| 1. |
- Sterky, Fredrik, et al.
(författare)
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The sequence of a 30 kb fragment on the left arm of chromosome XV from Saccharomyces cerevisiae reveals 15 open reading frames, five of which correspond to previously identified genes
- 1996
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Ingår i: Yeast. - 0749-503X .- 1097-0061. ; 12:10B, s. 1091-1095
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Tidskriftsartikel (refereegranskat)abstract
- We report the sequence of a 30 469 bp long DNA fragment on the left arm of chromosome XV of Saccharomyces cerevisiae. The fragment contains 15 open reading frames (ORFs) of al least 300 bp. Five previously sequenced yeast genes, PHO80, TIR2, SLG1, the gene encoding the subtilisin-like protease III precursor and the gene coding for ATP-dependent permease, are found among these ORFs. By DNA sequence comparison, two ORFs identified previously reported expressed sequence tags from yeast. Of the proteins encoded by the remaining eight ORFs, six show similarities to proteins from different organisms and two lack detectable similarity with any amino acid sequence described in public data banks. The DNA sequence has been deposited in GenBank under Accession Number U43491.
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| 2. |
- Astromskas, Eimantas, et al.
(författare)
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Tools and methods for genetic analysis of Saccharomyces castellii
- 2007
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 24:6, s. 499-509
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Tidskriftsartikel (refereegranskat)abstract
- The budding yeast species Saccharomyces castellii has provided important new insights into molecular evolution when incorporated in comparative genomics studies and studies of mitochondrial inheritage. Although it shows some diversity in the specific molecular details, several analyses have shown that it contains many genetic pathways similar to those of S. cerevisiae. Here we have investigated the possibility of performing genetic analyses in S. castellii. We optimized the LiAc transformation protocol to achieve 200-300 transformants/mu g plasmid DNA. We found that the commonly used plasmids for S. cerevisiae are stably maintained in S. castellii under selective conditions. Surprisingly, both 2 mu and CEN/ARS plasmids are kept at a high copy number. Moreover, the kanMX cassette can be used as a resistance marker against the selective drug geneticin (G418). Finally, we determined that the S. cerevisiae GAL] promoter can be used for the activation of transcription in S. castellii, thus enabling the controlled overexpression of genes when galactose is present in the medium. The availability of these tools provides the possibility or performing genetic analyses in S. castellii, and makes it a promising new model system in which hypotheses derived from bioinformatics studies can be experimentally tested. Copyright (C) 2007 John Wiley & Sons, Ltd.
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| 3. |
- Bendixsen, Devin P., et al.
(författare)
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Saccharomyces yeast hybrids on the rise
- 2022
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 39:1-2, s. 40-54
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Forskningsöversikt (refereegranskat)abstract
- Saccharomyces hybrid yeasts are receiving increasing attention as a powerful model system to understand adaptation to environmental stress and speciation mechanisms, using experimental evolution and omics techniques. We compiled all genomic resources available from public repositories of the eight recognized Saccharomyces species and their interspecific hybrids. We present the newest numbers on genomes sequenced, assemblies, annotations, and sequencing runs, and an updated species phylogeny using orthogroup inference. While genomic resources are highly skewed towards Saccharomyces cerevisiae, there is a noticeable movement to use wild, recently discovered yeast species in recent years. To illustrate the degree and potential causes of reproductive isolation, we reanalyzed published data on hybrid spore viabilities across the entire genus and tested for the role of genetic, geographic, and ecological divergence within and between species (28 cross types and 371 independent crosses). Hybrid viability generally decreased with parental genetic distance likely due to antirecombination and negative epistasis, but notable exceptions emphasize the importance of strain-specific structural variation and ploidy differences. Surprisingly, the viability of crosses within species varied widely, from near reproductive isolation to near-perfect viability. Geographic and ecological origins of the parents predicted cross viability to an extent, but with certain caveats. Finally, we highlight publication trends in the field and point out areas of special interest, where hybrid yeasts are particularly promising for innovation through research and development, and experimental evolution and fermentation.
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| 4. |
- Bengtsson, Oskar, et al.
(författare)
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Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.
- 2008
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:11, s. 835-847
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Tidskriftsartikel (refereegranskat)abstract
- Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.
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| 5. |
- Beskow, Anne, et al.
(författare)
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Comparative analysis of regulatory transcription factors in Schizosaccharomyces pombe and budding yeasts
- 2006
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 23:13, s. 929-935
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Tidskriftsartikel (refereegranskat)abstract
- Regulatory transcription factors (rTFs), which bind specific DNA sequences in the regulatory regions of genes and subsequently activate or repress transcription, play a central role in programming genomic expression. The number of rTFs in a species might therefore reflect its functional complexity. For simple organisms like yeast, a relatively small number of rTFs might be expected that is fairly constant between yeast species. We show that the budding yeast, Saccharomyces cerevisiae, contains 201 rTfs, which is one of the largest rTF numbers found in yeast species for which genome sequences are available. This is a much higher number than the 129 rTFs found in the fission yeast, Schizosaccharomyces pombe, which is currently the yeast with the lowest number of rTFs. Comparative analysis of several different budding yeast species shows that most of the 'extra' rTFs found in S. cerevisiae were probably acquired as a result of a whole genome duplication (WGD) event that occurred in an ancestor of a subset of budding yeast species. However, we also show that budding yeast species that have not been affected by the WGD contain a greater number of rTFs than S. pombe (mean = 145). Thus, two or more mechanisms have led to the 60% increase in rTFs in S. cerevisiae compared to S. pombe. This difference may correlate with a more extensive functional divergence in budding yeasts compared to fission yeasts. The relatively small number of rTFs in S. pombe make this organism an attractive model for global studies of mechanisms that programme gene expression.
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| 6. |
- Blomqvist, Johanna, et al.
