• Mycobiology
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• v.31 no.1
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• pp.32-35
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• 2003

Restriction enzyme-mediated integration(REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 ${\mu}g$ of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 ${\mu}g$ of linearized pTRura3-2 DNA was added into $1{\times}10^7$ protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.

• Archives of Pharmacal Research
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• v.15 no.1
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• pp.30-34
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• 1992

In order to obtain acetaminophen, a popular analgesic-antipyretic, through microbial p-hydroxylation and N-acetylation of aniline, various fungi and bacteria were secreened. Among them, Streptomyces species were chosen for strain improvement by the use of interspecific protoplast fusion technique. Two interspecific fused strains were developed between S. rimosus (N-cetylation function) and S. aureofaciens (p-hydroxylation function) and also between S. lividans and S. globisporus. For efficient protoplast fusion and cell wall regeneration, various conditions were examined. In a typical experiment of mixed S rimosus ($pro^- \;his^-$) and S. aureofaciens ($ilv^-$) protoplasts with 40% (w/v) polythylene glycol 3350 (PEG) for 3 min gave $8.3\times10^{-7}$ of fusion frequency. Treatment of mixed S. lividans (pant-) and S. globisporus (leu-) protoplasts with 50% (w/v) PEG for 3 min at $30^\circ{C}$ gave $1.2\times10^{-6}$ of frequency. Among the fused strains, up to 40-50% increase in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation power was observed. To investigate the possibility of plasmid involvement in p-hydroxylation of acetanilide, plasmid curing was attempted. We found that cells treated with acriflavine (at the frequency of 100%) and cells regenerated from protoplsts of S. auroefaciens (2% frequency) lost their p-hydroxylation function.

• Microbiology and Biotechnology Letters
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• v.19 no.1
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• pp.31-36
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• 1991

A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Eschurichia cwli plasmid pBELl which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSRl of Corynebacterium glutamicum. pCE1301 was transformed C. glutaicum by PEG-mediated protoplast method and its transformation efficiency was about $3.0\times 10^3$ transformants per $\mu g$ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRl, respectively. 'The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected. pCI31301 was also introduced into Brevibacterium flavum and E coil, and replicated in those strains. pCE1301 was proved to be useiul in cloning the homoscrine dehydrogenase gene from C. glutamicum.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.687-690
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• 1999

Bacillus stearothermophilus No. 236 isolated from the soil is a strong xylan degrader producing all the xylanolytic enzymes. However, the strain was discovered to be highly intractable to its transformation. In the present study, we have developed a reliable method for transformation of B. stearothermophilus No. 236 by a systematic examination of several factors which might have an influence on the efficiency of electrotransformation. Notably, we found that the most critical factor influencing the transformation efficiency (TE) was the incubation temperature after pulsing, with its optimum incubation of $37^{\circ}C.\; At\; 50^{\circ}C$, the optimum growth temperature of the B. stearothermophilus strain, the transformants could not be obtained at a recognizable level. The combination of field strength of 7.5 kV/cm along with pulse duration of 10 msec (resistance of $400{\Omega}\; and\; capacitance\; of\; 25{\mu}F$) was shown to be the best electrical parameters at the incubation temperature of $37^{\circ}$. A higher TE was obtained when the cells were harvested at an early-exponential phase. Twenty percent of PEG-8000 in a suspension buffer and an addition of 0.1% glycine in the growth medium resulted in about 4-fold and 3-fold increases in TE, respectively. We also found that the plasmid DNA which had been cycled through the host B. stearothermophilus cells enhanced TE by one order of magnitude higher. Under the presently described conditions, $2.5{\times}10^{5} transformants per ${\mu}g$ DNA was attained.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.125-131
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• 1986

In order to obtain the optimum conditions for intact yeast cell transformation in the various yeast host-vector systems, 3 yeast plasmid vectors, YRp7, YEpl3 and YIp5 were introduced into 5 yeast hosts, Saccaromyces cervisiae Dl3-1A, DKD-5D, DBY-746, MC-16 and S2022D with various transformation conditions, and plasmid stabilities in all the transformants were also observed. The highest transformation frequencies in all the host-vector system were obtained in the 16 hour Cultured cell (5.4 $\times$ 10$^6$ - 2.4 $\times$ 10$^8$cells/$m{\ell}$) treated with 0.1-0.2 M lithium chloride in 0.1 M tris-HCl (pH 7.6), 35% polyethylene glycol 4000, and heat-shocked at 42$^{\circ}C$ for 5 minutes after 60 minutes of induction. The intact cell transformation got more transformation frequency in DKD-5D (YRp7) and DBY-746 (YEpl3) than protoplast transformation, but reverse tendency was observed in DKD-5D (YEp13) and Dl3-lA (YRp7). The transformants, D13-1A (YRp7) and DKD-5D (YRp7) were very unstable in selective medium, with 80 to 85% of the transformants losing the plasmid after 70 generations, but the transformants, DKD-5D (YEpl3) and DBY-746 (YEpl3) were quite stable, with 35% of the transformants losing the plasmid.

• The Korean Journal of Mycology
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• v.17 no.4
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• pp.209-213
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• 1989

Transformation of an auxotrophic requirement for uracil in Pleurotus florida P101 has been achieved using chimeric vector containing Aspergillus nidulans ans 1, and Neurospora crassa pyr 4 DNA. Protoplasts of $Ura^-$strains of P. florida were incubated with plasmid pDJB3 containing the cloned pyr 4 gene in the presence of polyethylene glycol and $CaCl_2$. Transformants could grow on MMM showing mitotical stability. Southern hybridization analysis of DNA isolated from transformants showed that the Neurospora pyr 4 gene and vector sequence might be integrated into the P. florida chromosomes. As the transformants were monokaryon, each transformant was mated with the other monokaryon. Fruitbody shape of untransformant was eroded type but those of transformants were eroded type, funnel type, plane type and ungrowing cap type.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.988-998
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• 2015

The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl₂-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30^oC. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

• 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.

• Journal of Life Science
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• v.14 no.2
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• pp.368-373
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• 2004

We have obtained fertile transgenic rice plants resistant to the broad spectrum herbicide Bast $a^{(R)}$ (active ingredient phosphinothricin, PPT) by PEG-mediated transformation procedure. The plasmid pCaMV35S::Bar was used to deliver the bar gene into embryogenic suspension culture-derived protoplasts of rice (Oryza sativa L.). Transformed plants were regenerated and selected on medium containing 15 mg/l of phosphinothricin. Stable integration and expression of the bar gene in transgenic rice plants was confirmed by Southern and Northern blot analysis. Transgenic $R_1$ plants were also confirmed by assays for phosphinothricin acetyltransferase (PAT) activity. The bar gene was expressed in the primary transgenic rice plants and in the next generation progeny, in which it showed a 3 : 1 Mendelian inheritance pattern. Transgenic $R_1$ and $R_2$ plants were resistant to the herbicide Bast $a^{(R)}$ when sprayed at rates used in field practice.ice.