• Korean Journal of Microbiology
• /
• v.25 no.3
• /
• pp.198-204
• /
• 1987

Biparental clones and recombinant clones were obtained by spheroplast fusion of Pseudomonas putida KU218R-3 and P.putida KU428. Formation of the fusion product was the most effective when the Pseudomonas spheroplast mixture were treated with 40% plyethyleneglycol(PEG) 6000 for 10min at room temperature, The fusants which selected by indirect method were obtained at an average frequency of 10.8%. Most of the fusants were biparental clones (10.4%) and the recombinant clones were produced in low yield (0.42%). Fusants, at the frequency of 4% were obtained without PEG 6000, which shows that fusion is not strictly dependant on PEG. The stability of fusants were examined. Most of the biparental clones were segregated to parental form amd late recombinants were formed on further propagation of biparental clone but the recombinant clones were nery stable.

• Journal of Microbiology and Biotechnology
• /
• v.6 no.4
• /
• pp.286-291
• /
• 1996

A study was conducted to investigate the characteristics of segregation and alcohol fermentation of intergeneric fusants. The protoplast fusion of both Pichia stipitis CBS 5776 and Saccharomycess cerevisiae STV 89 was carried out. The fusion frequency was $5\times10^{-8}$ and among fusants selected, a fusant F5 showed the best results in ethanol production by sucrose and xylose fermentations. The performance of xylose fermentation by this fusant was better than that of P. stipitis CBS 5776 and fusant F5 exhibited sucrose fermentation patterns intermediate to the two parent strains. The fusant F5 was segregated into a pair of parental strains during the several culture passages. In the average, 91$%$ of colonies had a similar characteristics of P. stipitis while 7$%$ of colonies resembled S. cerevisiae. Only 2$%$ of colonies had the characteristics of the original fusants. At the sixth passage, all segregants resembled P. stipitis. From these results it is suggested that intergeneric protoplast fusion led to an integration of S. cerevisiae genes, rather than whole chromosomes, within the entire genome of P. stipitis.

• The Korean Journal of Mycology
• /
• v.21 no.4
• /
• pp.323-331
• /
• 1993

The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

• YAKHAK HOEJI
• /
• v.38 no.5
• /
• pp.595-601
• /
• 1994

Acetaminophen, a widely used analgesic, can be formed by N-acetylation and p-hydroxylation of aniline. Interspecific protoplast fusion technique was used to get acetaminophen directly from aniline and to increase the productivity of acetaminophen. Three auxotrophic mutants were obtained from S. griseus(ATCC 13273) and S. hygroscopicus(KCTC 1089) by N-methyl-N'-nitro-N-nitrosoguanidine(NTG) treatment. Regeneration frequencies of S. griseus$(his^-)$, S. griseus$(lys^-)$, S. hygroscopicus$(arg^-)$ were 42%, 45%, and 31%, respectively. Fusion of protoplasts carrying different auxotrophic markers was achieved by treatment with polyethylene glycol. When protoplasts were treated with 50% polyethylene glycol for 3 minutes, the fusion frequency between S. griseus$(his^-)$ and S. hygroscopicus$(arg^-)$ was $3.8{\times}10^{-5}$. The fusion frequency between S. griseus$(lys^-)$ and S. hygroscopicus$(arg^-)$ was $5.6{\times}10^{-4}$. When we checked the production of acetaminophen, thirty-four out of the fifty-six fusants produced larger amounts of acetaminophen than the parent strains did. Nine fusants produced twice more and twenty-five fusants produced one to two times more of acetaminophen than their parents.

