• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.232-237
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• 1993

A strain of yeast which can convert starch directly to ethanol was developed by the intergeneric protoplast fusion between Schwanniomyces alluvius possessing $\alpha$ amylase as well as glucoamylase with debranching activity and FSC-14-75 which previously had been formed from a heterologous transformation and subsequent intergeneric protoplast fusion. Fusants were selected on minimal medium after protoplasts of auxotrophic mutant of S. alluvius fused with heat-treated protoplasts of FSC-14-75 in the presence of 30%(w/v) PEG and 20 mM $CaCl_2$. The fusion frequency was in the range of $10^{-6}$ order. All fusants tested were intermediate types of parental strains for carbon compound assimilation, and their cell volumes were approximately 1.1 times larger than FSC-14-75 and 1.8 times larger than S. alluvius. The fusants were unable to sporulate like FSC-14-75, while S. alluvius could sporulate. In flask scale the most promising fusant, FSCSa-R10-6, produced 7.83%(v/v) and 10.17%(v/v) ethanol from 15% and 20% of liquefied potato starch, respectively, indicating that the fermetation efficiency of each case increased 1.2 times and 1.6 times than that of FSC-14-75. The elution pattern on DEAE-cellulose chromatography showed that FSCSa-R10-6 has four distinct amylase peaks of which two peaks originated from S. alluvius and the other two from FSC-14-75. These results suggest that the enhanced fermentation efficiency of the fusant might be due to almost-complemented parental amylases.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.311-318
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• 1986

Glucoamylase and ethanol productivities of HSDD-170 and HSDM-119 formed by S. cerevisiae and S. diastaticus protoplast fusion were investigated. For the production of the glucoamylase, soluble starch as carbon source, yeast extract and C. S. L as nitrogen source added into the basal medium were favorable. The production of the enzyme reached at maximum after cultivation of the fusant for 4 days at 3$0^{\circ}C$, aerobically. The properties of glucoamylase produced by fusants were very similar to those produced by S. diastaticus as based on optimum temperature, pH stability. In alcohol fermentation from starch, strain HSDD-170 fermented starch faster than either of its parental strains. The maximum of alcohol yield in 15% of liquefied potato starch was 7.5% (v/v).

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.46-49
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• 1992

The availability of Phaffia rhodozyma as an astaxanthin sources in the aquaculture industry is limited because of the low carotenoid content of natural isolate. In this study, we have used the protoplast fusion technique to construct cell hybrids with an increased content of astaxanthin from P. rhodozyma. Cell hybrids (F307 and F406) obtained were very stable and produced considerably more astaxanthin (> 1 mg/g yeast) than the wild parent. Karyogamy was confirmed by the isolation of recombinants after mitotic segregation of parental auxotrophic genetic markers, the increased amount of chromosomal DNA/cell and the presence of single nucleus/cell.

• KSBB Journal
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• v.5 no.3
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• pp.255-261
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• 1990

Cell fusion between complementary mutants isolated from astaxanthin-producing yeast, Phaffia rhodozyma, was carried out to obtain astaxanthin-overproducing strains by protoplast fusion technique. The frequency of protoplast fusion was ranged from 2.3$\times$10-5 to 6.0$\times$10-5, and nuclear fusion in the cells of hybrids was demonstrated by several techniques such as isolation of recombinants after mitotic segregation of parental genetic markers, estimation of DNA content, direct observation of nuclei with nuclear staining, and comparison of survival rate to UV exposure. One of several hybrids, Fl, showed approximately 3-fold increase in astaxanthin content when compared with wild parent.

• Korean Journal of Microbiology
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• v.37 no.4
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• pp.312-316
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• 2001

To improve the ethanol fermentability, four Saccharomyces yeast strains with efficient ethanol fermentability were subjected to rare-mating and protoplast fusion. Using these 4 strains, 5 different combinations of mating-pair or fusion-pair were constructed and their hybrids or fusants were obtained. From the statistical analysis of the results of the ethanol fermentation by the hybrids of the different mating-pair or fusion-pair, no difference was found in ethanol production, but [S. kluveri $khl{\times}S$ cerevisiae cp3] pair was shown to be the best combination which can produce high cell-viability. In fact, the clone No. 3 of the [S. kluveri $khl{\times}S$ cerevisiae cp3] pair was selected as the best strain which produced ethanol of 10.11% (w/v) or 12.81% (v/v) from 25% (w/v) glucose at $33^{\circ}C$ for 3 days with the residual sugar of 3.53% (w/v), viability of 62.65%, fermentation efficiency of 92.2%.