(författare)
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Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions
- 2012
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 29:7, s. 265-274
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Tidskriftsartikel (refereegranskat)abstract
- The effect of glucose and oxygen limitation on the growth and fermentation performances of Dekkera bruxellensis was investigated in order to understand which factors favour its propagation in ethanol or wine plants. Although D. bruxellensis has been described as a facultative anaerobe, no growth was observed in mineral medium under complete anaerobiosis while growth was retarded under severe oxygen limitation. In a continuous culture with no gas inflow, glucose was not completely consumed, most probably due to oxygen limitation. When an air/nitrogen mixture (O-2-content ca. 5%) was sparged to the culture, growth became glucose-limited. In co-cultivations with Saccharomyces cerevisiae, ethanol yields/g consumed sugar were not affected by the co-cultures as compared to the pure cultures. However, different population responses were observed in both systems. In oxygen-limited cultivation, glucose was depleted within 24 h after challenging with S. cerevisiae and both yeast populations were maintained at a stable level. In contrast, the S. cerevisiae population constantly decreased to about 1% of its initial cell number in the sparged glucose-limited fermentation, whereas the D. bruxellensis population remained constant. To identify the requirements of D. bruxellensis for anaerobic growth, the yeast was cultivated in several nitrogen sources and with the addition of amino acids. Yeast extract and most of the supplied amino acids supported anaerobic growth, which points towards a higher nutrient demand for D. bruxellensis compared to S. cerevisiae in anaerobic conditions. Copyright (c) 2012 John Wiley & Sons, Ltd.
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| 7. |
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| 8. |
- Boynton, Primrose, et al.
(författare)
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Yeast ecology and communities
- 2022
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 39:1-2, s. 3-3
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 9. |
- Broach, James R, et al.
(författare)
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The Msn2 mediated stress response : Survival based on "hedging your bet" and a dynamic interplay of transcription factor binding and nucleosome occupancy
- 2015
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Ingår i: Yeast. - : Wiley-Blackwell. - 0749-503X .- 1097-0061. ; 32:Suppl. 1, s. S221-S222
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Yeast cell subjected to many different stresses elicit an acute transcriptional stress response mediated by the Msn2 transcription factor, which alters expression of both a stress specific-cohort of genes as well as a common cohort of genes that changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have shown by dynamic single cell analysis that stresses regulate Msn2 activity through cytoplasm to nuclear relocalization but do so in an unusual way: stresses induce increased frequency of bursts of short-lived, recurrent periods of Msn2 nuclear localization with different stresses eliciting different patterns of bursts. Moreover, genetically identical cells subject to an identical stress can behave quite differently, with some cells mounting a robust nuclear occupancy of Msn2 while others show no nuclear localization at all. We have proposed that this idiosyncratic behavior allows populations of cells to “hedge their bet” as to what will be the optimum strategy for surviving the ensuing stress. We have used computational modeling and single cell analysis to determine that bursting is a consequence of noise in the stress signaling pathways amplified by the small number of Msn2 molecules in the cell. Moreover, we have applied genome wide chromatin immunoprecipitation and nucleosome profiling to address how different stresses determine where Msn2 binds under a particular stressful conditions, and thus what genes are regulated by that stress, and how that binding affects, and is affected by, nucleosome positioning and other transcription factor binding. These results provide in vivo validation of Widon's model of indirect cooperativity of transcription factor binding, mediated by partial unwinding of nucleosomes by one transcription factor to allow access for a second transcription factor to a previously occluded binding site. Finally, we have addressed the “bet hedging” hypothesis by showing that persistence of the Msn2-mediated stress response yields cell growth arrest and have identified the targets responsible for that growth arrest. We have applied experimental evolution paradigms to address the relative fitness of cells exhibiting stochastic stress responses versus those with a uniform response. In short, our results indicate that the stress response is complex and that complexity is critical for cell survival.
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| 10. |
- Carlquist, Magnus, et al.
(författare)
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Process engineering for bioflavour production with metabolically active yeast - a minireview.
- 2015
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Ingår i: Yeast. - 1097-0061. ; 32:1, s. 123-143
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Forskningsöversikt (refereegranskat)abstract
- Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industry. The production of flavours can take place by either extraction from plant materials, chemical synthesis, through biological conversion of precursor molecules or through de novo biosynthesis. The latter alternatives are gaining importance through the rapidly growing fields of systems biology and metabolic engineering giving efficient production hosts for the so-called "bioflavours", which are natural flavour and/or fragrance compounds obtained with cell factories or enzymatic systems. One potential production host for bioflavour is yeast. In this mini-review, we give an overview of bioflavour production in yeast from the process engineering perspective. Two specific examples - production of 2-phenylethanol and vanillin - are used to illustrate process challenges and strategies used.
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| 11. |
- Earp, Caroline, et al.
(författare)
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Acute sensitivity of DNA replication to reduction in dNTP pools following Mec1ATR inactivation
- 2015
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Ingår i: Yeast. - : Wiley-Blackwell. - 0749-503X .- 1097-0061. ; 32:Suppl. 1, s. S56-S56
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by a fatal mitotic catastrophe. The mec1 S phase arrest was proposed to stem from a defect in the Mec1-Rad53-Dun1 dependent removal of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), at the onset of S phase: According to this view, Sml1 removal and the ensuing RNR activation would promote the dNTP production necessary for genome duplication. In support for this view, dNTP levels in hypomorphic mec1 or rad53 mutants and a dun1∆ strain were shown to be reduced by as much as 46% compared to a MEC1 control strain. Notably however, nearly all analyses on a lethal mec1 allele (e.g. mec1∆ or mec1-kd [kinase dead]) have been performed in a strain background that was either deleted for SML1 or over-expressing RNR1, a requirement for maintaining viability of a mutant lacking Mec1's essential function. As a result, while it is clear that absence of Mec1 causes dNTP pool to decrease, the true extent of the reduction and whether it would be sufficient to account for the replication arrest remain elusive. Here, we addressed these questions utilizing a temperature sensitive mutant, mec1-4, which maintains its viability at permissive temperature in an otherwise wild-type background, circumventing the need to exogenously manipulate Sml1 and/or RNR activity. Results show that Mec1 inactivation leads to an S phase arrest and a ~17% reduction in dNTP pool; expression of a novel suppressor, GIS2 (glucose inhibition of gluconeogenic growth suppressor 2), rescues the arrest and partially restores the dNTP pool to ~ 93% of a control. Unexpectedly modest effects of mec1 and GIS2 on dNTP levels demonstrate that the arrest does result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to its availability
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| 12. |
- Flagfeldt, D. B., et al.