• Korean Journal of Microbiology
• /
• v.29 no.2
• /
• pp.117-122
• /
• 1991

In order to improve the removal ability of phosphate, Spheroplast fusions were performed among auxotrophic mutants of Aeromonas hydrophila isolated from waste water, named A13 and A14, Aci37 auxotrophic mutant of Acinetobactercalcoaceticus, and auxotrophic E. coli HR262/pCE27 carring pit gene. Eight fusants obtained from this experiment showed different biochemical characteristics. When the rate of phosphate uptake among fusants (F1-F8) was investigated in Phosphate Uptake Medium (PUM), F8 strain showed the highest rate for phosphate removal, 7 times as much as control after two hours incubation. The role of cations ($Mg^{++}$ ,$Ca^{++}$ , $K^{+}$ in phosphate uptade by F8 was also investigated in PUM without each salt. $K^{+}$ seemed to be crucial. Being compared with phosphate untake rate in PUM, that in PUM without $K^{+}$ was reduced 1.5 times. Therefore, by applying F8 strain and $K^{+}$ in practical environmental system, the increased efficiency in phosphate removal can be derived.

• Korean Journal of Microbiology
• /
• v.27 no.2
• /
• pp.98-107
• /
• 1989

Intraspecific fusants, produced by protoplast fusion of auxotrophic mutants from Trichoderma koningii ATCC 26113, were segregated into various strains including parental types, non-parental auxotrophic hybrids, and prototrophic hybrids on complete plate. Interestingly, some of non-parental prototrophic hybrids revealed to have enhanced cellulolytic activity incomparison with other strains of parents or hybrids derived thereafter. It was also evident that prototrophic hybrids of aneuploid could be constructed after the spontaneous segregation of complementing fusants produced through the protoplast fusion.

• The Korean Journal of Mycology
• /
• v.21 no.1
• /
• pp.9-15
• /
• 1993

ABSTRACT: The cell fusants were constructed from complementary auxotrophic mutants of Phffia rhodozyma. The nuclear fusion of the fusants was demonstrated by several techniques including comparision of cell volume, estimation of DNA content and nuclear staining. The cell fusants were very stable for succeeding transfer culture on complex medium for more than one year. Malt extract (1%, w/v) and abscisic acid(1 mM) increased the carotenoid formation whereas gibberellic acid(5mM) and riboflavin(0.1 mM) decreased the corresponding content.

• Journal of Microbiology and Biotechnology
• /
• v.3 no.4
• /
• pp.232-237
• /
• 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
• /
• v.17 no.1
• /
• pp.1-7
• /
• 1989

To enhance the capability of starch fermentation of the transformant TSD-14, the heat treated protoplasts of TSD-14 were fused with the protoplasts of C. tropicalis (lys$^-$) in the presence of 30% (w/ v) PEG and 20 mM CaC1$_2$. Fusants were selected by nutritional complementation on minium medium and the fusion frequency was 4.4$\times$10$^{-5}$. All fusants tested were possessed of complemented traits concerning carbon compound assimilation, and the cell volumes of the fusants were approximately 1.5 times larger than the parental strains. The fusants were genetically very stable, and were able to hydrolyze alpha 1,4-glucosidic linkage as well as alpha 1,6-linkage of starch contrary to one of parents TSD-14, The most promising fusant FSC-14-75 produced 8.7% (v/v) of ethanol from 15% liquefied potato starch medium, but the result was enhanced to 9.3% (v/v) by addition of 0.3% peptone. The corresponding fermentation efficiency was 86.0%.

• Korean Journal of Food Science and Technology
• /
• v.38 no.5
• /
• pp.712-716
• /
• 2006

Saccharomyces cerevisiae and Zygosaccharomyces rouxii were electrofused and fermented in germaniumfortified nutrients to produce high-yield, organic germanium. The conditions for the preparation of protoplasts from both strains and for electrofusion were studied. The protoplasts of both cells formed long pearl chains and the cell membranes were lysed and fused through cellulase and high frequency voltage $(450{\sim}750V/128{\sim}512\;{\mu}sec)$. The fusants with the fastest growth were selected, and then characterized for their carbohydrate usage and tolerance to glucose and salts. The glucose tolerance of the fusants was better than that of S. cerevisiae and similar to that of Z. rouxii. The fusants appeared to have resistance to 12% NaCl. The cell size of the fusants was greater than that of the parental strains. The fusant cells contained more gemlanium than the parental cells did. The electrofusion of S. cerevisiae and Z. rouxii increased the cell capacity and accumulation of germanium in the yeasts. This method was proved to be effective to produce a high quantity of organic germanium.