Flocculent yeasts are useful for alcohol production because of the easy and efficient separation of yeast cells from fermenting mash. Their use will result in energy-saving in the separation of yeast cells from fermentation mash, labor-saving in the preparation of seed mash for each fermentation batch, and a higher alcohol production rate. We had already proposed an alcohol production process that used flocculent yeasts, named repeated-batch fermentation system (Saiki et al., 1996). For this system, excellent flocculent yeast strains are required.The mechanism of flocculation in S. cerevisiae cells is explained by the lectin-like theory, which assumes that specific bondings occur between surface proteins (lectins) on flocculent cells and the sugar residues intrinsic to the mannan that comprises cell walls (Miki et al., 1982). The yeast flocculation is likely under genetic control in addition to physiological and environmental factors. It was reported by Straver et al. (1994) that two flocculation phenotypes were distinguished by sugar inhibition, and further, they suggested the existence of at least two distinct lectin-encoding flocculation genes. To date, many genetic studies on flocculation in S. cerevisiae have reported several chromosomal genes affecting flocculation (for reviews, see Stratford, 1992;Watari et al.,1994b).FLO1, which is one of the dominant flocculation genes, has been cloned and sequenced by three groups (Bidard et al., 1995;Teunissen et al., 1993;Watari et al., 1994b). Recently it was shown that FLO1 should be a structural gene coding for a cell surface protein, which has a key role in the mechanism of yeast flocculation, because of the localization of its product (Bidard et al., 1995), the similarity of the N-terminal sequence between the putative FLO1 product and a protein that was purified from flocculent yeast cell surface, and the stimulative effect on the flocculation ability of the yeast cells (Straver, 1994 Bunkyo-ku, Tokyo 113-8657, Japan (Received July 17, 1998; Accepted October 8, 1998) A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.
