• Journal of Microbiology and Biotechnology
• /
• v.19 no.9
• /
• pp.869-872
• /
• 2009

The cyclic undecapeptide cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. To increase CyA productivity by wild-type T. niveum (ATCC 34921), random mutagenesis was first performed using an antifungal agar-plug colony assay (APCA) selection approach. This generated a mutant strain producing more than 9-fold greater CyA than the wild-type strain. Additionally, a foreign bacterial gene, Vitreoscilla hemoglobin gene (VHb), was transformed via protoplast regeneration and its transcription was confirmed by RT-PCR in the UV-irradiated mutant cell. This led to an additional 33.5% increase of CyA production. Although most protoplast-regenerated T. niveum transformants tend to lose CyA productivity, the optimized combination of random mutagenesis and protoplast transformation described here should be an efficient strategy to generate a commercially valuable, yet metabolite low-producing, fungal species, such as CyA-producing T. niveum.

• Journal of Environmental Health Sciences
• /
• v.31 no.4 s.85
• /
• pp.327-331
• /
• 2005

Micromonosporas purpurea로부터 효율적 gentamicin 생산을 위해 protoplast fusion와 protoplast mutagenesis 방법이 검토 되었다. $CO^{60}\;irradiation\;(2.3{\times}10^5$ units, UV 3 min) 방법에 의해서 MP3-112, MP3-141, MP3-143을 분리 했다. 특히 MP3-143균주는 최대 gentamicin생산량이 얻어졌다. 개량된 MP3-143균주를 이용해서 탄소원 소비, 균체성장, 그리고 gentamicin 생산량이 batch culture에서 비교되었다. MP3-413와 parent 균주의 glucose 소비는 배양 2일과 3일 후에 각각 완전히 이루어졌다. 그러나 균체성장과 Soybean oil 소비는 비슷한 결과 얻어졌다. Gentamicin최대 생산량은 배양 5일 후 29756 U/ml였다. 이 결과는 parent 균주에 비해 생산량이 5.6배 증가했다.

• Archives of Pharmacal Research
• /
• v.12 no.4
• /
• pp.249-253
• /
• 1989

High producers or blocked mutants of aminoglycoside antibiotic-producing Streptomyces spp. were selected by application of an agar plug method and by culturing individual colonies in broth. The productivities of aminoglycoside antibiotic producing organisms were increased by selection of a high producer from colonies obtained by spreading spores of wild strain, or survived from treatment of a mutagen or from the colonies regenerated from protoplast-formation and cell-wall regenerations. Some mutagen treated colonies lost the ability to produce antibiotics (5-8%). Some A-factor negative and deostreptamine or streptidine negative mutants were obtained by N-methyl-N'-nitro-N-nitrosomethylguanidine (MNNG) treatment. Many of the survivors from the MNNG treatment lost the ability to produce antibiotics. Major colonies produced less amount of antibiotics ; only few survived colonies produced more antibiotics than the parent. Resistance of Streptomyces spp. against the antibiotics produced by itself was also markedly affected by mutagen treatment.

• Journal of environmental and Sanitary engineering
• /
• v.23 no.4
• /
• pp.23-29
• /
• 2008

This study was performed to develop mutant strain using a combination of UV irradiation procedures with protoplast mutagenesis in order to achieve an effective kasugamycin production from Streptomyceskasugaensis. Whenlessthan 1.0g/lof Linoleic acid was used, the cell growth was not inhibited. On the other hand, the cell growth was greatly inhibited when more than 1.6 g/l of linoleic acid was used. Among the various mutant strains, SK-12 was obtained in medium containing 1.6g/l of linoleic acid, showing the highest rate of both cell growth and kasugamycin production. In order to compare kasugamycin production with the SK-12 and the parent strain using soybean oil, cultures were performed in a flask. The production of kasugamycin was increased with the increase time. The maximum kasugamycin concentration was 1.2g/l after 6 days of culture. The product yield from soybean oil was 0.05g/l/g consumed carbon source, which was roughly 5.0 fold higher than the parent strain. These results show that it was effective method for obtaining a mutant resistant to linoleic acid for the effective production of kasugamycin from soybean oil.

• The Korean Journal of Mycology
• /
• v.40 no.2
• /
• pp.93-97
• /
• 2012

Gamma ray irradiation mutagenesis was employed to get variants of Pleurotus eryngii with functionally enhanced and improved characteristics. Protoplasts released from P. eryngii were treated with gamma ray radiation under 0.25-1.25 KGy. Protoplast sample that showed fatality rate of 80% at the 0.25 KGy was spreaded on YPMGA (yeast, peptone, malt-extract, glucose, agar) and 500 mycelial colonies were randomly selected from the medium. Of them, 100 mutant strains with mycelial morphology and growth rate that differ to control strain were observed on PDA. The cellulase and laccase activity of 67 gamma ray-irradiated P. eryngii isolates with morphological variation were investigated. Among these, 5 isolates were higher cellulase. In addition, the genetic variation of the mutant strains was analyzed by PCR fingerprinting.