(författare)
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Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae
- 2009
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 26:10, s. 545-551
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Tidskriftsartikel (refereegranskat)abstract
- The construction of mitotically stable yeast strains for heterologous gene or pathway expression often requires chromosomal integration. However, transcription levels vary between different chromosome regions. We therefore characterized 20 different integration sites of the Sacchromyces cerevisiae genome by inserting lacZ as a reporter gene under the control of two different promoters and determining expression levels through enzyme activity measurement. An up to 8.7-fold difference was detected between the sites conferring lowest and highest expression, respectively. This opens the opportunity for modulating gene expression levels while retaining promoter and culture conditions. Copyright (C) 2009 John Wiley & Sons, Ltd.
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| 13. |
- Fredlund, Elisabeth, et al.
(författare)
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Exploring the mode of action of Pichia anomala - a postharvest biocontrol yeast
- 2001
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Ingår i: Yeast. - : John Wiley & Sons. - 0749-503X .- 1097-0061. ; 18, s. S212-S212
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The ascomycetous yeast Pichia anomala J121, inhibits mould growth in malfunctioning airtight storage systems for moist animal feed grain. Extensive studies of P. anomala J121 have given detailed knowledge of growth physiology and limiting environmental factors, which is necessary to understand the inhibitory activity. Our main objective is to identify the mechanisms behind the inhibitory activity. We have two non-exclusive working hypothesis: I)P. anomala produces antifungal substances and II)P. anomala out-competes the mould for space, nutrients, and oxygen. We have found that volatile metabolites restrict radial growth and sporulation of moulds in mouth-to-mouth assays where agar plates are placed facing each other with the yeast inoculated on the upper plate and the mould on the lower. Previous studies of P. anomala have shown that it produces high quantities of ethyl acetate - a mould-inhibitory substance. We are working to identify homologous genes in P. anomala J121 to the acetyltransferase encoding genes ATF1 and ATF2 in Saccharomyces cerevisiae. Another approach has been to identify intra- and extracellular metabolites during aerobic and oxygen limited growth. Intracellular metabolites were identified by Magic Angle Spinning-High Resolution-Nuclear Magnetic Resonance (MAS-HR- NMR) that allows analysis of living cells. Extracellular metabolites were analysed with HPLC. Glycerol, well known for its role during osmotic stress, is accumulated in response to oxygen stress.
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| 14. |
- Fredlund, Elisabeth, et al.
(författare)
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Oxygen and carbon source-regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala
- 2006
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Ingår i: Yeast. - West Sussex, USA : John Wiley & Sons. - 0749-503X .- 1097-0061. ; 23:16, s. 1137-1149
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Tidskriftsartikel (refereegranskat)abstract
- We amplified, sequenced and studied the transcriptional regulation of genes of the alcoholic fermentation pathway in the biocontrol and non-Saccharomyces wine yeast, Pichia anomala. Two ADH isogenes, PaADH1 and PaADH2, and one PDC gene, PaPDC1, were amplified from genomic P. anomala DNA by a two-step PCR approach, using degenerated primers against conserved regions of the respective genes for cloning core regions, and PCR-based gene walking for cloning the respective 5' and 3'-ends. According to sequence analysis, ADHI and PDC1 are most likely cytoplasmatic proteins, while ADH2 is most probably localized in the mitochondria. PaADH1 was expressed during aerobic growth on glucose, ethanol and succinate, but was ninefold upregulated in response to oxygen limitation when grown on glucose. The gene seems to be involved in both production and consumption of ethanol. Only low expression of PaADH2 was detected during growth on glucose and ethanol, but it was highly expressed during growth on the non-fermentable carbon source succinate and repressed by the addition of glucose. PaPDC1 was expressed during aerobic growth on glucose and was upregulated four-fold in response to oxygen limitation. PaPDC1 expression was lower in cells grown on ethanol and succinate than on glucose and was up- regulated two- and four-fold, respectively, after glucose addition. Our results demonstrate that transcription of genes of the fermentative pathway is regulated by hypoxia and carbon source but posttranscriptional regulation may play a major role in regulating the metabolic flux.
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| 15. |
- Fredlund, Elisabeth, et al.
(författare)
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The correlation of oxygen and sugar dependent regulation of glycolysis to the biocontrol activity of the yeast Pichia anomala
- 2003
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Ingår i: Yeast. - : John Wiley & Sons. - 0749-503X .- 1097-0061. ; 20, s. S352-S352
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Pichia anomala inhibits growth of mould during airtight storage of moist cereal grain for animal feed. The cereal grain is stored in large airtight containers where the anaerobic environment prevents growth of mould. Air can leak into the system due to the removal of grain or technical difficulties in keeping completely anaerobic conditions. This subsequently enables growth of spoilage moulds. Addition of P. anomala cells inhibits the growth of mould making the system more robust [1]. We analysed the physiological basis of the biocontrol activity. P. anomala is a Crabtree negative yeast but in contrast to other Crabtree negative organisms it can grow under anaerobic conditions [2]. The ability to switch between respiratory and fermentative growth in response to O2-availability is essential for its survival in the airtight system and for its biocontrol activity. End products of the sugar metabolism had inhibitory effects on mould growth. Addition of glucose to a model biocontrol system enhanced biocontrol activity without increasing yeast biomass, suggesting the involvement of a product of glycolysis in biocontrol. Sugar consumption, production of ethanol and other metabolites and the activity of key enzymes were investigated in cells grown under defined conditions of oxygen and nutrient supply. The impact of the different parameters on biocontrol activity is discussed. [1] Druvefors et al. (2002) FEMS Yeast Res. 2: 389-394 [2] Fredlund et al. (2002) FEMS Yeast Res. 2: 395-402
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| 16. |
- Gilchrist, Ciaran, et al.
(författare)
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Aneuploidy in yeast : Segregation error or adaptation mechanism?
