• Journal of Microbiology and Biotechnology
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• v.3 no.1
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• pp.24-30
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• 1993

In order to develop a novel yeast which produces the charateristic aroma of soy sauce, a protoplast fusion between Zygosaccharomyces rouxii WFS4 and Torulopsis versatilis IAM 4993 was carried out. Auxotrophic mutants as selective markers were obtained from Zygosaccharomyces rouxii and Torulopsis versatilis by treatment of N-methyl-N -nitro-N-nitrosoguanidine. The conditions of the protoplast formation and the regeneration for fusion were examined. The protoplast fusion using polyethylene glycol 4000 led to the fusion frequency of $4~5{\times}10^{-7}\;cells/ml$. Among fusants, a fusant ST723-F31 presented the best results in terms of the aromaticity of fragrance, the growth pattern, the resistance against salt and the degree of growth according to pH. It makes easy to control the production and the balance of aroma components so that it gives a good flavor, shortens the fermentation period and, simplifies the preparation process when using a bioreactor into which fusant is immobilized.

• Korean Journal of Microbiology
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• v.27 no.4
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• pp.422-429
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• 1989

Auxotrophic mutant were isolated from wild types by the treatment with NTG as a mutagen, and the conditions of protoplast formation for them were established. The protoplasts of killer yeast Saccharomyces cerevisiae K52 were formed to the level of above 70% when cells grown for 20 hr in PM medium were treated with 200 unit/ml Lyticase 50,000 at $30^{\circ}C$ for 60 min after pretreatment of 50 mM 2-mercaptoethanol in 10mM potassium phosphate buffer (pH 7.5) containing EDTA and 0.6 M sorbitol for 15 min. Also, the protoplast of the recipient S. cerevisiae S 29 were formed to the level of above 85% as it was cultured to the log phase of 24 hr in PM medium under the same conditions. The fusion frequency between the protoplast of killer yeast S. cerevisiae K 52 and the protoplast of recipient S. cerevisiae S 29 was reached to $8.2\times 10^{-6}$ when the hypertonic regeneration medium embeded with the fused protoplasts after mixing the parental protoplasts to 10$^{8}$ cells/ml in SP buffer containing 20 mM $CaCl_{2}$ and 30% PEG 6,000 for 15 min at $30^{\circ}C$ were incubated.

• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.151-156
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• 1991

In order to construct a yeast strain having high ethanol tolerance together with good lactose fermentation ability, the protoplast fusion using Saccharomyces cerevisiae STV 89 and Kluyveromyces fragilis CBS 397 was carried out. Auxotrophic mutants of K. fragilis were obtained as a selection marker by treatment of ethylmethane sulfonate. The best mutant for protoplast fusion was selected based on the capabilities of ${\beta}-galactosidase$ production and lactose fermentation. The protoplast fusion using polyethylene glycol and calcium chloride solution led to the fusion frequence of $3{\times}10^{-6}$ and a number of fusants were obtained. Among these fusants, a fusant F-3-19 showed the best results in terms of ethanol tolerance, ${\beta}-galactosidase$ activity and lactose fermentation. The performance of lactose fermentation and ethanol tolerance by this fusant were better than those of K. fragilis. Study on the ethanol tolerance having relation to fatty acid composition and intracellular ethanol concentration revealed that the fusant F-3-19 had a higher unsaturated fatty acids content and accumulated less amount of intracellular ethanol compared with a parent of K. fragilis.

• Journal of Life Science
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• v.29 no.3
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• pp.376-381
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• 2019

To construct useful yeast strain for bioethanol production, we improved yeast harboring various phenotypes by using yeast protoplast fusion method. In this study, S. cerevisiae BYK-F11 strain which have ethanol tolerance, thermotolerance and ${\beta}-glucanase$ activity and P. $stipitis{\Delta}ura$ strain which has xylose metabolism pathway were fused by genome shuffling. P. $stipitis{\Delta}ura$ strain was constructed for protoplast fusion by URA3 gene disruption, resulting in uracil auxotroph. By protoplast fusion, several fused cells were selected and BYKPS-F8 strain (fused cell) showing both karyotypes from two parent strains (S. cerevisiae BYK-F11 and P. $stipitis{\Delta}ura$ strain) among 22 fused cells was finally selected. Sequentially, various phenotypes such as ${\beta}-glucanase$ activity, xylose utility, ethanol tolerance, thermotolerance and ethanol productivity were analyzed. The BYKPS-F8 strain obtained ${\beta}-glucanase$ activity from BYK-F11 strain and 1.2 fold increased xylose utility from P. $stipitis{\Delta}ura$ strain. Also, the BYKPS-F8 strain showed thermotolerance at $40^{\circ}C$ and increased ethanol tolerance in medium containing 8% ethanol. In this fused cell, 7.5 g/l ethanol from 20 g/l xylose was produced and the multiple phenotypes were stably remained during long term cultivation (260 hr). It was proved that novel biological system (yeast strains) is easily and efficiently bred by protoplast fusion among yeasts having different genus.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.359-363
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• 1986

