Hisashi Yazawa

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae.

Screening of the homozygous diploid yeast deletion pool of 4741 non‐essential genes identified two null mutants (Δura7 and Δgal6) that grew faster than the wild‐type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild‐type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild‐type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly β‐1,3‐glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild‐type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild‐type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 °C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively. Copyright

Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance.

Saccharomyces cerevisiae produces saturated and monounsaturated fatty acids of 16‐ and 18‐carbon atoms and no polyunsaturated fatty acids (PUFAs) with more than two double bonds. To study the biological significance of PUFAs in yeast, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase (KlFAD2) and ω3 fatty acid desaturase (KlFAD3) genes into S. cerevisiae to produce linoleic and α‐linolenic acids in S. cerevisiae. The strain producing linoleic and α‐linolenic acids showed an alkaline pH‐tolerant phenotype. DNA microarray analyses showed that the transcription of a set of genes whose expressions are under the repression of Rim101p were downregulated in this strain, suggesting that Rim101p, a transcriptional repressor which governs the ion tolerance, was activated. In line with this activation, the strain also showed elevated resistance to Li+ and Na+ ions and to zymolyase, a yeast lytic enzyme preparation containing mainly β‐1,3‐glucanase, indicating that the cell wall integrity was also strengthened in this strain. Our findings demonstrate a novel influence of PUFA production on transcriptional control that is likely to play an important role in the early stage of alkaline stress response. The Accession No. for microarray data in the Center for Information Biology Gene Expression database is CBX68. Copyright

Heterologous Production of Dihomo-γ-Linolenic Acid in Saccharomyces cerevisiae

ABSTRACT To make dihomo-γ-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Δ12 fatty acid desaturase, rat Δ6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15°C than in those grown at 20°C, and no DGLA production was observed in the cells grown at 30°C. In NSD at 15°C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15°C, the yield of DGLA reached 2.19 μg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.

Efficient accumulation of oleic acid in Saccharomyces cerevisiae caused by expression of rat elongase 2 gene (rELO2) and its contribution to tolerance to alcohols

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.

Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe

Triglycerides (TG) are major storage lipids for eukaryotic cells. In this study, we characterized three genes of fission yeast Schizosaccharomyces pombe, SPCC1450.16c, SPAC1786.01c, and SPAC1A6.05c, that show high homology to Saccharomyces cerevisiae TG lipase genes, TGL3, TGL4, and TGL5. Deletion of each gene increased TG content by approximately 1.7-fold compared to the parental wild-type strain, and their triple deletion mutant further increased TG content to 2.7-fold of the wild-type strain, suggesting that all three genes encode TG lipase and are functioning in S. pombe. The triple deletion mutant showed no growth defect in rich and synthetic medium, but its growth was sensitive to cerulenin, an inhibitor of fatty acid synthesis. This growth defect by cerulenin was restored by adding oleic acid in media, suggesting that these genes were involved in the mobilization of TG in S. pombe. When ricinoleic acid was produced in the triple mutant by introducing CpFAH12 fatty acid hydroxylase gene from Claviceps purpurea, percent composition of ricinoleic acid increased by 1.1-fold compared to the wild-type strain, in addition to a 1.6-fold increase in total fatty acid content per dry cell weight (DCW). In total, the ricinoleic acid production per DCW increased by 1.8-fold in the triple deletion mutant.