• Journal of Photoscience
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• v.7 no.3
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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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• v.19 no.9
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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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• v.20 no.12
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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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• v.57 no.1
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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.

• Journal of Life Science
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• v.29 no.8
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• pp.916-921
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• 2019

In this study, we constructed a biological system that exhibited thermotolerance, ethanol tolerance, and increased ethanol productivity using a random mutagenesis method. We attempted to isolate a thermotolerant mutant using proofreading-deficient DNA polymerase ${\delta}$ and ${\varepsilon}$ encoded by the pol3 and pol2 genes, respectively, in Saccharomyces cerevisiae. To obtain mutants that could grow at high temperatures ($38^{\circ}C$ and $40^{\circ}C$), random mutagenesis of AMY410 (pol2-4) and AMY126 (pol3-01) strains was induced. The parental strains (AMY410 and AMY126) grew poorly at temperatures higher than $38^{\circ}C$. By stepwise elevation of the incubation temperature, AMY410-Ht (heat tolerance) and AMY126-Ht strains that proliferated at $40^{\circ}C$ were obtained. These strains were further incubated in medium containing 6% and 8% ethanol and then AMY410-HEt (heat and ethanol tolerance) and AMY126-HEt strain with ethanol tolerance at an 8% ethanol concentration was obtained. The AMY126-HEt strain grew even at an ethanol concentration of 10%. Furthermore, following the addition of high concentrations of glucose (5% and 10%), an AMY126-HEt3 strain with increased ethanol productivity was isolated. This strain produced 24.7 g/l of ethanol (95% theoretical conversion yield) from 50 g/l of glucose. The findings demonstrate that a new biological system (yeast strain) showing various phenotypes can be easily and efficiently bred by random mutagenesis of a proofreading- deficient mutant.

• Proceedings of the Korean Society of Potoscience Conference
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• 2002.06a
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• pp.41-41
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• 2002

• The Plant Pathology Journal
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• v.25 no.3
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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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• v.5 no.2
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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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• v.28 no.1
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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.

• Korean Journal of Agricultural Science
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• v.50 no.2
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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.