S. diastaticus hydrolysised $\alpha$-1.4 linkage of the starch and was known fermenting yeast strain, but poorly hydrolized $\alpha$-1.6 linkage of the starch. To improve the starch fermentation ability of yeast, we tried that protoplast fusion between S. diastaticus and C. tropicalis and finally two starins of fusant (FPDC42, FPDC43) were obtained. C. tropicalis well hydrolysis both $\alpha$-1.4 and $\alpha$-1.6 linkanges in the starch. The protoplast of parental auxotrophic cells were fused in the presence of 10mM CaCl$_2$ and 35% of polyethylene glycol (M. W. 4,000). The fusion frequency was 10$^{-5}$ to 10$^{-6}$. Properties of the fusants(genetic stability, assimilation of carbon sources, random spore formation, copper resistance, NaCl tolerance, DNA content, cell size and growth rate) were investigated.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.88-93
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• 1989

To improve a new yeast strain capable of converting xylan to ethanol directly, we tried protoplast fusion between xylose fermenting yeast (Candida sp. X-6-41) and xylan assimilating yeast (Crypto-coccus sp. XB-33), finally selected the most promising two fusants (XFU-1 and XFU-2). As the optimum conditions for protoplast formation, the yeast cells were cultured to exponential phase in YPD and YPX containing 0.6M KCI, respectively, and then treated with zymolyase (0.25mg/$m\ell$), cellulase(4mg/$m\ell$) and 100mM 2-mercaptoethanol at pH 8 and 3$0^{\circ}C$. The protoplasts of parental auxotrophs were fused in the presence of 20mM CaCl$_2$and 40% polyethylene glycol(M.W.4000). The physiological and morphological characteristics of the fusants, such as assimilation of carbon sources, cell size, growth rate, xylanase activity and xylan fermentation ability were investigated. Xylanase activity of fusants that cultured in chemically minimal medium was higher than that of fusants that cultured in completed medium, because xylanase producing activity of xylose fermenting yeast(X-6-41) was inhibited by isoleucine.

• Korean Journal of Microbiology
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• v.24 no.4
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• pp.364-369
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• 1986

A procedure for the preparation, regeneration and fusion of protoplasts of Streptomyces tubercidicus was confirmed. Also, protoplast releasingprocesses from mycelia were observed by scanning electron microscope. Three types of protoplasts releasing processes-from the hyphal tip, hyphal end regions and lateral regions of the hyphae-were observed. More than 17% regeneration efficiency was obtained by regeneration medium that is composed of tryptone-yeast extract-sodium acetate-$MgCl_2-CaCl_2$-sucrose. Optimal concentrations of $Ca^{++},\;Mg^{++}$ and sucrose in the regeneration medium were 50mM, 0.4-0.5M respectively. Above 30% of fusion frequency of the protoplasts derived from two auxotrophic strains of S. tubercidicus was induced by polyethylene glycol 4000(60% w/v).

• Journal of Life Science
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• v.23 no.10
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• pp.1192-1198
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• 2013

To improve yeast strains for bioethanol production, yeasts with ethanol tolerance, thermotolerance, and ${\beta}$-1,3-glucanase activity were bred using yeast genome shuffling. Saccharomyces cerevisiae $BY4742{\Delta}exg1$/pAInu-exgA, which has extracellular ${\beta}$-1,3-glucanase activity, and the Aspergillus oryzae and S. cerevisiae YKY020 strains, which exhibit ethanol tolerance and thermotolerance, were fused by yeast protoplast fusion. Following cell fusion, four candidate cells (No. 3, 9, 11, and 12 strains) showing thermotolerance at $40^{\circ}C$ were selected, and their ethanol tolerance (7% ethanol concentration) and ${\beta}$-1,3-glucanase activity were subsequently analyzed. All the phenotypes of the two parent cells were simultaneously expressed in one (No. 11) of the four candidate cells, and this strain was called BYK-F11. The BYK-F11 fused cell showed enhanced cell growth, ethanol tolerance, ${\beta}$-1,3-glucanase activity, and ethanol productivity compared with the $BY4742{\Delta}exg1$/pAInu-exgA and YKY020 strains. The results prove that a new yeast strain with different characters and the same mating type can be easily bred by protoplast fusion of yeasts.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.221-224
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• 1989

Amylolytic filamentous fungus, Aspergillus oryzae and nonamylolytic sugar fermentable yeast, Saccharomyces cerevisiae were fused by protoplast fusion in order to develope microorganisms having their intergrated function. Aminoacid auxotrophic properties were used as a genetic marker of protoplast fusion, and 35% PEG 4000 was used as a fusogenic agent. Complementation frequengy of fusion was $4.6\times 10^{-6}$ Obtained fusants showed the morphology of yeast strains, the amylase activity and the ethanol productivity. Among the properties of the fusants, morphology and prototrophic property were sustained stably but their ethanol productivity from starch was reduced. Although fusant strains had 0.5-fold ethanol productivity compared to that of S. cerevisiae in glucose medium, they produced ethanol from strach by direct fermentation.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.305-310
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• 1986

To improve the starch fermentation ability of yeast, hybrids were introduced by protoplast fusion of S. cerevisiae and S. diastaticus. The protoplasts of parental auxotrophic cells were fused in the presence of 10 mM CaCl$_2$and 30% of polyethyleneglycol (M.W 4, 000). The frequencies of fusant formation varied depending upon the strains used and were 3.51$\times$10$^{-4}$ to 5.04$\times$10$^{-4}$ for the regenerated protoplasts. The strains capable of extensive starch hydrolysis produce only 10% to total fusants. The 4 strains were finally selected by the results of starch fermentation and genetic stability test. The DNA content and cell volume of the fusants were greater than those of the parental strains.