• KSBB Journal
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• 제24권3호
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• pp.279-286
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• 2009

방선균은 다양한 생리활성 물질을 이차대사산물로 생산하는 산업적으로 매우 유용한 미생물이다. 이에 따라 많은 연구진들이 방선균에 대한 분자생물학적 연구와 산업적 이용에 대한 연구들을 수행하고 있다. 방선균 중에서도 S. avermitilis는 강력한 구충효과가 있는 avermectin을 생산하지만, 또한 포유동물 세포의 미토콘드리아에서 산화적 인산화반응을 억제하는 oligomycin도 함께 생성된다. 따라서 S. avermitilis에서 oligomycin의 생성을 제거시키기 위하여 oligomycin synthetase gene을 disruption 시키기 위한 연구를 수행하였다. 이를 위하여 S. avermitilis로부터 cloning 한 oligomycin synthetase gene (olmA5)의 중앙부분에 apramycin resistance gene을 끼워 넣어 integration vector로 구축한 후에 S. avermitilis의 chromosomal DNA와의 homologous recombination에 의하여 olmA5 gene의 disruption을 유도하였다. Disruption mutants (olmA5::apra)는 PCR을 통해 olmA5 gene의 위치에 apramycin resistance gene이 존재하는 것으로 확인하였고, 또한 HPLC 분석을 통해 oligomycin 생합성이 완전히 제거된 것임을 확인하였다. 그러나 disruption mutant (olmA5::apra)를 이용하여 avermectin 만을 생산할 수 있었으나, avermectin의 생산량에는 거의 변화가 없었다. 이러한 mutants는 산업적으로 avermectin을 생산하기 위한 균주 개량의 훌륭한 source가 될 수 있을 것이다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권5호
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• pp.327-331
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• 2000

The application of LFH-PCR(long flanking homology region-PCR) for Bacillus subtilis gene disruption is presented. Without plasmid- or phage-vector construction, only by PCR, based on a DNA sequence retrieved from B. subtilis genome data base, kanamycin resistance gene was inserted into two genes of B. subtilis involved in sporulation, spoIIIE and spoIIIG. The effect of gene disruption on subtilisin expression was examined and the sporulation frequency of two mutants was compared to that of the host strain. For this purpose, only 2 or 3 rounds of PCR were required with 4 primers. We first demonstrated the possibility of LFH-PCR for rapid gene disruption to characterize an unknown functional gene of B. subtilis or other prokaryote in the genomic era.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1472-1476
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• 2006

Previous study demonstrated that the restriction barrier of Streptomyces griseus is almost completely bypassed by the Streptomyces-E. coli shuttle vectors passed through the E. coli GM161 strain and methylated with AluI and HpaII methyltransferases. The same DNA methylation of the genomic DNA fragments cloned the nonreplicative vectors generated integrative transformation and gene disruption of their chromosomal counterparts at high efficiencies in S. griseus. This result indicated that the efficiency of gene disruption depends on the efficient transfer of the incoming DNA into bacterial hosts.

• The Plant Pathology Journal
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• 제32권3호
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• pp.173-181
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• 2016

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1), which is associated with fumonisin B1 bio-synthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

• Journal of Microbiology and Biotechnology
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• 제21권1호
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• pp.14-19
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• 2011

A cyclic undecapeptide-family natural product, cyclosporin A (CyA), which is one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex in a filamentous fungal strain named Tolypocladium niveum. Previously, structural modifications of cyclosporins such as a regionspecific hydroxylation at the $4^{th}$ N-methyl leucine in a rare actinomycetes called Sebekia benihana were reported to lead to dramatic changes in their bioactive spectra. However, the reason behind this change could not be determined since a system to genetically manipulate S. benihana has not yet been developed. To address this limitation, in this study, we utilized the most commonly practiced gene manipulation techniques including conjugation-based foreign gene transfer-and-expression as well as targeted gene disruption to genetically manipulate S. benihana. Using these optimized genetic manipulation systems, a putative cytochrome P450 hydroxylase (CYP) gene named CYP506, which is involved in CyA hydroxylation in S. benihana, was specifically disrupted and genetically complemented. The S. benihana${\Delta}$CYP506 exhibited a significantly reduced CyA hydroxylation yield as well as considerable yield restoration by functional complementation of the S. benihana CYP506 gene, suggesting that the genetically manipulated S. benihana CYP mutant strains may serve as a more efficient bioconversion host for various valuable metabolites including CyA.