• KSBB Journal
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• v.25 no.6
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• pp.565-571
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• 2010

To develop thermostable ethanol fermentative yeast strain for lignocellulosic simultaneous saccharification and fermentation, high ethanol producing yeast, Saccharomyces cerevisiae CHY1012 and thermostable yeast, Kluyveromyces marxianus CHY1703 were fused by protoplast fusion. The thermostable fusant, CHY1612 was identified as a Kluyveromyces marxianus by phenotypic and physiological characteristics, as well as molecular analysis based on the D1/D2 domains of the large subunit (26S) rDNA gene and the internally transcribed spacer (ITS) 1 + 2 regions. For lignocellulosic ethanol production, AFEX pretreated barley straw at $150^{\circ}C$ for 90 min was used in a simultaneous saccharification and fermentation (SSF) process using thermotolerant CHY1612. The SSF from 16% pretreated barley straw at $43^{\circ}C$ gave a saccharification ratio of 90.5%, a final ethanol concentration of 38.5 g/L, and a theoretical yield of 91.2%. These results show that K. marxianus CHY1612 has potential for lignocellulosic ethanol production through simultaneous saccharification and fermentation with further development of process.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.383-389
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• 1985

Optimum conditions of PEG treatment for the intergeneric fusion of yeast protoplasts were investigated. Fusants were selected by nutritional complementation on minimal medium. The intergeneric fusion frequency between pro-toplasts of S. cerevisiae and C. tropicalis was distributed 10$^{-4}$ to 10$^{-6}$, depending on the combination of parental strains. PEG 4000 or 6000 are equally effective. 30%(w/v) PEG 4000 was found to be optimum and below 20% its stabilizing effect was lost, resulting in protoplast lysis, and optimum pH was 8.0. The efficiency of PEG was enhanced by higher temperature of the PEG solution, and by the addition of Ca ions. The stimulating effect of Ca ions in the range of 1 mM to 100 mM proved similar.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.158-164
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• 2008

The fusants which contain starch utilizing ability and alcohol fermenting ability were developed by protoplast fusion of Saccharomyces cerevisiae KOY-1 and Saccharomyces diastaticus KCTC 1804. Sacharomyces cerevisiae KH-12 was obtained by haploid induction from Saccharomyces cerevisiae KOY-1. The auxotropic mutants of yeast were obtained by using an ethylmethane sulfonate (EMS). The frequency of protoplast formation in Saccharomyces cerevisiae KOY-1 $(Met^-)$ and Saccharomyces diastaticus KCTC 1804 $(Trp^-)$ were 90.5% and 97.7%, respectively. The frequency of fusant formation was $1.79{\times}10^{-4 }$ for the regenerated protoplast and the 1,000 fusants were obtained. Fusant FA 776 was selected as a potential yeast which contain an alcohol fermenting ability in the starch medium. The genetic stability was 4.64% for 10 passages of generation. Fusant FA 776 produced 13mg/ml of alcohol in 24% starch medium and showed 1.86-fold higher alcohol fermenting ability than Saccharomyces diastaticus KCTC 1804.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.546-552
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• 1992

Ethanol-tolerant strain, S. eerevisiae BUI a26 ($alc^r thr^-$) and gJucoamylase-producing strain, S diastatieus AI5a6 (STA+ hom-) were prepared by means of genetic manipulation, Protoplast fusion was carried out to introduce STA gene from AI5a6 strain to BUla26 strain, Protoplast formation was shown at 0,8 M sorbitol and 200 Jig/ml to 400 Jig/ml zymolyase treatment for 2 hours incubation, Fusion frequency was $ 3.25 {\times} 10^{-3}$ to the regenerated protoplast number using PEG 6000 for 90 min incubation. The excellent fusants with genotype of STA- $alc^r thr^-$ hom+/STA+ ($alc^s thr^+$ hom- (2n), F7 and FIO, were selected by ethanol-tolerant, ethanol fermentation, and glucoamylase production tests, Glucoamylase production of AI5a6 showed 2,7 units, but 4.2 or 8.4 units for F7 or FIO fusant at $30^{\circ}C$, Ethanol fermentation from 32% glucose by BUla26 was 14,0%(v/v) in fermentaion medium for 5 days incubation, but 14.5% or 15,0% for F7 or FIO strain, respectively. Ethanol fermentation from 5% starch was 2,0% by F7, or 1.8% by FIO strain in fermentation medium for 5 days fermentation.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.377-382
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• 1985

As an essential previous step towards the development of cell fusion to breed a new brewing yearst strain, several factors predicted to affect the protoplast formation of S. cerevisiae, C. tropicalis and E. fibuligera were investigated in order to obtain the protoplasts in high yields. The optimum pH and temperature for the protoplas formation were 7.5 and 35$^{\circ}C$, respectively. Pretreatment of the yeast cells with 2-mercaptoethanol stimulated the protoplast formation and 50mM of the reagent was found as effective. Among several osmotic stabilizers tested for their effect on protoplas formation, 0.6M KCI was comparatively favorable.