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PubMed1999

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface.

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and alpha-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha factor, respectively, were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and alpha-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and alpha-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase.

Agglutinins↗
PubMed2000

Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development.

Diploid strains of the budding yeast Saccharomyces cerevisiae change the pattern of cell division from bipolar to unipolar when switching growth from the unicellular yeast form (YF) to filamentous, pseudohyphal (PH) cells in response to nitrogen starvation. The functions of two transmembrane proteins, Bud8p and Bud9p, in regulating YF and PH cell polarity were investigated. Bud8p is highly concentrated at the distal pole of both YF and PH cells, where it directs initiation of cell division. Asymmetric localization of Bud8p is independent of the Rsr1p/Bud1p GTPase. rsr1/bud1 mutations are epistatic to bud8 mutations, placing Rsr1p/Bud1p downstream of Bud8p. In YF cells, Bud9p is also localized at the distal pole, yet deletion of BUD9 favours distal bud initiation. In PH cells, nutritional starvation for nitrogen efficiently prevents distal localization of Bud9p. Because Bud8p and Bud9p proteins associate in vivo, we propose Bud8p as a landmark for bud initiation at the distal cell pole, where Bud9p acts as inhibitor. In response to nitrogen starvation, asymmetric localization of Bud9p is averted, favouring Bud8p-mediated cell division at the distal pole.

Cell Polarity↗
PubMed2005

Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose.

A decreased fermentation rate due to inhibition is a significant problem for economic conversion of acid-pretreated lignocellulose hydrolysates to ethanol, since the inhibition gives rise to a requirement for separate detoxification steps. Together with acetic acid, the sugar degradation products furfural and 5-hydroxymethyl furfural are the inhibiting compounds found at the highest concentrations in hydrolysates. These aldehydes have been shown to affect both the specific growth rate and the rate of fermentation by yeast. Two strains of Saccharomyces cerevisiae with different abilities to ferment inhibiting hydrolysates were evaluated in fermentations of a dilute acid hydrolysate from spruce, and the reducing activities for furfural and 5-hydroxymethyl furfural were determined. Crude cell extracts of a hydrolysate-tolerant strain (TMB3000) converted both furfural and 5-hydroxymethyl furfural to the corresponding alcohol at a rate that was severalfold higher than the rate observed for cell extracts of a less tolerant strain (CBS 8066), thereby confirming that there is a correlation between the fermentation rate in a lignocellulosic hydrolysate and the bioconversion capacity of a strain. The in vitro NADH-dependent furfural reduction capacity of TMB3000 was three times higher than that of CBS 8066 (1,200 mU/mg protein and 370 mU/mg protein, respectively) in fed-batch experiments. Furthermore, the inhibitor-tolerant strain TMB3000 displayed a previously unknown NADH-dependent reducing activity for 5-hydroxymethyl furfural (400 mU/mg protein during fed-batch fermentation of hydrolysates). No corresponding activity was found in strain CBS 8066 (<2 mU/mg). The ability to reduce 5-hydroxymethyl furfural is an important characteristic for the development of yeast strains with increased tolerance to lignocellulosic hydrolysates.

Cellulose↗
PubMed2005

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteins in vivo.

All organisms contain thioredoxin (TRX), a regulatory thiol:disulfide protein that reduces disulfide bonds in target proteins. Unlike animals and yeast, plants contain numerous TRXs for which no function has been assigned in vivo. Recent in vitro proteomic approaches have opened the way to the identification of >100 TRX putative targets, but of which none of the numerous plant TRXs can be specifically associated. In contrast, in vivo methodologies, including classical yeast two-hybrid (Y2H) systems, failed to reveal the expected high number of TRX targets. Here, we developed a yeast strain named CY306 designed to identify TRX targets in vivo by a Y2H approach. CY306 contains a GAL4 reporter system but also carries deletions of endogenous genes encoding cytosolic TRXs (TRX1 and TRX2) that presumably compete with TRXs introduced as bait. We demonstrate here that, in the CY306 strain, yeast TRX1 and TRX2, as well as Arabidopsis TRX introduced as bait, interact with known TRX targets or putative partners such as yeast peroxiredoxins AHP1 and TSA1, whereas the same interactions cannot be detected in classical Y2H strains. Thanks to CY306, we also show that TRXs interact with the phosphoadenosine-5-phosphosulfate (PAPS) reductase MET16 through a conserved cysteine. Moreover, interactions visualized in CY306 are highly specific depending on the TRX and targets tested. CY306 constitutes a relevant genetic system to explore the TRX interactome in vivo and with high specificity, and opens new perspectives in the search for new TRX-interacting proteins by Y2H library screening in organisms with multiple TRXs.