• The Plant Pathology Journal
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• 제35권2호
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• pp.91-99
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• 2019

Mitogen-activated protein kinase (MAPK) cascades in fungi are ubiquitously conserved signaling pathways that regulate stress responses, vegetative growth, pathogenicity, and many other developmental processes. Previously, we reported that the AbSte7 gene, which encodes a mitogen-activated protein kinase kinase (MAPKK) in Alternaria brassicicola, plays a central role in pathogenicity against host cabbage plants. In this research, we further characterized the role of AbSte7 in the pathogenicity of this fungus using ${\Delta}AbSte7$ mutants. Disruption of the AbSte7 gene of A. brassicicola reduced accumulation of metabolites toxic to the host plant in liquid culture media. The ${\Delta}AbSte7$ mutants could not efficiently detoxify cruciferous phytoalexin brassinin, possibly due to reduced expression of the brassinin hydrolase gene involved in detoxifying brassinin. Disruption of the AbSte7 gene also severely impaired fungal detoxification of reactive oxygen species. AbSte7 gene disruption reduced the enzymatic activity of cell walldegrading enzymes, including cellulase, ${\beta}$-glucosidase, pectin methylesterase, polymethyl-galacturonase, and polygalacturonic acid transeliminase, during host plant infection. Altogether, the data strongly suggest the MAPKK gene AbSte7 plays a pivotal role in A. brassicicola during host infection by regulating multiple steps, and thus increasing pathogenicity and inhibiting host defenses.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2869-2873
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• 2014

Background: Human papillomavirus (HPV) integration within the E2 gene has been proposed as a critical event in cervical carcinogenesis. This study concerned whether HPV16 status and E2 gene intactness are predictive of radiation response in patients with cervical cancer. Materials and Methods: Biopsies of 44 patients with cervical cancer were collected before or after radiotherapy. The presence of HPV16 was assessed by polymerase chain reaction (PCR) using specific primers for the L1 region. E2 disruption was detected by amplifying the entire E2 gene. Results: HPV16 DNA was found in 54.5% of the clinical samples. Overall, 62.5% of the HPV16 positive tumors had integrated viral genome and 37.5% had episomal genome. There was a tendency of increase of HPV16 E2 negative tumors compared with HPV16 L1 ones in advanced stages (75% versus 20% in stage III respectively). Detection of E2 gene appeared influenced by the radiotherapy treatment, as the percentage of samples containing an intact HPV16 E2 was more frequent in pretreated patients compared to radiotherapy treated patients (66.6% versus 20%). The radiation therapy caused an eight-fold [OR= 8; CI=1.22-52.25; p=0.03] increase in the risk of HPV16 genome disruption. The integration status is influenced by the irradiation modalities, interestingly E2 disruption being found widely after radiotherapy treatment (75%) with a total fractioned dose of 50Gy. Conclusions: This study reveals that the status of the viral DNA may be used as a marker to optimize the radiation treatment.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1170-1175
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• 2004

The involvement of the relA and rsh genes in the morphological and physiological differentiation of Streptomyces clavuligerus was evaluated with the relA and rsh genes mutants. The morphological differentiation of S. clavuligerus was greatly affected by the disruption of the relA gene, but not very much by the disruption of the rsh gene. The altered morphological characteristics were completely restored by the complementation of the corresponding disrupted genes. Thus, it was apparent that the mycelial morphology and clavulanic acid production were severely affected by the disruption of the relA gene. Production of clavulanic acid in the submerged batch culture and glycerol-limited chemostat showed that production was inversely related to the specific growth rate in the wild-type strain. However, the production of clavulanic acid in the ${\Delta}relA$ and ${\Delta}rsh$ null mutants was completely abolished. Therefore, it seems plausible that the stringent response of S. clavuligerus to starvation for amino acids is governed mainly by ReIA, rather than Rsh, and that the (p)ppGpp synthesized immediately after the depletion of amino acids triggers the initiation of pathways for both morphological and physiological differentiation in this species.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.793-799
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• 2012

A gene related to high pristinamycin yield in Streptomyces pristinaespiralis was selected by amplified fragment length polymorphism (AFLP) and its functions were investigated by gene disruption. First, a 561 bp polymorphic sequence was acquired by AFLP from high-yield recombinants compared with the S. pristinaespiralis ancestor ATCC25486, indicating that this approach is an effective means of screening for valuable genes responsible for antibiotic yield. Then, a 2,127 bp open reading frame of a gene designated spy1 that overlaps with the above fragment was identified and its structure and biological functions were investigated. In silico analysis of spy1 encoding a deduced 708-amino-acid-long serine/threonine protein kinase showed that it only contains a catalytic domain in the N-terminal region, which is different from some known homologs. Gene inactivation of chromosomal spy1 indicated that it plays a pleiotropic regulatory function in pristinamycin production, with a positive correlation to pristinamycin I biosynthesis and a negative correlation to pristinamycin II biosynthesis.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2004년도 생명공학 실용화를 위한 비젼
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• pp.55-55
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• 2004