- 2019
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 36:9, s. 525-539
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Tidskriftsartikel (refereegranskat)abstract
- Aneuploidy is the loss or gain of chromosomes within a genome. It is often detrimental and has been associated with cell death and genetic disorders. However, aneuploidy can also be beneficial and provide a quick solution through changes in gene dosage when cells face environmental stress. Here, we review the prevalence of aneuploidy in Saccharomyces, Candida, and Cryptococcus yeasts (and their hybrid offspring) and analyse associations with chromosome size and specific stressors. We discuss how aneuploidy, a segregation error, may in fact provide a natural route for the diversification of microbes and enable important evolutionary innovations given the right ecological circumstances, such as the colonisation of new environments or the transition from commensal to pathogenic lifestyle. We also draw attention to a largely unstudied cross link between hybridisation and aneuploidy. Hybrid meiosis, involving two divergent genomes, can lead to drastically increased rates of aneuploidy in the offspring due to antirecombination and chromosomal missegregation. Because hybridisation and aneuploidy have both been shown to increase with environmental stress, we believe it important and timely to start exploring the evolutionary significance of their co-occurrence.
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| 17. |
- Granath, Katarina, 1971, et al.
(författare)
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The YIGI (YPL201c) encoded protein is involved in regulating anaerobic glycerol metabolism in Saccharomyces cerevisiae
- 2005
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 22:16, s. 1257-1268
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Tidskriftsartikel (refereegranskat)abstract
- Under anaerobic conditions S. cerevisiae produces glycerol to regenerate NAD+ from the excess NADH produced in cell metabolism. We here report on the role of an uncharacterized protein, Yig1p (YpI201cp), in anaerobic glycerol production. Yig1p was previously shown to interact in two-hybrid tests with the GPP1 and GPP2 encoded glycerol 3-phosphatase (Gpp), and we here demonstrate that strains overexpressing YIG1 show strongly decreased Gpp activity and content of the major phosphatase, Gpp1p. However, cells overexpressing YIG1 exhibited only slightly decreased GPP1 transcript levels, suggesting that Yig1p modulates expression on both transcriptional and post-transcriptional levels. In agreement with such a role, a GFP-tagged derivate of Yig1p was localized to both the cytosol and the nucleus. Deletion or overexpression of YIG1 did not, however, significantly affect growth yield or glycerol yield in anaerobic batch cultures, which is consistent with the previously proposed low flux control exerted at the Gpp level. Copyright (c) 2005 John Wiley & Sons, Ltd.
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| 18. |
- Guaragnella, Nicoletta, et al.
(författare)
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Acetic acid stress in budding yeast: From molecular mechanisms to applications
- 2021
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 38:7, s. 391-400
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Tidskriftsartikel (refereegranskat)abstract
- Acetic acid stress represents a frequent challenge to counteract for yeast cells under several environmental conditions and industrial bioprocesses. The molecular mechanisms underlying its response have been mostly elucidated in the budding yeast Saccharomyces cerevisiae, where acetic acid can be either a physiological substrate or a stressor. This review will focus on acetic acid stress and its response in the context of cellular transport, pH homeostasis, metabolism and stress-signalling pathways. This information has been integrated with the results obtained by multi-omics, synthetic biology and metabolic engineering approaches aimed to identify major cellular players involved in acetic acid tolerance. In the production of biofuels and renewable chemicals from lignocellulosic biomass, the improvement of acetic acid tolerance is a key factor. In this view, how this knowledge could be used to contribute to the development and competitiveness of yeast cell factories for sustainable applications will be also discussed.
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| 19. |
- Görgens, Johann F, et al.
(författare)
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Comparison of three expression systems for heterologous xylanase production by S-cerevisiae in defined medium
- 2004
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:14, s. 1205-1217
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Tidskriftsartikel (refereegranskat)abstract
- The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 furI autoselection system, significantly improved production levels of heterollogous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated. Copyright (C) 2004 John Wiley Sons, Ltd.
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| 20. |
- Jeppsson, Marie, et al.
(författare)
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The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains.
- 2003
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 20:15, s. 1263-1272
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Tidskriftsartikel (refereegranskat)abstract
- Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD+ into NADP+ and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H2O2 than the control strain TMB3001. Copyright © 2003 John Wiley & Sons, Ltd.
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| 21. |
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| 22. |
- Johansson, Björn, et al.
(författare)
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Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae
- 2002
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 19:3, s. 225-231
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Tidskriftsartikel (refereegranskat)abstract
- Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.
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| 23. |
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| 24. |
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| 25. |
- Karademir Andersson, Ahu, et al.
(författare)
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Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii)
- 2016
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 33:12, s. 633-646
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Tidskriftsartikel (refereegranskat)abstract
- The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies, functional analyses of centromere DNA elements, and was shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or MATα) was determined for each hoΔ mutant, and was stable under sporulation inducing conditions, showing that the switching system was totally non-functional. The hoΔ strains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔ strains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii. These haploid strains containing genetic markers will be useful tools for doing genetic analyses in N. castellii. Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci.
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| 26. |
- Karademir Andersson, Ahu, et al.
(författare)
-
Naumovozyma castellii: an alternative model for budding yeast molecular biology
- 2017
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 34:3, s. 95-109
-
Tidskriftsartikel (refereegranskat)abstract
- Naumovozyma castellii (Saccharomyces castellii) is a member of the budding yeast family Saccharomycetaceae. It has been extensively used as a model organism for telomere biology research and has gained an increasing interest as a budding yeast model for functional analyses owing to its amenability to genetic modifications. Due to the suitable phylogenetic distance to S. cerevisiae the whole genome sequence of N. castellii has provided unique data for comparative genomic studies, and it played a key role in the establishment of the timing of the whole genome duplication and the evolutionary events that took place in the subsequent genomic evolution of the Saccharomyces lineage. Here we summarize the historical background of its establishment as a laboratory yeast species, and the development of genetic and molecular tools and strains. We review the research performed on N. castellii, focusing on areas where it has significantly contributed to the discovery of new features of molecular biology and to the advancement of our understanding of molecular evolution.