Phenotype↗
PubMed2004

Identification of sumoylated proteins by systematic immunoprecipitation of the budding yeast proteome.

The identification of post-translational modifications to proteins is critical for understanding many important aspects of biology. Utilizing a collection of epitope-tagged yeast strains, we developed a novel approach to determine which proteins are modified by the small ubiquitin-related modifier (SUMO). We crossed traits useful for the detection of SUMO conjugation into 4246 tandem affinity purification-tagged strains and successfully immunoprecipitated and screened 2893 of these proteins for association with SUMO ( approximately 70% of the expressed proteome detectable by immunoblot analysis). We found 82 proteins associated with SUMO, including many of low abundance. Because our screen was performed under non-denaturing conditions, we were able to identify multiple members of four complexes that were associated with SUMO: the RSC chromatin remodeling complex, the mediator complex, the TFIID complex, and the septin complex. In addition, we describe five new direct conjugates of SUMO, and we mutated SUMO conjugation sites in four proteins. This is the first attempt to immunoprecipitate a large fraction of the proteome of a eukaryote, and it demonstrates the utility of this method to identify post-translational modifications in the yeast proteome.

Cysteine Endopeptidases↗
PubMed2006

Xylitol dehydrogenase from Candida tropicalis: molecular cloning of the gene and structural analysis of the protein.

Yeasts can metabolize xylose by the action of two key enzymes: xylose reductase and xylitol dehydrogenase. In this work, we present data concerning the cloning of the XYL2 gene encoding xylitol dehydrogenase from the yeast Candida tropicalis. The gene is present as a single copy in the genome and is controlled at the transcriptional level by the presence of the inducer xylose. XYL2 was functionally tested by heterologous expression in Saccharomyces cerevisiae to develop a yeast strain capable of producing ethanol from xylose. Structural analysis of C. tropicalis xylitol dehydrogenase, Xyl2, suggests that it is a member of the medium-chain dehydrogenase (MDR) family. This is supported by the presence of the amino acid signature [GHE]xx[G]xxxxx[G]xx[V] in its primary sequence and a typical alcohol dehydrogenase Rossmann fold pattern composed by NAD(+) and zinc ion binding domains.

Amino Acid Sequence↗
PubMed2002

Identifying regulators of pheromone signalling in the fission yeast Schizosaccharomyces pombe.

The rate and extent of a cell's response to an extracellular stimulus is influenced by regulators that act on the intracellular signalling machinery. Although not directly involved in propagating the intracellular signal, regulators control the activity of the proteins that transmit the signals. To understand this aspect of cell signalling, we studied the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, a relatively simple signalling system in a genetically tractable organism. Here, we describe the development of yeast strains containing ura4 and lacZ reporter genes under the control of the pheromone-regulated sxa2 promoter and the use of these strains to isolate mutants defective in their ability to regulate signalling. Several different types of mutant were identified. Some mutants were defective in proteins already known to regulate the pheromone-signalling pathway (Rgs1, Map1, Map2). Our approach also identified the MAP kinase phosphatase Pmp1 as a regulator of the pheromone-response pathway. Although previously shown to regulate other MAP kinase pathways in Sz. pombe, this is the first demonstration of a role for Pmp1 in pheromone signalling.

Cell Cycle↗
PubMed1984

Regulated expression of a human interferon gene in yeast: control by phosphate concentration or temperature.

