• Journal of Photoscience
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• 제7권3호
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• pp.103-108
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• 2000

We have evaluated a new mutagenesis strategy called random insertional mutagenesis with subtracted cDNA fragments. The cDNAs from long day Arabidopsis plants were subtracted by cDNAs from short day plants using PCR based cDNA subtraction. The subtracted cDNAs were inserted between 35S promoter and 3'-NOS terminator regardless of orientation. When the cDNA library was used for the random insertion into Arabidopsis genome by Agrobacterium-mediated transformation, approximately 15% of transformants showed abnormal development in leaf, floral organ, shoot apex. When 20 mutants were analyzed, 12 mutants showed single cDNA fragment insertion and 8 mutants showed more than 2 transgene insertions. Only two mutants among 12 mutants that have single cDNA insert showed consistent phenotype at T2 generation, suggesting the genetic instability of the mutants.

• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.869-872
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• 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 Microbiology and Biotechnology
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• 제20권12호
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• pp.1696-1701
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• 2010

The ${\alpha}$-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis ${\alpha}$-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.

• BMB Reports
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• 제57권1호
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• pp.30-39
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• 2024

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements.

• 생명과학회지
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• 제29권8호
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• pp.916-921
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• 2019

본 연구는 내열성, 에탄올내성 및 에탄올생산성이 향상된 새로운 생물시스템을 육종하기 위해 DNA 복제 시 교정기능이 결여된 변이주(proofreading-deficient mutant)를 이용한 무작위적 돌연변이(random mutagenesis)를 유도하였다. DNA polymerase ${\delta}$와 ${\varepsilon}$의 catalytic subunit를 코드하고 있는 POL3와 POL2 유전자가 변이된 AMY410 균주(pol2-4)와 AMY126 균주(pol3-01)를 이용하여 $30^{\circ}C$, $38^{\circ}C$, $48^{\circ}C$에서 내열성 변이를 유도하였다. AMY410 균주와 AMY126 균주는 $38^{\circ}C$ 이상의 온도에서는 거의 자랄 수가 없기 때문에, 배양 온도를 점차 높여가며 변이를 유도한 결과, $40^{\circ}C$에서도 잘 자라는 AMY410-Ht와 AMY126-Ht 균주를 선별할 수 있었다. 또한 AMY410-Ht와 AMY126-Ht 균주의 에탄올내성을 추가로 획득하기 위해 6%와 8% 에탄올이 함유된 배지에서 변이를 유도하였고, 8% 에탄올 농도에서 내성을 가지는 AMY410-HEt 균주와 AMY126-HEt 균주를 분리하였다. 특히 AMY126-HEt 균주는 10% 에탄올 농도에서도 내성을 가짐을 확인할 수 있었다. AMY126-HEt 균주의 에탄올생산성의 증가를 위한 변이는 고농도 당(glucose)의 첨가에 의해 유도되었고, 50 g/l의 고농도 glucose를 함유한 배지에서 24.7 g/l의 에탄올(95% 이론적 전환수율)을 생산할 수 있는 고효율 에탄올 생산균주(AMY126-HEt3)를 최종 분리하였다. 따라서, 본 연구를 통해서 교정기능이 결여된 균주(proofreading-deficient mutant)를 사용한 돌연변이법으로 산업적으로 유용한 다양한 형질을 가진 생물시스템의 구축이 가능함을 확인하였다.

• 한국광과학회:학술대회논문집
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• 한국광과학회 2002년도 정기총회 및 제9회 광과학 학술심포지움 논문초록
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• pp.41-41
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• 2002

• The Plant Pathology Journal
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• 제25권3호
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• pp.205-212
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• 2009

Colletotrichum acutatum was the main cause of the recent outbreaks of anthracnose on pepper fruit in Korea. To facilitate molecular analysis of C. acutatum, we generated an arginine auxotrophic mutant of the C acutatum strain JC24 using a targeted gene replacement strategy. A 3.3-kb genomic region carrying an ortholog (designated CaARG2) of the fungal gene encoding N-acetylglutamate synthase, the first enzyme of arginine biosynthesis in fungi, was deleted from the fungal genome. The mutant exhibited normal growth only when arginine was exogenously supplied into the culture medium. Transformation of the arginine auxotrophic mutant with a plasmid DNA carrying an intact copy of CaARG2, which was smaller than the deleted region in the mutant, not only caused random vector insertions in the fungal genome, but also recovered both hyphal growth and pathogenicity of the mutant to the wild-type level. Using this new selection system, we have successfully developed a restriction enzyme-mediated integration procedure, which would provide an economically efficient random mutagenesis method in C. acutatum.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.110-113
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• 1995

Transposon Tn5 was used to induce random insertional mutations in Bradyrhizobium japonicum, a soybean endosymbiont. By genomic Southern blot analysis, transposition events were found to have occurred randomly throughout the B. japonicum genome. After screening 3, 626 mutants by auxotrophy test, a histidine auxotroph was isolated. Upon plant infection test, the His mutant showed a 3~4 day delay in nodule formation.

• Journal of Microbiology and Biotechnology
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• 제28권1호
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• pp.95-104
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• 2018

Biosurfactants or microbial surfactants are surface-active biomolecules that are produced by a variety of microorganisms. Biodegradability and low toxicity have led to the intensification of scientific studies on a wide range of industrial applications for biosurfactants in the field of environmental bioremediation as well as the petroleum industry and enhanced oil recovery. However, the major issues in biosurfactant production are high production cost and low yield. Improving the bioindustrial production processes relies on many strategies, such as the use of cheap raw materials, the optimization of medium-culture conditions, and selecting hyperproducing strains. The present work aims to obtain a mutant with higher biosurfactant production through applying mutagenesis on Bacillus subtilis SPB1 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on blood agar and subsequent formation of halos, the mutated strains were examined for emulsifying activity of their culture broth. A mutant designated B. subtilis M2 was selected as it produced biosurfactant at twice higher concentration than the parent strain. The potential of this biosurfactant for industrial uses was shown by studying its stability to environmental stresses such as pH and temperature and its applicability in the oil recovery process. It was practically stable at high temperature and at a wide range of pH, and it recovered above 90% of motor oil adsorbed to a sand sample.

• 농업과학연구
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• 제50권2호
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• pp.241-247
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• 2023

Erwinia amylovora , which causes fire blight disease on apple and pear trees, is one of the most important phytopathogens because of its devastating impact. Currently, the only way to effectively control fire blight disease is through the use of antibiotics such as streptomycin, kasugamycin, or oxytetracycline. However, problems with the occurrence of resistant strains due to the overuse of antibiotics are constantly being raised. It is therefore necessary to develop novel disease control methods through an advanced understanding of the pathogenesis mechanism of E. amylovora . To better understand the pathogenesis of E. amylovora , we investigated unknown virulence factors by random mutagenesis and screening. Random mutants were generated by Tn5 transposon insertion, and the pathogenicity of the mutants was assessed by inoculation of the mutants on apple fruitlets. A total of 17 avirulent mutants were found through screening of 960 random mutants. Among them, 14 mutants were already reported as non-pathogenic strains, while three mutants, TS3128_M2899 (ΔSUFU ), TS3128_M2939 (ΔwcaG ), and TS3128_M3747 (ΔrecB ), were not reported. Further study of the association between E. amylovora pathogenicity and these 3 novel genes may provide new insight into the development of control methods for fire blight disease.