• Journal of Radiation Industry
• /
• v.5 no.4
• /
• pp.285-295
• /
• 2011

Mushrooms belonging to macrofungi have been consumed by humans for their nutritional and medicinal values for centuries throughout the world. Mushroom farming is practiced in more than 100 countries of the world, with production increasing at a rate of 7% per annum. High yield and good quality are always the principal goals for agriculturally important crops, including mushrooms. Several breeding methods are employed for strain improvement such as mass selection based on the natural chance mutation and induced mutation (mutation breeding), protoplast fusion technology, cross breeding and transgenic breeding. However, mutation breeding has shown prominent success in crop plant improvement. Though several-hundred mutant crop varieties have been developed around the world, the mutation breeding of mushrooms is limited. This review paper explores the potential application of radiation on the development of mutant varieties of mushrooms for breeding with desired traits such as better quality and productivity.

• The Korean Journal of Mycology
• /
• v.15 no.2
• /
• pp.80-86
• /
• 1987

The author isolated high cellulolytic fungi from natural sources and determined optimal condition of protoplast formation and fusion as fundamental step for improvement of the isolated it's cellulolytic ability. Three different cellulolytic fungi, such as Aspergillus sp., Penicillium sp. and Trichoderma sp., were isolated from soil. Their cellulolytic activities were compared with that of Aspergillus niger which was useful industrially and had cellulase activity. It was Aspergillus sp. that showed the highest activity of all these four fungi. And then it was followed by Penicillium sp., Trichoderma sp., and Aspergillus niger in order. An auxotrophic mutant of Aspergillus sp. was obtained by UV mutagenesis method. Having try to produce protoplast from mycelia, the author found that ${\beta}-glucuronidase$, at pH 6.0, was effective cell-wall lytic enzyme. And the optimal concentration of this enzyme was 5,000 unit/ml. Regeneration rates of wild type, met. auxotroph and arg. auxotroph, in presence of osmotic stabilizer, were 7. 0%, 7. 5% and 5.2%, respectively. PEG with M.W. 6,000 was effective stimulator for protoplast fusion in the concentration of 30% (W IV). In such a condition, we obtained 1.2% cell fusion rate.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 2003.10a
• /
• pp.144.1-144
• /
• 2003

Attenuated viruses can protect their hosts against challenge to their related viruses. Increasing evidence shows that mutations of the tobamoviral 126/183 kDa protein play a major role in the viral attenuation and contribute to the cross protection mechanism. In this study, four mutants of Pepper mild mottle virus (PMMoV) have been constructed by mutagenesis; two mutants, pTPpoly348 and pTPpoly762, were substituted in the middle of replicase gene, and the others, pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219, were deletion mutants made by deleting some parts of pseudoknot structures of the 3' noncoding region (NCR) of the virus. Progeny viruses generated from the four mutants were infectious on N. benthamiana plants with symptomless or mild mosaic symptom. Replication efficiency and viral product accumulations of four mutants were assessed by Northern and Western blot analyses on BY-2 protoplast cells. Accumulation of CP for the pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219 were lower than that of other mutants and wild type virus. These data suggest that the 3'-NCR mutations contribute to the viral gene expression in host tissues, while mutants of replicase gene rather govern the symptom expression.

• The Korean Journal of Mycology
• /
• v.14 no.2
• /
• pp.185-188
• /
• 1986

After treatment of basidiospores of P. cornucopiae with ultraviolet light, 84 putative mutants from 4671 isolates were obtained. The highest proportion of auxotrophic mutants was obtained from the isolates irradiated to give $0.4{\sim}1.0%$ survival. Fourteen auxotrophs were selected for protoplast fusion and each genetic marker was identified.

• The Korean Journal of Mycology
• /
• v.13 no.2
• /
• pp.75-78
• /
• 1985

Mutagenesis of protoplast could serve a great potential tool for improvement of strains and genetics in higher fungi. For the isolation of auxotrophic mutants from protoplasts of Pleurotus ostreatus and Pleurotus florida, viability levels of ultraviolet lights were determined. Seven auxotrophs were obtained from protoplasts irradiating UV to give $0.83{\sim}15%$ survival. The mutants showed a single requirement for each of Arg, Ribo-l, Ribo-2 or Phen for growth. Some of them showed two or three kinds of requirements, Gly Ser, Ade Hypo or Ala Om Tryp for growth.