The promoter/regulator region from the yeast repressible acid phosphatase gene was used to construct a vector for the regulated expression of cloned genes in yeast. The gene for human leukocyte interferon was inserted into this vector. Yeast cells transformed with the resulting plasmid produced significant amounts of interferon only when grown in medium lacking inorganic phosphate. Mutants in two acid phosphatase regulatory genes (coding for a defective repressor and a temperature-sensitive positive regulator) were used to develop a yeast strain that grew well at a high temperature (35 degrees C) but produced interferon only at a low temperature (23 degrees C), independent of phosphate concentration.

Acid Phosphatase↗
PubMedDate not supplied

[Increase in the yield of recombinant proteins in Saccharomyces cerevisiae yeasts as a result of optimizing their culture conditions].

The optimal one-stage scheme of the cultivation of the recombinant yeast strains was developed. In these strains, the expression of the foreign gene is induced at the medium depletion by phosphorus. The scheme was tested for the producers of human interleukin-2 and bovine gamma-interferon. In all the strains, the yield of cell biomass and the production of the foreign proteins increased as compared to the standard two-stage cultivation. The use of the developed scheme resulted in the threefold increase in the yield of the target proteins and in the productivity of the biotechnological process.

Animals↗
PubMed2006

Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors.

One major barrier to the economic conversion of biomass to ethanol is inhibitory compounds generated during biomass pretreatment using dilute acid hydrolysis. Major inhibitors such as furfural and 5-hydroxymethylfurfural (HMF) inhibit yeast growth and subsequent fermentation. The ethanologenic yeast Saccharomyces cerevisiae demonstrated a dose-dependant inhibition by the inhibitors and has the potential to transform furfural and HMF into less toxic compounds of furfuryl alcohol and 2,5-bis-hydroxymethylfuran (also termed as furan-2,5-dimethanol (FDM)), respectively. For a sustainable and cost-competitive biomass-to-ethanol industry, it is important to develop more tolerant yeast strains that can, in situ, detoxify the inhibitors and produce ethanol. This study summarizes current knowledge and our understanding of the inhibitors furfural and HMF and discusses metabolic conversion pathways of the inhibitors and the yeast genomic expression response to inhibitor stress. Unlike laboratory strains, gene expression response of the ethanologenic yeast to furfural and HMF was not transient, but a continued dynamic process involving multiple genes at the genome level. This suggests that during the lag phase, ethanologenic yeasts undergo a genomic adaptation process in response to the inhibitors. The findings to date provide a strong foundation for future studies on genomic adaptation and manipulation of yeast to aid more robust strain design and development.

Antifungal Agents↗
PubMed1998

Development of a yeast trihybrid screen using stable yeast strains and regulated protein expression.

We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling.

Clone Cells↗
PubMed2000

A baker's yeast mutant (fil1) with a specific, partially inactivating mutation in adenylate cyclase maintains a high stress resistance during active fermentation and growth.

The initiation of fermentation in the yeast Saccharomyces cerevisiae is associated with a rapid drop in stress resistance. This is disadvantageous for several biotechnological applications, e.g. the preparation of freeze doughs. We have isolated mutants in a laboratory strain which are deficient in fermentation-induced loss of stress resistance ('fil' mutants) using a heat shock selection protocol. We show that the fil1 mutant contains a mutation in the CYR1 gene which encodes adenylate cyclase. It causes a change at position 1682 of glutamate into lysine and results in a tenfold drop in adenylate cyclase activity. The fil1 mutant displays a reduction in the glucose-induced cAMP increase, trehalase activation and loss of heat resistance. Interestingly, the fil1 mutant shows the same growth and fermentation rate as the wild type strain, as opposed to other mutants with reduced activity of the cAMP pathway. Introduction of the fil1 mutation in the vigorous Y55 strain and cultivation of the mutant under pilot scale conditions resulted in a yeast that displayed a higher freeze and drought resistance during active fermentation compared to the wild type Y55 strain. These results show that high stress resistance and high fermentation activity are compatible biological properties. Isolation of fil-type mutations appears a promising avenue for development of industrial yeast strains with improved stress resistance during active fermentation.

Adenylyl Cyclases↗
PubMed1997

Development of pseudohyphae by embedded haploid and diploid yeast.

