• 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.

• Korean Journal of Food Science and Technology
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• v.20 no.2
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• pp.164-169
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• 1988

Protoplast susion between Zygosaccharmoyces rouxii M-12 and Saccharimyces cerevusuare M-43 were investigated for breeding of a new brewing yeat strain for soy sauce. Auxotrophic mutants of Zygosaccharomyces rouxii ZRM-83 ($Met^-,\;Thr^-$) and Saccharomyces cerevisiae SCM-46 ($Lys^-,\;Arg^-$) were selected by treatment of 3.0% ethylmethane sulfonate and nutritional complementary method. Protoplast of both strains were more effective by treatment of 0.05mg/ml zymolase 20T for 60min. Fusion effeciency was much higher by treatment of 30% PEG 6,000 for 30min and fusion frequencies were $10^{-4}{\sim}10^{-5}$. These fusants originated from two protoplasts had properties of big cell size and much DNA content.

• Journal of the Korean Society of Tobacco Science
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• v.20 no.1
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• pp.40-49
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• 1998

Protoplasts were isolated from mesophyll of tobacco(Nicotiana glauca) transformed with kanamycin-resistant gene (NPT II gene) and potato hairy root callus containing Ri plasmid of Agrobacterium rhiEogenes, and protoplasm fusion was made between the isolated protoplasts. The transgenic tobacco leaf tissue could grow on the media containing high concentrations of kanamycin, but not on the phytohormone-free media. On the other hand, the potato hairy root calli could be cultured on the phytohormone-free media but not on media containing more than 40 ㎍/ml kanamycin. In these conditions, the viability of both protoplasts were above 90%, These selection markers were used for the selection of protoplasts fused between the two, i.e. protoplast fusion was detected using selection media containing 100㎍/ml kanamycin and with no phytohormone. The mixture of 1.0% cellulase, 0.3% macerozyme, and 0.7M mannitol was best for the maximum protoplast production for tobacco, and that of 2.0% cellulase, 2.0% macerozyme, 1.0% dricelase, and 0.5M mannitol for potato. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution on the selectable medium. Cell walls were regenerated after 5 days in this medium, and colonies were alive until 4 weeks after cultural, but died after 6 weeks.

• Korean Journal of Microbiology
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• v.24 no.3
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• pp.243-250
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• 1986

Protoplast formation and regeneration from wild-type and auxotrophic mutants of Candida pseudotropicalis CBS 607 as well as fusion between complementary mutants were carried out. Frequencies of protoplast formation from wild-type and histidine or adenine requiring mutants ranged from 96 to 100% whereas those from methionine or tryptophan auxotrophs were 52 and 72%, respectively. When bovine serum albumin(4mg/ml, BSA) was added to protoplasting buffer for cells of methionine or tryptophan auxotrophs grown in a medium supplemented with myoinositol(0.5mg/ml), 96-99 % of cells were converted to protoplasts. Protoplasts were regenerated at the frequencies ranging from 18 to 20%. However, the addition of BSA to protoplasting buffer and the supplement of myoinositol to a medium of cell growth doubled the regeneration rate except adenine auxotroph in which such an improvement was not observed. It was found that optimal concentrations of polyethylene glycol and $CaCl_2$ are 20% and 100mM while optimal pH and exposure time are 6.0 and 30min. The fusion frequencies between complementary mutants ranged from $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ and were enhanced by the improvement in the rate of protoplast regeneration. When histidine auxotroph was fused with tryptophan mutant, several fusion products were obtained which were found to be in the state of aneuploid or diploid, judging from DNA content and the presence of a large nucleus in the products.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.175-181
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• 1988

To produce L-lysine from cellulosic substrate, the intergeneric protoplast fusion between Cellulomonas flavigena and Corynebacterium glutamicum, Cellulomonas flavigena and Brevibacterium flavum was performed. The fusion frequencies were 1.9$\times$10$^{-6}$ to 2.1$\times$10$^{-6}$ for the regenerated protoplasts when two parental strains were treated with 30% of polyethyleneglycol (M.W.6000) containing 5 mM EDTA at 3$0^{\circ}C$ for 30 min. Two fusants, FCB3 and FCC 19 were finally selected by comparision of their genetic stability and L-lysine productivity. The properties of fusants-DNA con-tent, G＋C content and L-lysine productivity-were investigated. The DNA content of fusants was greater than those of the parental strain and their G＋C contents are equal to half of total G＋C con-tent of two parental strains. The fusants showed high productivity of L-lysine from carboxy methyl cellulose as substrate.

