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  • 1.
    Fredlund, Elisabeth
    et al.
    Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Beerlage, Christiane
    Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Melin, Petter
    Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Schnürer, Johan
    Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Passoth, Volkmar
    Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Oxygen and carbon source-regulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala2006In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 23, no 16, p. 1137-1149Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Fredlund, Elisabeth
    et al.
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Boysen, Marianne
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Schnürer, Johan
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Broberg, Anders
    Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Exploring the mode of action of Pichia anomala - a postharvest biocontrol yeast2001In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 18, p. S212-S212Article in journal (Other academic)
    Abstract [en]

    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.

  • 3.
    Fredlund, Elisabeth
    et al.
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Druvefors, Ulrika Ädel
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Passoth, Volkmar
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Schnürer, Johan
    Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    The correlation of oxygen and sugar dependent regulation of glycolysis to the biocontrol activity of the yeast Pichia anomala2003In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 20, p. S352-S352Article in journal (Other academic)
    Abstract [en]

    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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