• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.528.2-528
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• 1986

섬유소 분해효소를 생산하는 P. verruculosum으로부터 유도된 영 양요구성 돌연변이주의 원형질체 융합을 위한 조건을 검토하였다. 18-20시간 배양한 각 영양요구성 돌연변이주 균사체에 Novozym 234를 처리하여 원형질체를 추출할 수 있었으며, 원형질체 생성량은 각 영양요구성 돌연변이주의 균사체 40mg (dry weight) 당 2.4-3.0$\times$10/suo 7/수준이었고, 원형질체 재생 완전 배지상에서의 환원율은 26.6-42.4% 수준이었다. 원형질체 융합을 위한 Polyethylene glycol (PEG) 6000의 최적농도는 20%였으며, PEG 최적 처리시간은 10분, CaCl$_2$의 최적 첨가농도는 10mM, 최적 pH는 5.5였고, 원형질체 융합 최적조건 하에서의 융합율은 1.8$\times$$10^{-3}$ 수준으로 나타났다. 모균주와 융합체로부터 측정된 DNA 양의 차이로 보아 융합체의 염색체는 aneuploid 상태임을 알 수 있었으며, filter paper를 기질로 한 Cellulase 활성측정에서 융합체가 야생형 균주보다 2배이상, 모균주 보다는 1-3배 이상 향상되었다.

• Microbiology and Biotechnology Letters
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• v.28 no.2
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• pp.80-86
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• 2000

To develop thermotolerant ethanol producing yeast strains, the protoplasts of Saccharomyces carlsbergensis having good fermentability at $30^{\circ}C$ and Kluyveromyces marxianus able to grow at $42^{\circ}C$ were fused. Under the optimal conditions for protoplast formation, the frequency of protoplast formation of S. carlsbergensis was 92 - 94% and that of K. marxianus was 98%. Fusion frequency between S. carlsbergensis and K. marxianus was $1.4\times10^{-6}-4.8$\times10^{-7}$. Among the 27 fusants obtained, 6 fusants were able to grow at $42^{\circ}C$. While the parental strains produced 3.2-3.4%(w/v) ethanol after 3 days from the fermentation medium containing glucose, fusants SK41-4 and SK53-22 produced 5.2%(w/v) ethanol in the same condition. The thermotolerance of SK53-22 was not high, but that of SK41-4 was quite high.

• The Korean Journal of Mycology
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• v.14 no.1
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• pp.9-15
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• 1986

Interspecific hybrids of Pleurotus ostreatus and Pleurotus florida were formed by using protoplasts of complementing auxotrophs. The genetic markers were shown to segregate and recombine in the first generation of monosporus isolates from basidiocarp of seven fusion products. The analysis provides proof of heterokaryosis and strong evidence for haploidy of vegetative nuclei, a sexual cycle consisting of nuclear fusion and meiosis. In all the crosses there was no evidence of linkage between the genetic markers. Clamp connections were formed in monosporus mycelia from basidiocarp of fusion products.

• 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.25 no.4
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• pp.274-281
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• 1987

The effects of bovine serum albumin, myoinositol and ergosterol on protoplast formation, regeneration and fusion from auxotrophic mutants of Candida pseudotropicalis were examined. Frequency of protoplast formation ranged from 48 to 98% depending on auxotrophic types. When myoinositol (0.5mg/ml) and ergosterol (0.1mg/ml) were supplemented in the medium of cell growth, and bovine serum albumin (4mg/ml)was added to protoplasting buffer, 50-100% of cells were converted to protoplasts. Such a treatment of three additives improved 2.2-3.0 fold of regeneration rate of protoplasts. The fusion frequencies between complementary auxotrophs ranged from $7.0\times 10^{-4}$ to $1.5\times 10^{-3}$ in the optimal conditions. These values showed 1.9-2.3 fold increase when compared with fusion frequencies obtained without the treatment of additives. These results suggested that these comsion frequencies obtained without the treatment of additives. These results suggested that these xompounds may improve protoplast regeneration and fusion between complementary auxotrophs used in this study.

