).

Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme.

Abstract: A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.

Yeast cell-surface display—applications of molecular display

Abstract: In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.

Rapid isolation of yeast genomic DNA: Bust n' Grab

Abstract: Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae

Abstract: Objectives: The aim of this study was to investigate the mechanism of action of tea tree oil and its com- ponents against Candida albicans, Candida glabrata and Saccharomyces cerevisiae. Methods: Yeast cells were treated with tea tree oil or components, at one or more concentrations, for up to 6 h. During this time, alterations in permeability were assessed by measuring the leakage of 260 nm absorbing materials and by the uptake of Methylene Blue dye. Membrane fluidity was measured by 1,6-diphenyl-1,3,5- hexatriene fluorescence. The effects of tea tree oil on glucose-induced medium acidification were quantified by measuring the pH of cell suspensions in the presence of both tea tree oil and glucose. Results: The treatment of C. albicans with tea tree oil and components at concentrations of between 0.25 and 1.0% (v/v) altered both permeability and membrane fluidity. Membrane fluidity was also increased when C. albicans was cultured for 24 h with 0.016%-0.06% (v/v) tea tree oil, as compared with control cells. For all three organisms, glucose-induced acidification of the external medium was inhibited in a dose-dependent manner in the presence of 0.2%, 0.3% and 0.4% tea tree oil. Conclusions: Data from this study support the hypothesis that tea tree oil and components exert their anti- fungal actions by altering membrane properties and compromising membrane-associated functions.

Direct Production of Ethanol from Raw Corn Starch via Fermentation by Use of a Novel Surface-Engineered Yeast Strain Codisplaying Glucoamylase and α-Amylase

Abstract: Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

Abstract: Aims: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. Methods and Results: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0·2 and 6·7 g l−1) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. Conclusions: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. Significance and Impact of the Study: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.

Ferulic acid release and 4-vinylguaiacol formation during brewing and fermentation: indications for feruloyl esterase activity in Saccharomyces cerevisiae.

Abstract: The release of ferulic acid and the subsequent thermal or enzymatic decarboxylation to 4-vinylguaiacol are inherent to the beer production process. Phenolic, medicinal, or clove-like flavors originating from 4-vinylguaiacol frequently occur in beer made with wheat or wheat malt. To evaluate the release of ferulic acid and the transformation to 4-vinylguaiacol, beer was brewed with different proportions of barley malt, wheat, and wheat malt. Ferulic acid as well as 4-vinylguaiacol levels were determined by HPLC at several stages of the beer production process. During brewing, ferulic acid was released at the initial mashing phase, whereas moderate levels of 4-vinylguaiacol were formed by wort boiling. Higher levels of the phenolic flavor compound were produced during fermentations with brewery yeast strains of the Pof(+) phenotype. In beer made with barley malt, ferulic acid was mainly released during the brewing process. Conversely, 60-90% of ferulic acid in wheat or wheat malt beer was hydrolyzed during fermentation, causing higher 4-vinylguaiacol levels in these beers. As cereal enzymes are most likely inactivated during wort boiling, the additional release of ferulic acid during fermentation suggests the activity of feruloyl esterases produced by brewer's yeast.

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered Saccharomyces yeast capable of cofermenting glucose and xylose.

Abstract: Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

Abstract: The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction.

Specific and differential inhibition of very-long-chain fatty acid elongases from Arabidopsis thaliana by different herbicides

Abstract: In higher plants, very-long-chain fatty acids (VLCFAs) are the main constituents of hydrophobic polymers that prevent dessication at the leaf surface and provide stability to pollen grains. Of the 21 genes encoding VLCFA elongases (VLCFAEs) from Arabidopsis thaliana, 17 were expressed heterologously in Saccharomyces cerevisiae. Six VLCFAEs, including three known elongases (FAE1, KCS1, and KCS2) and three previously uncharacterized gene products (encoded by At5g43760, At1g04220, and At1g25450) were found to be enzymatically active with endogenous yeast fatty acid substrates and to some extent with externally supplied unsaturated substrates. The spectrum of VLCFAs accumulated in expressing yeast strains was determined by gas chromatography/mass spectrometry. Marked specificity was found among elongases tested with respect to their elongation products, which encompassed saturated and monounsaturated fatty acids 20–30 carbon atoms in length. The active VLCFAEs revealed highly distinct patterns of differential sensitivity to oxyacetamides, chloroacetanilides, and other compounds tested, whereas yeast endogenous VLCFA production, which involves its unrelated elongase (ELO) in sphingolipid synthesis, was unaffected. Several compounds inhibited more than one VLCFAE, and some inhibited all six active enzymes. These findings pinpoint VLCFAEs as the target of the widely used K3 class herbicides, which have been in commercial use for 50 years, provide important clues as to why spontaneous resistance to this class is rare, and point to complex patterns of substrate specificity and product spectrum among members of the Arabidopsis VLCFAE family.

Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast

Abstract: We have developed recombinant Saccharomyces yeasts that can effectively co-ferment glucose and xylose to ethanol. However, these yeasts still ferment glucose more efficiently than xylose. The transport of xylose could be one of the steps limiting the fermentation of xylose. In this study, we characterized the changes in the expression pattern of the hexose transporter and related genes during co-fermentation of glucose and xylose using one of our recombinant yeasts, Saccharomyces cerevisiae 424A(LNH-ST). The transcription of the hexose transporter and related genes was strongly influenced by the presence of glucose; HXT1, HXT2 and HXT3 were greatly activated by glucose and HXT5, HXT7 and AGT1 were significantly repressed by glucose. We also examined the effectiveness of individual transporters encoded by HXT1, HXT2, HXT4, HXT5, HXT7 and GAL2 genes for transporting xylose during co-fermentation of glucose and xylose in a Saccharomyces hxt degrees mutant (RE700A). We compared these hxt degrees derivatives to RE700A wild-type strain (S. cerevisiae MC996A) where all of them contained the same xylose metabolizing genes present in our xylose-fermenting yeasts such as 424A(LNH-ST). Our results showed that recombinant RE700A containing the cloned HXT7 or HXT5 were substantially more effective for fermenting xylose to ethanol. In addition, we found that the efficiency of transporters for intracellular accumulation of xylose was as follows: HXT7 > HXT5 > GAL2 > WT > HXT1 > HXT4 > > > RE700A. Furthermore, we provided evidence that the Saccharomyces galactose transporter system could be a highly effective xylose transporter. The information reported here should be of great importance for improving the Saccharomyces yeast transport of xylose.

A Novel Family of Cys-Rich Membrane Proteins Mediates Cadmium Resistance in Arabidopsis

Abstract: Cadmium (Cd) is a widespread pollutant that is toxic to plant growth. However, only a few genes that contribute to Cd resistance in plants have been identified. To identify additional Cd(II) resistance genes, we screened an Arabidopsis cDNA library using a yeast (Saccharomyces cerevisiae) expression system employing the Cd(II)-sensitive yeast mutant ycf1. This screening process yielded a small Cys-rich membrane protein (Arabidopsis plant cadmium resistance, AtPcrs). Database searches revealed that there are nine close homologs in Arabidopsis. Homologs were also found in other plants. Four of the five homologs that were tested also increased resistance to Cd(II) when expressed in ycf1. AtPcr1 localizes at the plasma membrane in both yeast and Arabidopsis. Arabidopsis plants overexpressing AtPcr1 exhibited increased Cd(II) resistance, whereas antisense plants that showed reduced AtPcr1 expression were more sensitive to Cd(II). AtPcr1 overexpression reduced Cd uptake by yeast cells and also reduced the Cd contents of both yeast and Arabidopsis protoplasts treated with Cd. Thus, it appears that the Pcr family members may play an important role in the Cd resistance of plants.

Systematic investigation of Saccharomyces cerevisiae enzymes catalyzing carbonyl reductions.

Abstract: Eighteen key reductases from baker's yeast (Saccharomyces cerevisiae) have been overproduced in Escherichia coli as glutathione S-transferase fusion proteins. A representative set of α- and β-keto esters was tested as substrates (11 total) for each purified fusion protein. The stereoselectivities of β-keto ester reductions depended both on the identity of the enzyme and the substrate structure, and some reductases yielded both l- and d-alcohols with high stereoselectivities. While α-keto esters were generally reduced with lower enantioselectivities, it was possible in all but one case to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. Taken together, the results demonstrate not only that individual yeast reductases can be used to supply important chiral building blocks, but that GST-fusion proteins allow rapid identification of synthetically useful biocatalysts (along with their corresponding genes).