The process of ethanol fermentation has a long history in the production of alcoholic drinks, but much larger scale production of ethanol is now required to enable its use as a substituent of gasoline fuels at 3%, 10%, or 85% (referred to as E3, E10, and E85, respectively). Compared with fossil fuels, the production costs are a major issue for the production of fuel ethanol. There are a number of possible approaches to delivering cost-effective fuel ethanol production from different biomass sources, but we focus in our current report on high-temperature fermentation using a newly isolated thermotolerant strain of the yeast Kluyveromyces marxianus. We demonstrate that a 5 degrees C increase only in the fermentation temperature can greatly affect the fuel ethanol production costs. We contend that this approach may also be applicable to the other microbial fermentations systems and propose that thermotolerant mesophilic microorganisms have considerable potential for the development of future fermentation technologies.

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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A chitinase gene (Chi3K) was cloned from the genomic DNA of Vitis vinifera cv. Koshu. The structural gene comprised 891 by without introns and encoded 297 amino acids. The Chi3K product showed high similarity to the class III chitinase of V. vinifera cv. Pinot noir. Chi3K was expressed using a bacterial expression vector for purification and enzymatic characterization of its gene product. The recombinant chitinase exhibited hydrolytic activity toward glycol chitin and its optimum pH was 4.0. It also inhibited the growth of Botrytis cinerea, which causes grey mold disease in grapes.

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Industrial yeast strains are generally diploid and are often defective in sporulation. Such strains are hence thought to be less tractable for manipulation by genetic engineering. To facilitate more reliable genetic manipulation of the diploid yeast Japanese sake, we constructed variants of this strain that were homozygous for a URA3 deletion, homozygous for either MATa or MATalpha, and homozygous for either the his3 or the lys4 mutation. A ura3-null genotype enabled gene targeting to be undertaken more easily. The TDH3 promoter was inserted upstream of six yeast genes that have been implicated in flavor control to drive their constitutive overexpression. The homozygous MAT alleles, combined with the non-complementary auxotrophic mutations in the targeted transformants, allowed for tetraploid selection through mating. This resulted in the combinatorial construction of tetraploid strains that overexpress two different genes simultaneously. In addition, a recessive mutant gene, sah1-1, that is known to overproduce S-adenosylmethionine, was introduced into the diploid sake strain by the replacement of one wild-type allele and subsequent disruption of the other. The resulting sah1-1/sah1Delta::URA3 strain produced higher amounts of S-adenosylmethionine than the wild type. The novel sake yeast diploid strains we generated in this study can thus undergo simple PCR-mediated gene manipulation and mating in a manner analogous to established laboratory strains. Moreover, none of these sake strains had extraneous sequences, and they are thus suitable for use in commercial applications.

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