• The Korean Journal of Mycology
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• v.20 no.4
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• pp.324-336
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• 1992

On the five interspecific protoplast fusants of Ganoderma lucidum and G. applanatum was the antitumor test performed. The fusant F-2 was selected, to examine the cultured mycelia (protein bound polysaccharide) as antitumor components. When a dose of 20 mg/kg/day of each components purifed from F-2 fusant was, i.p., injected into ICR mice, the inhibition ratio of Fr. II against the solid form of sarcoma 180 increased to 1.5 times as compared with that of their parents. When Fr. II was examined for immunopotentiation activity, it increased the amount of the superoxide anion in activated macrophages to 1.2 times and the count of hemolytic plaque forming cells in the spleen to 4.3 times as compared with that of each control group. Its chemical analysis showed 85.2% polysaccharide which consisted of glucose, galactose, mannose, fucose and xylose, and 0.39% protein of 15 amino acids. The content of hexosamine was 0.39% and the molecular weight of Fr. V was $5.6{\times}10^4$ dalton.

• 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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.30 no.3
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• pp.395-402
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• 2001

A fusant FG-21 was selected on the basis of higher ${\gamma}-GTP$ activity following fusion process between SM-2 and SM-10 of Bacillus subtilis mutants. ${\gamma}-GTP$ activity of the mutant FG-21 was increased up to 612 U/mL when grown for 36 hr at $37^{\circ}C$ in culture media containing 1% glycerol 1% glycerol, 1% peptone, 0.1% citric acid, 5 mM $K_2HPO_4$, 1 mM $FeCl_3$, 1 mM $MgCl_2$, 1 mM $NH_4Cl$, pH 7.0. In fusnat FG-21, the ratio of protein to total sugar contents for biopolymer A was 38 to 59. for biopolymer B from parental strains it was 19 to 78. Fructose contents determined by HPLC were $573.7\;\mu\textrm{g}/mg\;and\;764.4\;\mu\textrm{g}/mg$ for biopolymer A and B, respectively. And glutamic acid content were $163.7\;\mu\textrm{g}/mg\;and\;94.6\;\mu\textrm{g}/mg$ for biopolymer A and B, respectively. In fusant FG-21, the ratio of fructose to glutamic acid contents for biopolymer A was 78 to 22. For biopolymer B from parental strains it was 89 to 11.

• The Korean Journal of Mycology
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• v.22 no.4
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• pp.378-385
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• 1994

Interspecific somatic hybrids were obtained by protoplast fusion between Pleurotus ostreatus and Pleurotus sajor-caju. The fusion products between incompatible strains did not form clamp connections. Fruiting body of the clampless fusants was induced by light-dark cycle on saw-dust-rice bran substrate in glass bottles. Out of them, seven somatic hybrids produced fruiting bodies of intermediate morphology of the two species. Light and low temperature were the initiating factors for the development of clamped hyphae from the clampless mycelial colonies. All of these basidiocarps had clamp connections. Eight fusants from the six crosses were analysed with the segregation of genetic characters by random spore isolates. In the three combinations, unexpected alleles were shown. Somatic hybrid between P188 (P. ostreatus 2-1 + P. sajor-caju 2-53) and P. florida 2-3 by triple cross produced fruiting bodies similar to those of fusant between P. ostreatus and P. florida. All the genetic charaters from the three strains were shown to segregate and recombine.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.303-309
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• 1988

In order to establish the process of interspecific protoplast fusion of the genus Cellulomonas capable of utilizing of cellulose, C. flavigena NCIB 12901 and Cellulomonas sp. CSI-1, the optimum conditions for the regeneration and fusion were examined. The condition of suitable osmotic stabilizer for the protoplast regeneration of C. flavigena was established by using 0.4M sorbitol. And then, by addition of 3% po]yvinyl pyrrolidone (PVP) to cell wall regeneration medium, regeneration frequency was increased 3 times higher than that without PVP addition. The optimum conditions for the interspecific protoplast fusion between auxotrophic and antibiotics resistant mutants were obtained with 40%(W/V) of PEG (polyethylene glycol) 6000 as the fusogenic agent and 25mM of CaCl$_2$on treating time for 15 min. The fusion frequency between mutants was from 2.0$\times$10$^{-4}$ to 4.0$\times$10$^{-4}$ under the optimum conditions. The fusants were confirmed to revert from protoplast to cells of rod type during regeneration process and the aggregation of protoplast by PEG was observed. Also the progress of fusion was observed by scanning electron microscopy, Many isolated fusants were shown to be complement clones of both parents which occured at a high frequency among the isolated clones.