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| 27. |
- Karhumaa, Kaisa, et al.
(författare)
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Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering
- 2005
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 22:5, s. 359-368
-
Tidskriftsartikel (refereegranskat)abstract
- A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae. Copyright (c) 2005 John Wiley & Sons, Ltd.
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| 28. |
- Karhumaa, Kaisa, et al.
(författare)
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Proteome analysis of the xylose-fermenting mutant yeast strain TMB 3400.
- 2009
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 26:7, s. 371-382
-
Tidskriftsartikel (refereegranskat)abstract
- Xylose fermentation in yeast has been a target of research for years, yet not all the factors that may affect xylose fermentation perfomance of yeast strains are known. In this study, the mutant S. cerevisiae strain TMB 3400, which has good xylose fermentation properties, was compared with its parental strain to examine the factors behind the improved xylose utilization at protein level. The proteome of the parental and the mutant strains were characterized by difference in gel electrophoresis (DiGE) to quantitatively identify proteins that are expressed at altered levels in the mutant. The most significant changes detected by proteome analysis were the 6-10-fold increased levels of xylose reductase, xylitol dehydrogenase and transketolase (Tkl1) in the mutant, which is in accordance with previous knowledge about xylose metabolism in yeast. The level of acetaldehyde dehydrogenase (Ald6) was also significantly increased. In addition, several proteins homologous to proteins from yeast species other than S. cerevisiae were identified in both strains, demonstrating the genetic heterogeneity of industrial yeast strains. The results were also compared with a previously reported transcription analysis performed with identical experimental set-up; however, very little correlation between the two datasets was observed. The results of the proteome analysis were in good agreement with a parallel study in which rationally designed overexpression of XR, XDH and the non-oxidative pentose phosphate pathway resulted in similar improvement in xylose utilization, which demonstrates the usefulness of proteome analysis for the identification of target genes for further metabolic engineering strategies in industrial yeast strains. Copyright (c) 2009 John Wiley & Sons, Ltd.
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| 29. |
- Katz, Michael, et al.
(författare)
-
Mild detergent treatment of Candida tropicalis reveals a NADPH-dependent reductase in the crude membrane fraction, which enables the production of pure bicyclic exo-alcohol
- 2004
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:15, s. 1253-1267
-
Tidskriftsartikel (refereegranskat)abstract
- This study demonstrated the occurrence of a NADPH-dependent exo-alcohol reductase in the crude membrane fraction of Candida tropicalis. Cytosolic endo-alcohol reductase activity could be separated from the membrane-bound exo-alcohol activity by means of detergent treatment, enabling the preparation of pure exo-alcohol via the enzymatic conversion of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione. The exo-alcohol reductase is, to our knowledge, the first membrane-bound NADPH-dependent reductase accepting a xenobiotic carbonyl substrate that was not a steroid. When C. tropicalis was grown on D-sorbitol, a two-fold increase in the exo-reductase activity was observed as compared to when grown on glucose. An in silico comparison at the protein level between putative xenobiotic carbonyl reductases in Candida albicans, C. tropicalis and Saccharomyces cerevisiae was performed to explain why Candida species are often encountered when screening yeasts for novel stereoselective reduction properties. C. albicans contained more reductases with the potential to reduce xenobiotic carbonyl compounds than did S. cerevisiae. C. tropicalis had many membrane-bound reductases (predicted with the bioinformatics program, TMHMM), some of which had no counterpart in the two other organisms. The exo-reductase is suspected to be either a -hydroxysteroid dehydrogenase or a polyol dehydrogenase from either the short chain dehydrogenase family or the dihydroflavonol reductase family.
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| 30. |
- Kettner, K., et al.
(författare)
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Schizosaccharomyces pombe ER oxidoreductin-like proteins SpEro I a p and SpEro I b p
- 2004
-
Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 21:12, s. 1035-1044
-
Tidskriftsartikel (refereegranskat)abstract
- Endoplasmic reticulum oxidoreductins (Ero proteins) are essential for oxidation of protein disulphide isomerase (Pdi), which introduces disulphide bonds in target proteins. Contrary to the situation in Saccharomyces cerevisiae, with a single Ero protein (Ero1p), the genomes of Schizosaccharomyces pombe and of humans encode two Ero-like proteins. Here we show that both Sz. pombe proteins (SpEro1a p and SpEro1b p) are N-glycosylated and firmly associated with membranes of the secretory pathway. Surprisingly, only expression of SpEro1b p completely restores growth of the temperature-sensitive S. cerevisiae ero1-1 mutant, whereas SpEro1a p only partially complements this mutation. Upon expression in S. cerevisiae wild-type cells, SpEro1b p leads to a significantly increased resistance to reductive stress by dithiothreitol, whereas SpEro1a p has only a marginal effect. These data suggest that SpEro1b p is a functional homologue of the S. cerevisiae Ero1p
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| 31. |
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| 32. |
- Kuzmenko, Anton, et al.
(författare)
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Aim23 is an yeast mitochondrial translation initiation factor 3 which is unnecessary for protein synthesis
- 2015
-
Ingår i: Yeast. - : Wiley-Blackwell. - 0749-503X .- 1097-0061. ; 32:Suppl. 1, s. S192-S193
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are essential organelles of virtually all eukaryotic cells. They have their own genome and are able to transcribe and translate their genetic material. The system of mitochondrial protein synthesis is organized in a manner close to that of prokaryotes. However, mitochondrial DNA contains just a few protein-coded genes (9 in yeast, 13 in humans), so the mitochondrial translation system deals with a limited number of mRNAs. The mitochondrial translation machinery is also somewhat lineage-specific, with various components being gained and lost in different taxonomic groups. The classical bacterial initiation factors (IFs) IF1, IF2 and IF3 are universal in prokaryotes, but only IF2 is universal in mitochondria (mIF2). No IF1 has been identified in mitochondria of any organism. An insertion in mIF2 has been suggested to functionally compensate for the absence of mIF1. Mitochondrial IF3 (mIF3), although known to be present in various eukaryotes, has not been identified for many years in budding yeast Saccharomyces cerevisiae, the model organism for studying mitochondrial translation in vivo. In 2012, we have proven that IF3 does present in yeast mitochondria, and it is Aim23 protein. In the present study, we have characterized the effects of AIM23 gene deletion on yeast mitochondrial function. One could suggest that such a deletion would lead to a complete loss of respiration, translation and other molecular processes in mitochondria. However, this was not the case: the growth of AIM23∆ yeast on clycerol-containing media was suppressed in first 1-2 days only and reached the levels of wild-type in 3-4 days. AIM23∆ cells also were able to respire. Interestingly, we observed a very unusual pattern of mitochondrially-synthesized proteins in the ΔAIM23 strain. The amount of several proteins is decreased in the mutants compared to the wild-type but the amount of some others is increased. We conclude that the yeast cells are able to adapt somehow to the absence of Aim23p.
