• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.215-218
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• 2000

Aspergillus oryzae is a filamentous fungus classified in the group Aspergillaceae Ascomycetes. It is an important microorganism for industrial production of enzymes and fermented food productions. The genetic transformation system in A. oryzae was used to protoplast mediated transformation with PEG/$CaCl_2$. When the protoplast was used, the regeneration efficiency was decreased and then transformation frequence was also effected. In this study, fungal transformation was carried out by bypassing the protoplast isolation step, changing enzymes, such as hemicellulase and celluclast, and decreasing the culturing time for the increment of the transformation efficiency. 83 transformants/10ug of DNA with hemicellulase were obtained, compared with less than 10 transformants with novozyme234 and celluclast.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.10a
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• pp.85-91
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• 2000

Aspergillus oryzae is a filamentous fungus classified in the group Aspergillaceae Ascomycetes. It is an important microorganism for industrial production of enzymes and fermented food productions. It secrets large quantities of proteins or enzymes into the culture medium which makes this organism appealing for the production of heterologous proteins. Recently Electric field-mediated transformation method, electroporation, has been applied to fungal transformation. In this study, fungal transformation was carried out by bypassing the protoplast isolation step, decreasing the culturing time and non-protoplast transformation for the increment of transformation efficiency. Transformants were obtained with electroporation in optimal condition 2,500 voltage, 1,540 ohm and 0.50 capacitance. More than 1,000 transform ants were obtained with 6-10 hrs cultured mycelia without enzyme treatment, called non-protoplast transformation.

• Microbiology and Biotechnology Letters
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• v.21 no.5
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• pp.453-460
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• 1993

The extracellular bacteriolytic enzyme from alkalophilic Bacillus sp. YJ-451 was endopeptidase which hydrolyzes the peptide bond at the amino group of D-glutamic acid in the peptidoglycan. Protoplast transfomation system of B. subtilis by the lytic enzyme that differs, in mechanisms, from lysozyme which was used to transformation of B. subtilis was investigated. High protoplast yield was obtained from cells cultured in PAB at the late logarithmic growth phase.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.683-688
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• 2005

A transformation system mediated by Agrobacterium tumefaciens is routinely used for the genetic engineering of plants. Here, we report an efficient and stable method for transformation of heterogeneous genes into an industrial Cephalosporium acremonium by using a similar transformation system established in plants. Both the phleomycin-resistant gene and vgb gene were used as screening markers to confirm the success of transformation by either Southern hybridization or PCR amplification. It was found that acetosyringone (AS) was necessary only for protoplast transformation and the heterogeneous genes transferred were integrated into the genome of C. acremonium. The transformation efficiency obtained with this system was much higher than the conventional techniques used for transformation of C. acremonium.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.345-348
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• 1985

Recombinant chimeric plasmid constructed with Xba I digested pUBl10 and -pE194 was transformed by polyethylene glycol induced protoplast transformation system into Bacillus subtilis BR 151 on the mannitol regeneration media, and two genes of antibiotics resistance were expressed simultaneously in the transfromant. Transformation frequency of the recombinant plasmid was 6.5 $\times$ 10$^{-5}$ on the mannitol regeneration agar plate containing neomycin and erythromycin. The replication of recombinant plasmid in the recipient cells was confirmed by the alkaline extraction method and agarose gel electrophoresis.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.575-584
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• 2019

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH₄Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 ㎍/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.

• Microbiology and Biotechnology Letters
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• v.17 no.3
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• pp.213-218
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• 1989

To investigate the possibility of using alkalophilic Bacillus sp. K-11 as a host for molecular cloning, plasmid pUB110 and pBD64 were introduced into alkalophilic Bacillus sp. K-17 by protoplast transformation system. Protoplasts of Bacillus sp. K-11 were prepared by treatment with 200 $\mu\textrm{g}$/$m\ell$ Iysozyme in SMM buffer containing 0.4M sucrose. Optimal temperature, pH and culture time for protoplast formation were 4$0^{\circ}C$, 7.0 and 4hrs, respectively. Cell wall was regenerated efficiently on DM3 medium containing 0.8% agar and 0.5M sodium succinate. Under these conditions for protoplast formation and regeneration, the highest transformation efficiency was obtained with cells incubated for 4hrs, and using 30%(V/V) of 40%(W/V) PEG6,000, In characteristics of transfer-mants, plasmid pUB110 was more stable than plasmid pBD64 in Bacillus sp. K-17. Maximum xylanase production of both transformants carrying pUB110 and pBD64, respectively was similar, but under the same conditions, enzyme secretion by transformant carrying pUB110 was earlier than that of transformant carrying pBD64.

• Microbiology and Biotechnology Letters
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• v.23 no.2
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• pp.156-163
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• 1995

Bacillus sphaericus 1593 is pathogenic to the larvae of a number of mosquito species that are known as important vectors for the transmission of certain human and animal diseases. As a preliminary experiment for developing a multfunctional B. sphaericus 1593 as a potent antagonist, we investigated the conditions for the protoplast transformation system of B. sphaericus 1593 using the plasmid pGB215-110$\Delta$B. The protoplast of B. sphaericus 1593 were obtained most efficiency by treating the cells with 500 $\mu$g/ml of lysozyme in the SMM buffer containing 0.5 M sucrose at pH 8.0 and 40$\circ$C for 60 minutes. The cell wall was regenerated on the plate containing 1.2% agar and 0.8 M mannitol. Under the best condition for protoplast formation and regeneration established in the work the highest frequency of transformation was achieved with the 40% PEG (M.W 4,000) treatment for 15 minutes of incubation at 4$\circ$C, and subsequently for 120 minutes incubation at 30$\circ$C for phenotypic expression. The highest transformation efficiency were observed at 1.0 $\mu$g/ml of the final concentration of the plasmid DNA and the plasmids were found to be fairly stable since about 70% of the plasmids were maintained after 8 successive daily transfers onto the fresh medium.

• 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.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.356-361
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• 1991

- To investigate the possibility of genetic development for a multi-purpose strain of Bacillus subtilis YBL-7 against Fusat-iurn solani causing root rot of many impotant corps, the plasmid pGU66 inserting urease gene of Bacillus pasteurii had been introduced into Bacillus subtilis YBL-7 by PEG-induced protoplast (PIP) transformation system. Protoplasts of B. subtilis YBL-7 were prepared by treating the cells with lysozyme (200 $\mu g$/ml) in hypertonic buffer (SMMP). The highest transformation frequency was achieved when cells of the strain with lysozyme at $42^{\circ}C$ for 90 minutes. Optimal transformation was obtained using polyethylene glycol (MW 4000) at final concentration of 30% (V/V). The transformation frequency was increased proportionally to 1.2 $\mu g$ of plasmid DNA. At best condition, the transformation frequency (transformants/ regenerants/$\mu g$ of DNA) for pGU66 was appoximately $4 \times 10^{-3}$. Also, the urease gene was strongly expressed in the transformants of B. subtilis YBL-7 and maintained steadily. The antifungal ability of transformant was very similar to that of B. ssubtilis YBL-7.