Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants.

Abstract

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.

Construction of FlocculentKluyveromyces marxianusStrains Suitable for High-Temperature Ethanol Fermentation

“…[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. 11) However, there have been no previous reports on the construction of flocculent S. cerevisiae strains using the FLO9 or the FLO10 gene.…”

“…The introduction of the FLO1 gene into non-flocculent brewery yeast strains has been found to confer a flocculation phenotype. 23) Similarly, there have been many attempts to express FLO genes under the regulation of heterologous promoters in nonflocculent S. cerevisiae strains, resulting in the generation of flocculating strains, 1,24,25) but producing flocculation strains in other yeast species by introducing FLO genes of S. cerevisiae has not been previously reported.The thermotolerant yeast K. marxianus has received considerable attention because of its ability to grow and initiate fermentation at high temperatures, and also because of its native utilization of xylose, arabinose, and cellobiose, which are not fermented by S. cerevisiae. 26,27) In particular, the K. marxianus DMKU3-1042 strain, isolated in Thailand, has shown the most potent high-temperature growth among 17 strains that were examined, and displays comparable ethanol fermentation properties to a S. cerevisiae strain used in Brazil.…”

“…The introduction of the FLO1 gene into non-flocculent brewery yeast strains has been found to confer a flocculation phenotype. 23) Similarly, there have been many attempts to express FLO genes under the regulation of heterologous promoters in nonflocculent S. cerevisiae strains, resulting in the generation of flocculating strains, 1,24,25) but producing flocculation strains in other yeast species by introducing FLO genes of S. cerevisiae has not been previously reported.…”

“…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.…”

125th Anniversary Review: Yeast Flocculation and Sedimentation in Brewing

“…In the first attempts to genetically engineer the flocculation ability of industrial brewer's yeasts, two non-flocculent strains were transformed with a FLO1 gene under the control of an ADH1 promoter 28,111 . Strong flocculation was achieved with apparently a single, integrated copy of FLO1.…”