Construction of Flocculent Yeast Cells (<i>Saccharomyces cerevisiae</i>) by Mating or Protoplast Fusion Using a Yeast Cell Containing the Flocculation Gene<i>FL05</i>

Watari, Junji; Kudo, Masayoshi; Nishikawa, Norio; Kamimura, Minoru

doi:10.1080/00021369.1990.10870222

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“…The protoplast fusion technique has also been used in the construction of hybrids between nonflocculent and flocculent strains. [35][36][37] Transformation of plasmids containing FLO1 under the regulation of a constitutive ADH1 promoter or a HSP30 promoter was shown to confer a flocculation phenotype on nonflocculent S. cerevisiae strains. 1,23,25) Moreover, the FLO5 gene was also used in the construction of flocculating strains in laboratory S. cerevisiae by transforming multicopy plasmids containing an ADH2-FLO5 fusion gene.…”

Section: Discussionmentioning

confidence: 99%

Construction of FlocculentKluyveromyces marxianusStrains Suitable for High-Temperature Ethanol Fermentation

Nonklang

Ano²,

Abdel-Banat

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Flocculating yeasts are highly useful in fermentation processes because these cells can be separated easily from the fermentation mash. However, native yeasts are usually non-flocculating, including Kluyveromyces marxianus, which exhibits a potent high-temperature ethanol fermentation ability. We describe here the construction of flocculent K. marxianus strains via the introduction of the FLO1, FLO5, FLO9, and FLO10 genes from Saccharomyces cerevisiae. The S. cerevisiae FLO genes were overexpressed by upstream insertion of the constitutive TDH3 promoter, resulting in flocculent S. cerevisiae strains. These TDH3p-FLO sequences were then amplified by PCR and introduced directly into a K. marxianus strain. These K. marxianus strains showed a flocculation phenotype, indicating that the introduced S. cerevisiae TDH3 promoter and all FLO genes were functional in this strain. Moreover, a flocculating K. marxianus strain showed the same ethanol production profile as that of its wild-type parent. The K. marxianus flocculating strains we generated should be useful in the future development of cost-effective fed-batch and continuous fermentation systems at high temperatures.Key words: Kluyveromyces marxianus; yeast; flocculation; ethanol fermentationFlocculation is an attractive property in industrial yeasts because the cells can be easily separated from a fermentation mash. The application of flocculating strains to industrial fermentation processes removes the need for extensive and costly cell separation equipment, and these yeasts are thus suitable for both fedbatch and continuous fermentation. [1][2][3][4] In addition, flocculating yeast cells are useful in immobilized cell systems with no inert support materials. 5) However, although flocculation mechanisms and the introduction of this property to non-flocculent yeast strains have been extensively studied in S. cerevisiae, few studies of this nature have been undertaken in other yeast species. 6,7) The mechanism of yeast flocculation involves the interaction of lectin-like proteins, called flocculins, with receptors on a neighboring cell wall, resulting in the formation of aggregates. [8][9][10][11] Environmental factors such as pH, calcium, and organic stress also affect flocculation. 9,[12][13][14] The five FLO genes, FLO1, FLO5, FLO9, FLO10, and FLO11, have been described in S. cerevisiae thus far. [15][16][17][18][19] The FLO1, 5, 9, and 10 genes confer cell-cell adhesion (flocculation) ability, whereas FLO11 is responsible for substrate adhesion. 20) FLO1, FLO5, and FLO9 are highly similar to each other, but FLO10 has lower similarity. The mechanisms of flocculation in other yeasts appear to be lectin-based but of differing specificities. For instance, galactose and its derivatives have been found to inhibit flocculation of K. bulgaricus and K. lactis, suggesting that this interaction involves a galactose-specific lectin. 21,22) The advantages of using flocculating yeast cells in fermentation processes have led to many studies on the construction of flocculent s...

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