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| 33. |
- Laadan, Boaz, et al.
(författare)
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Identification of an NADH-dependent 5-hydroxymethylfurfural-reducing alcohol dehydrogenase in Saccharomyces cerevisiae.
- 2008
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:3, s. 191-198
-
Tidskriftsartikel (refereegranskat)abstract
- We report on the identification and characterization of a mutated alcohol dehydrogenase 1 from the industrial Saccharomyces cerevisiae strain TMB3000 that mediates the NADH-dependent reduction of 5-hydroxymethylfurfural (HMF) to 2,5-bis-hydroxymethylfuran. The co-factor preference distinguished this alcohol dehydrogenase from the previously reported NADPH-dependent S. cerevisiae HMF alcohol dehydrogenase Adh6. The amino acid sequence revealed three novel mutations (S109P, L116S and Y294C) that were all predicted at the vicinity of the substrate binding site, which could explain the unusual substrate specificity. Increased biomass production and HMF conversion rate were achieved in a CEN.PK S. cerevisiae strain overexpressing the mutated ADH1 gene. Copyright (c) 2008 John Wiley & Sons, Ltd.
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| 34. |
- Linder, Tomas
(författare)
-
A genomic survey of nitrogen assimilation pathways in budding yeasts (sub-phylum Saccharomycotina)
- 2019
-
Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 36, s. 259-273
-
Tidskriftsartikel (refereegranskat)abstract
- Sequenced genomes of 149 species of budding yeast (including 62 species with draft genomes that currently lack gene annotations) were surveyed for the presence of 24 genes associated with the assimilation of amines, uracil, dihydropyrimidines, purines, uric acid, allantoin, and nitrate as nitrogen sources. Genes for the assimilation of primary amines were distributed broadly across the Saccharomycotina while choline assimilation appeared to be mostly restricted to the families Debaryomycetaceae, Metschnikowiaceae, and Pichiaceae. Conversely, the uracil catabolic pathway was completely absent in Debaryomycetaceae and Metschnikowiaceae but present in the majority of the remaining Saccharomycotina. The super-pathway for assimilation of purines, uric acid, and allantoin was present in the majority of surveyed species. Genes for the assimilation of nitrate were restricted to a minority of species in families Phaffomycetaceae, Pichiaceae, and Trichomonascaceae as well as some currently unassigned genera. This study also successfully identified yeast homologs of all six previously known eukaryotic genes involved in the biosynthesis of the molybdenum cofactor, which is required for the activity of the nitrogen assimilation-associated enzymes nitrate reductase and xanthine oxidoreductase. Analysis of 1,187 upstream intergenic regions identified three novel putative regulatory motifs for the assimilation of uracil, purines, and uric acid as well as a possible role for the MADS-box transcription factor Mcm1 in the regulation of amine assimilation genes.
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| 35. |
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| 36. |
- Marques, Wesley Leoricy, et al.
(författare)
-
Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter
- 2018
-
Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 35:12, s. 639-652
-
Tidskriftsartikel (refereegranskat)abstract
- Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (mu = 0.19 h(-1) and mu = 0.08 h(-1), respectively). All five mutants displayed sucrose-induced proton uptake (13-50 mu mol H+ (g biomass)(-1) min(-1)). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1(I209F C265F G326C)) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h(-1)) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.
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| 37. |
- Masser, Anna E., et al.
(författare)
-
Luciferase NanoLuc as a reporter for gene expression and protein levels in Saccharomyces cerevisiae
- 2016
-
Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 33:5, s. 191-200
-
Tidskriftsartikel (refereegranskat)abstract
- Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo (R) is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate.
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| 38. |
- Meza, Eugenio, 1975, et al.
(författare)
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Development of a method for heat shock stress assessment in yeast based on transcription of specific genes
- 2021
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 38:10, s. 549-565
-
Tidskriftsartikel (refereegranskat)abstract
- All living cells, including yeast cells, are challenged by different types of stresses in their environments and must cope with challenges such as heat, chemical stress, or oxidative damage. By reversibly adjusting the physiology while maintaining structural and genetic integrity, cells can achieve a competitive advantage and adapt environmental fluctuations. The yeast Saccharomyces cerevisiae has been extensively used as a model for study of stress responses due to the strong conservation of many essential cellular processes between yeast and human cells. We focused here on developing a tool to detect and quantify early responses using specific transcriptional responses. We analyzed the published transcriptional data on S. cerevisiae DBY strain responses to 10 different stresses in different time points. The principal component analysis (PCA) and the Pearson analysis were used to assess the stress response genes that are highly expressed in each individual stress condition. Except for these stress response genes, we also identified the reference genes in each stress condition, which would not be induced under stress condition and show stable transcriptional expression over time. We then tested our candidates experimentally in the CEN.PK strain. After data analysis, we identified two stress response genes (UBI4 and RRP) and two reference genes (MEX67 and SSY1) under heat shock (HS) condition. These genes were further verified by real-time PCR at mild (42°C), severe (46°C), to lethal temperature (50°C), respectively.
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| 39. |
- Mountain, Harry A, et al.