Diploid strains of S. cerevisiae are known to develop pseudohyphae in response to starvation for nitrogen. We report that both haploid and diploid yeast grow in a filamentous form when embedded in solid media. This is not a response to starvation, since yeast grown on rich media and overlaid with rich agar grow within the agar as pseudohyphae. While we find that the only element of diploidy required for formation of pseudohyphae in response to nitrogen starvation is the a1/alpha 2 repressor, pseudohyphal development by embedded cells does not require a1/alpha 2. Deletion of BUD 5 prevented the formation of pseudohyphae by embedded cells, suggesting that these structures are the result of ordered filament formation rather than agar penetration. Deletion of STE 12 prevented the formation of pseudohyphae by all cell types, showing that the same signal transduction pathway is used by embedded cells as by those responding to nitrogen starvation. Different cell types of yeast thus form filaments in response to several kinds of environmental stimuli.

Diploidy↗
PubMed2003

Constitutive and UV-inducible synthesis of photoprotective compounds (carotenoids and mycosporines) by freshwater yeasts.

Twelve yeasts isolated from lakes of Northwestern Patagonia, Argentina, belonging to eight genera (Sporobolomyces, Sporidiobolus, Rhodotorula, Rhodosporidium, Cystofilobasidium, Cryptococcus, Torulaspora, and Candida) were analysed for their ability to produce photoprotective compounds. For this purpose, three laboratory experiments were performed to study the effect of photosynthetically active radiation (PAR) and PAR in combination with UV radiation (PAR + UVR) on the production of carotenoids and mycosporines. The synthesis of carotenoid compounds was clearly stimulated in six out of nine red yeast strains tested upon exposure to PAR or PAR + UVR; however, the latter conditions produced a stronger response than PAR alone. The increase in carotenoids in the red strains under PAR + UVR irradiation showed a negative exponential relationship with their basal carotenoid content, suggesting that cells with higher constitutive levels of carotenoids are less responsive to induction by PAR + UVR. Three red yeasts, Rhodotorula minuta, Rh. pinicola, and Rhodotorula sp., and the colourless Cryptococcus laurentii produced a UV-absorbing compound when exposed to PAR or PAR + UVR. This compound showed an absorption maximum at 309-310 nm and was identified as mycosporine-glutaminol-glucoside (myc-glu-glu). In these strains, exposure to PAR or PAR + UVR resulted in elevated concentrations of both carotenoids and myc-glu-glu. This is the first report on the production of mycosporines by yeasts. All strains that developed under PAR + UVR were able to synthesise carotenoids either constitutively or in response to PAR exposure, and a few of them also produced myc-glu-glu when exposed to PAR. Collectively, our results suggest that the presence of carotenoids, either alone or in combination with mycosporines, are required for sustaining growth under exposure to PAR + UVR in the freshwater yeast strains studied.

Carotenoids↗
PubMed2000

Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking.

Yeasts are predominant in the ancient and complex process of winemaking. In spontaneous fermentations, there is a progressive growth pattern of indigenous yeasts, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the 'wine yeast' and is widely preferred for initiating wine fermentations. The primary role of wine yeast is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. However, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialized wine yeast strains possessing a wide range of optimized, improved or novel oenological properties. This review highlights the wealth of untapped indigenous yeasts with oenological potential, the complexity of wine yeasts' genetic features and the genetic techniques often used in strain development. The current status of genetically improved wine yeasts and potential targets for further strain development are outlined. In light of the limited knowledge of industrial wine yeasts' complex genomes and the daunting challenges to comply with strict statutory regulations and consumer demands regarding the future use of genetically modified strains, this review cautions against unrealistic expectations over the short term. However, the staggering potential advantages of improved wine yeasts to both the winemaker and consumer in the third millennium are pointed out.

Consumer Behavior↗
PubMed2002

Protein expression and secretion in the yeast Yarrowia lipolytica.

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.

Bacterial Proteins↗
PubMedDate not supplied

[The antibiotic properties of macrocyclic trichothecene mycotoxins].

Some trichotecenic mycotoxins (verrucarine A, roridines A and H, T-2-toxin) have been studied for their antibiotic effect on a wide spectrum of the yeast cultures (761 strains). The studied substances differ both in their activity and the action character. The yeast strains promising for development of microbiological methods of indication and detoxification of mycotoxins have been revealed.

Antifungal Agents↗