• 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.

• Applied Biological Chemistry
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• v.34 no.2
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• pp.174-179
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• 1991

Protoplast fusion was carried out between Brevibacterium flavum and Corynebacterium glutamicum. For the protoplast fusion, various mutants were isolated from Brevibacterium flavum ATCC 21493 and Corynebacteriurn glutamicum ATCC 21831. The optimum conditions for protoplast fusion of these mutants were examined. In the present work, the authors obtained a fusant, MWF 9031, by the intergeneric protoplast fusion between Brevibacterium flavum 108-125 and Corynebacterium glutamicum 41-214A, which was excellent in L-arginine fermentation. Fusant MWF 9031 was found to accumulate a large amount of L-arginine reached 32.5 mg/ml with a medium containing 10% glucose. The fusant possessed intermediate characteristics between the parental strains and the stability was found to retain for 60 days.

• 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.

• Korean Journal of Microbiology
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• v.26 no.2
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• pp.82-87
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• 1988

The genetic analysis and characterization of protoplast fusion hybrids between complementary auxotrophic mutants of Candida pseudotropicalis were carried out. Nuclear fusion appeared to occur in fusion hybrids (e.g., F15 and F33), as strongly suggested by isolation of recombinants after mitotic segregation of parental genetic markers. This was confirmed by KNA content, nuclear staining and comparison of survival rate to UV light. After keeping fusion hybrids for approximately one year, the frequency of spontaneous mitotic segregation was $3.0\times 10^{-4}$ - $8.1\times 10^{-4}$ while that of induced mitotic segregation was $1.4\times 10^{-3}$- $1.7\times 10^{-3}$. These results suggested that they maintained stable karyogamy state. It was also found that the production of $\beta$-D-galactosidase from F15, F33 and F158 was somewhat increased when compared with that from either auxotrophic parents or wild type.

• YAKHAK HOEJI
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• v.35 no.1
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• pp.30-37
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• 1991

Streptomyces actuosus DMCJ-49 and Streptomyces minoensis DMCJ-144 produce the .alpha.-amylase inhibitor. Inerspecific protoplast fusion technique was used to increase the productivity of .alpha.-amylase inhibitor. Four auxotrophic mutants were obtained respectively from two strains by N-methyl-N'-nitor-N-nitrosoguanidine(3mg/ml) treatment. The optimum conditions for the protoplast formation of Streptomyces actuosus DMCJ-49 ade was as follows; 1.2% w/v of glycine, 3mg/ml of lysozyme, and 30 min of lysozyme treatment followed by 36 hr. incubation in the protop-last formation medium. In case of DMCJ-144-his those were 1.2%w/v, 3 mg/ml, 30 minutes and 60 hours, respectively. Regeneration was accomplished with hypertonic soft agar medium that contained 0.4M sucrose, 20mM CaCl$_2$, 50 mM MgCl$_2$ and low levels of phosphate. Fusion of protoplasts carrying different auxotrophic markers was achieved by treatment with polyethylene glycol. The optimum concentration of polyethylene glycol 1450 for the production of recombinants was 40%w/v. When the protoplasts was treated with 40% polyethylene glycol for 30 minutes, the frequency of recombinants was 6.5$\times$$10^{-3}$ and the $\alpha$-amylase inhibition activity of $ade^-his^-$ No. 4, which is the fusant with the most improved activity increased from 33 to 125 I.U./ml.