(författare)
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Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae
- 1991
-
Ingår i: Yeast. - : John Wiley & Sons. - 0749-503X .- 1097-0061. ; 7:8, s. 781-803
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Tidskriftsartikel (refereegranskat)abstract
- Genes encoding enzymes in the threonine/methionine biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.
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| 40. |
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| 41. |
- Nielsen, Jens
(författare)
-
Global regulation of yeast metabolism
- 2015
-
Ingår i: Yeast. - : Wiley-Blackwell. - 0749-503X .- 1097-0061. ; 32, s. S33-S33
-
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The yeast Saccharomyces cerevisiae is widely used for production of fuels, chemicals, pharmaceuticals andmaterials. Through metabolic engineering of this yeast a number of novel new industrial processes have beendeveloped over the last 10 years. Besides its wide industrial use, S. cerevisiae serves as an eukaryal modelorganism, and many systems biology tools have therefore been developed for this organism. Despite ourextensive knowledge of yeast metabolism and its regulation we are still facing challenges when we want tointegrate this information into mathematical models. In this presentation examples of studies on global regulationof yeast metabolism will be provided. This will include analysis of how yeast rewire its metabolism at the globallevel when exposed to various types of stress and how global regulators like Snf1 and Sir2 controls yeastmetabolism
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| 42. |
- Nielsen, Jens
(författare)
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Mathematical modeling of yeast : a driver for innovation in biotechnology and human medicine
- 2015
-
Ingår i: Yeast. - 0749-503X .- 1097-0061. ; 32, s. S50-S51
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Saccharomyces cerevisiae is the most studied organism and is for this reason used widely as a model organismfor studying molecular mechanisms of relevance for human disease. Thus, several Nobel Prizes have been givento yeast researchers. Among many other discoveries studies of yeast resulted in identification of cyclins andcyclin dependent kinases that play a central role in the cell cycle of eukaryal cells and mapping of the proteinsecretory pathway in eukaryal cells. This yeast is also used for production of fuels, chemicals, pharmaceuticalsand materials, and the annual revenue derived from processes based on S. cerevisiae fermentations by farexceeds 200 billion EURO. Furthermore. through metabolic engineering of this yeast a number of novel newindustrial processes are under development resulting in an even more important role of this cell factory in thefuture. In order to advance our fundamental understanding of this important organism, but in human healthresearch and industrial biotechnology, it is important to advance our ability to integrate novel experimental datain quantitative framework. Mathematical models represent an excellent scaffold for this as they allowreconciliation of data and at the same time enable generation of novel hypothesis concerning specific molecularprocesses. Furthermore, in the field of industrial biotechnology mathematical models may be used for advancingmetabolic engineering, which will result in a reduction in development costs and hereby advance towards biosustainable production of fuels and chemicals
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| 43. |
- Nilsson, Annika, et al.
(författare)
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The catabolic capacity of Saccharomyces cerevisiae is preserved to a higher extent during carbon compared to nitrogen starvation
- 2001
-
Ingår i: Yeast. - Chichester : Wiley. - 0749-503X .- 1097-0061. ; 18:15, s. 1371-1381
-
Tidskriftsartikel (refereegranskat)abstract
- A comparison of catabolic capacity was made between S. cerevisiae cells subjected to 24 h carbon or nitrogen starvation. The cells were shifted to starvation conditions at the onset of respiratory growth on ethanol in aerobic batch cultures, using glucose as the carbon and energy source. The results showed that the catabolic capacity was preserved to a much larger extent during carbon compared to nitrogen starvation. Nitrogen starvation experiments were made in the presence of ethanol (not glucose) to exclude the effect of glucose transport inactivation (Busturia and Lagunas, 1986). Hence, the difference in catabolic capacity could not be attributed to differences in glucose transport capacity during these conditions. In order to understand the reason for this difference in starvation response, measurement of protein composition, adenine nucleotides, inorganic phosphate, poly phosphate and storage carbohydrates were performed. No clear correlation between any of these variables and catabolic capacity after starvation could be obtained. However, there was a positive correlation between total catabolic activity and intracellular ATP concentration when glucose was added to starved cells. The possible mechanism for this correlation, as well as what determines the ATP level, is discussed.
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| 44. |
- Norbeck, Joakim, 1965, et al.
(författare)
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A QPCR-based reporter system to study post-transcriptional regulation via the 3' untranslated region of mRNA in Saccharomyces cerevisiae.
- 2009
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 26:7, s. 407-413
-
Tidskriftsartikel (refereegranskat)abstract
- Post-transcriptional regulation via the 3' untranslated region (3' UTR) of mRNA is an important factor in governing eukaryotic gene expression. Achieving detailed understanding of these processes requires highly quantitative systems in which comparative studies can be performed. To this end, we have developed a plasmid reporter system for Saccharomyces cerevisiae, in which the 3' UTR can be easily replaced and modified. Accurate quantification of the tandem affinity purification tag (TAP)-reporter protein and of TAP-mRNA is achieved by immuno-QPCR and by RT-QPCR, respectively. We have used our reporter system to evaluate the consequences on gene expression from varying the 3' UTR, a problem often encountered during C-terminal tagging of proteins. It was clear that the choice of 3' UTR was a strong determinant of the reporter expression, in a manner dependent on the growth conditions used. Mutations affecting either decapping (lsm1Delta) or deadenylation (pop2Delta) were also found to affect reporter gene expression in a highly 3' UTR-dependent manner. Our results using this set-up clearly indicate that the common strategy used for C-terminal tagging, with concomitant replacement of the native 3' UTR, will very likely provide incorrect conclusions on gene expression.
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| 45. |
- Partow, Siavash, 1979, et al.
(författare)
-
Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae
- 2010
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 27:11, s. 955-964
-
Tidskriftsartikel (refereegranskat)abstract
- The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose-containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose-induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC-URA plasmid except that gene expression is mediated by constitutive promoters.
