Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.     

Enhancement of Protein Secretion in Saccharomyces cerevisiae by Overproduction of Sso Protein,a Late-acting Component of the Secretory Machinery

Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins, Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus α-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted α-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the α-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiency obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast. © 1997 John Wiley & Sons, Ltd.     

Cloning and sequencing of the gene for apocytochrome b of the yeast Kluyveromyces lactis strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140)

The apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced. The coding sequences in strains WM27 (NRRL Y-17066) and WM37 (NRRL Y-1140) were identical but the upstream noncoding regions were slightly different. The sequences demonstrated the presence of a continuous open reading frame with no introns. The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273-10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50. CUN and CGN codon families were absent from the K. lactis gene. Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position. There were two unusual features. All threonines were coded by ACA(U) and all arginines by AGA.     

Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae

The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.     

Production of the STA2-encoded glucoamylase in Saccharomyces cerevisiae is subject to feed-back control

Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified; repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven-fold differences in the rate of GA production) can be displayed by either glucose-limited or glycerol-plus ethanol-consuming cultures; the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated GA. It is proposed that the feed-back control involved in GA production can be attenuated by starch which can thus 'induce' higher rates of GA production compared to the basal mode.     

Inhibitory Effect of Fusarium Mycotoxins on Growth of Brewing Yeasts. 1 Zearalenone and Fumonisin B1*

The Fusarium mycotoxins zearalenone (ZEA) and fumonisin B₁ (FB₁) added to the growth medium in low and high concentrations, were investigated as a possible cause of inhibition of growth of Saccharomyces cerevisiae lager and ale strains. Toxic effects were assessed by measurement of dry weight or growth relative to controls, cell number, viability and conductance changes of the growth medium using indirect and direct methods. The Fusarium mycotoxins studied affected growth on brewing yeasts, but the inhibitory effect was dependent on concentration. Low concentrations (0.1–2 μg/ml) had no significant effect on growth compared to controls. Although high concentrations of both mycotoxins strongly affected growth, the inhibitory effect depended on toxin concentration and type, yeast strain, length of incubation and method used to assess growth. The lowest concentrations of mycotoxin causing significant inhibition on growth of these brewing yeasts were 50 μg/ml ZEA for both yeast strains, and 10 μg/ml FB₁ for the lager strain and 50 μg/ml for the ale strain.     

酿酒活性干酵母在酒精生产中的应用

酿酒活性干酵母应用于酿酒行业和超级酿酒酵母应用于酒精浓醪发酵,不仅是我国酿酒工业的技术革命,而且极大地推动了酒精行业的技术快速发展。目前,酿酒活性干酵母广泛应用于酿酒工业和酒精的浓醪发酵以及燃料酒精工业。针对酒精生产原料的变化,酒精生产工艺的不同,酿酒活性干酵母的适应性和发酵浓度将会提高;酵母对温度、渗透压以及对产物、底物的抑制的敏感度将降低;酒精生产工艺的控制要求将进一步提高,对酿酒活性干酵母对酵母质量的要求也将更高。（孙悟）