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| 46. |
- Passoth, Volkmar
(författare)
-
A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration
- 2011
-
Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 28, s. 309-320
-
Tidskriftsartikel (refereegranskat)abstract
- Scheffersomyces stipitis PJH was mutagenized by random integrative mutagenesis and the integrants were screened for lacking the ability to grow with glutamate as sole carbon source. One of the two isolated mutants was damaged in the COX5 gene, which encodes a subunit of the cytochrome c oxidase. BLAST searches in the genome of Sc. stipitis revealed that only one singular COX5 gene exists in Sc. stipitis, in contrast to Saccharomyces cerevisiae, where two homologous genes are present. Mutant cells had lost the ability to grow with the amino acids glutamate, proline or aspartate and other non-fermentable carbon sources, such as acetic acid and ethanol, as sole carbon sources. Biomass formation of the mutant cells in medium containing glucose or xylose as carbon source was lower compared with the wild-type cells. However, yields and specific ethanol formation of the mutant were much higher, especially under conditions of higher aeration. The mutant cells lacked both cytochrome c oxidase activity and cyanide-sensitive respiration, whereas ADH and PDC activities were distinctly enhanced. SHAM-sensitive respiration was obviously essential for the fermentative metabolism, because SHAM completely abolished growth of the mutant cells with both glucose or xylose as carbon source. Copyright. (C) 2011 John Wiley & Sons, Ltd.
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| 47. |
- Passoth, Volkmar, et al.
(författare)
-
Analysis of the hypoxia-induced ADH2-promoter of the respiratory yeast Pichia stipitis reveals a new mechanism for sensing of oxygen limitation in yeast
- 2003
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 20:1, s. 39-51
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Tidskriftsartikel (refereegranskat)abstract
- We introduced a reporter gene system into Pichia stipitis using the gene for the artificial green fluorescent protein (GFP), variant yEGFP. This system was used to analyse hypoxia-dependent PsADH2 regulation. Reporter gene activity was only found under oxygen limitation on a fermentable carbon source. The promoter was not induced by oxygen limitation in the Crabtree-positive yeast Saccharomyces cerevisiae. Promoter deletions revealed that a region of 15 bp contained the essential site for hypoxic induction. This motif was different from the known hypoxia response elements of S. cerevisiae but showed some similarity to the mammalian HIF-1 binding site. Electrophoretic mobility shift assays demonstrated specific protein binding to this region under oxygen limitation. Similar to the S. cerevisiae heme sensor system, the promoter was induced by Co2+. Cyanide was not able to mimic the effect of oxygen limitation. The activation mechanism of PsADH2 also, in this respect, has similarities to the mammalian HIF-1 system, which is inducible by Co2+ but not by cyanide. Thus, the very first promoter analysis in P. stipitis revealed a hitherto unknown mechanism of oxygen sensing in yeast.
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| 48. |
- Passoth, Volkmar
(författare)
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C- and N-catabolic utilization of tricarboxylic acid cycle-related amino acids by Scheffersomyces stipitis and other yeasts
- 2011
-
Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 28, s. 375-390
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Tidskriftsartikel (refereegranskat)abstract
- Scheffersomyces stipitis and the closely related yeast Candida shehatae assimilated the L-amino acids glutamate, aspartate and proline as both carbon and nitrogen sole sources. We also found this rarely investigated ability in ascomycetous species such as Candida glabrata, C. reukaufii, C. utilis, Debaryomyces hansenii, Kluyveromyces lactis, K. marxianus, Candida albicans, L. elongisporus, Meyerozyma guilliermondii, C. maltosa, Pichia capsulata and Yarrowia lipolytica and in basidiomycetous species such as Rhodotorula rubra and Trichosporon beigelii. Glutamate was a very efficient carbon source for Sc. stipitis, which enabled a high biomass yield/mole, although the growth rate was lower when compared to growth on glucose medium. The cells secreted waste ammonium during growth on glutamate alone. In Sc. stipitis cultures grown in glucose medium containing glutamate as the nitrogen source the biomass yield was maximal, and ethanol concentration and specific ethanol formation rate were significantly higher than in glucose medium containing ammonium as the nitrogen source. Mainly C-assimilation of glutamate but also N-assimilation in glucose-containing medium correlated with enhanced activity of the NAD-dependent glutamate dehydrogenase 2 (GDH2). A Delta gdh2 disruptant was unable to utilize glutamate as either a carbon or a nitrogen source; moreover, this disruptant was also unable to utilize aspartate as a carbon source. The mutation was complemented by retransformation of the GDH2 ORF into the Delta gdh2 strain. The results show that Gdh2p plays a dual role in Sc. stipitis as both C- and N-catabolic enzyme, which indicates its role as an interface between the carbon and nitrogen metabolism of this yeast. Copyright (C) 2011 John Wiley & Sons, Ltd.
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- Passoth, Volkmar, et al.
(författare)
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Oleaginous yeasts for biochemicals, biofuels and food from lignocellulose-hydrolysate and crude glycerol
- 2023
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Ingår i: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 40
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Forskningsöversikt (refereegranskat)abstract
- Microbial lipids produced from lignocellulose and crude glycerol (CG) can serve as sustainable alternatives to vegetable oils, whose production is, in many cases, accompanied by monocultures, land use changes or rain forest clearings. Our projects aim to understand the physiology of microbial lipid production by oleaginous yeasts, optimise the production and establish novel applications of microbial lipid compounds. We have established methods for fermentation and intracellular lipid quantification. Following the kinetics of lipid accumulation in different strains, we found high variability in lipid formation even between very closely related oleaginous yeast strains on both, wheat straw hydrolysate and CG. For example, on complete wheat straw hydrolysate, we saw that one Rhodotorula glutinis strain, when starting assimilating D-xylosealso assimilated the accumulated lipids, while a Rhodotorula babjevae strain could accumulate lipids on D-xylose. Two strains (Rhodotorula toruloides CBS 14 and R. glutinis CBS 3044) were found to be the best out of 27 tested to accumulate lipids on CG. Interestingly, the presence of hemicellulose hydrolysate stimulated glycerol assimilation in both strains. Apart from microbial oil, R. toruloides also produces carotenoids. The first attempts of extraction using the classical acetone-based method showed that beta-carotene is the major carotenoid. However, there are indications that there are also substantial amounts of torulene and torularhodin, which have a very high potential as antioxidants.
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