• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1370-1375
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• 2023

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

• Genomics & Informatics
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• v.14 no.4
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• pp.255-264
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• 2016

The plethora of genome sequence information of bacteria in recent times has ushered in many novel strategies for antibacterial drug discovery and facilitated medical science to take up the challenge of the increasing resistance of pathogenic bacteria to current antibiotics. In this study, we adopted subtractive genomics approach to analyze the whole genome sequence of the Fusobacterium nucleatum, a human oral pathogen having association with colorectal cancer. Our study divulged 1,499 proteins of F. nucleatum, which have no homolog's in human genome. These proteins were subjected to screening further by using the Database of Essential Genes (DEG) that resulted in the identification of 32 vitally important proteins for the bacterium. Subsequent analysis of the identified pivotal proteins, using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automated Annotation Server (KAAS) resulted in sorting 3 key enzymes of F. nucleatum that may be good candidates as potential drug targets, since they are unique for the bacterium and absent in humans. In addition, we have demonstrated the three dimensional structure of these three proteins. Finally, determination of ligand binding sites of the 2 key proteins as well as screening for functional inhibitors that best fitted with the ligands sites were conducted to discover effective novel therapeutic compounds against F. nucleatum.

• Journal of Microbiology and Biotechnology
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• v.26 no.4
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• pp.725-729
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• 2016

A novel genome-engineering tool in Clostridium acetobutylicum was developed based on single-crossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1 plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp recombinase, leaving a single flippase recognition target sequence in the middle of the targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed the segregationally unstable pAMβ1 replicon, the plasmid was rapidly cured from the mutant C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.

• Genomics & Informatics
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• v.12 no.4
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• pp.216-224
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• 2014

Gastritis is a common but a serious disease with a potential risk of developing carcinoma. Helicobacter pylori infection is reported as the most common cause of gastritis, but other genetic and genomic factors exist, especially single-nucleotide polymorphisms (SNPs). Association studies between SNPs and gastritis disease are important, but results on epistatic interactions from multiple SNPs are rarely found in previous genome-wide association (GWA) studies. In this study, we performed computational GWA case-control studies for gastritis in Korea Associated Resource (KARE) data. By transforming the resulting SNP epistasis network into a gene-gene epistasis network, we also identified potential gene-gene interaction factors that affect the susceptibility to gastritis.

• The Korea Genome Conference
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• 2002.08a
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• pp.42.2-42.2
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• 2002

• Korean Journal of Microbiology
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• v.55 no.1
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• pp.74-76
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• 2019

Gram-positive anaerobic bacilli Actinomyces spp. commonly reside on mucosal surfaces of the oropharynx, gastrointestinal tract, and urogenital tract. Here, we first report the draft genome sequence of Actinomyces georgiae KHUD_A1, isolated from dental plaque of a Korean elderly woman. The genome is 2,652,059 bp in length and has a GC content of 68.06%. The genome includes 2,242 protein-coding genes, 9 rRNAs, and 64 tRNA. We identified 157 KHUD_A1 strain-specific genes, including genes encoding CPBP family intramembrane metalloprotease, bile acid: sodium symporter family protein, Txe/YoeB family addiction module toxin and Phd/YefM family antitoxin. The sequence information of A. georgiae KHUD_A1 will help understand the general characteristics of the bacterial species and the genome diversity of the genus Actinomyces.

• Genomics & Informatics
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• v.20 no.4
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• pp.43.1-43.18
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• 2022

Atropa belladonna is a valuable medicinal plant and a commercial source of tropane alkaloids, which are frequently utilized in therapeutic practice. In this study, bioinformatic methodologies were used to examine the pattern of coding sequences and the factors that might influence codon usage bias in the chloroplast genome of Atropa belladonna and other nightshade genomes. The chloroplast engineering being a promising field in modern biotechnology, the characterization of chloroplast genome is very important. The results revealed that the chloroplast genomes of Nicotiana tabacum, Solanum lycopersicum, Capsicum frutescens, Datura stramonium, Lyciumbarbarum, Solanum melongena, and Solanum tuberosum exhibited comparable codon usage patterns. In these chloroplast genomes, we observed a weak codon usage bias. According to the correspondence analysis, the genesis of the codon use bias in these chloroplast genes might be explained by natural selection, directed mutational pressure, and other factors. GC12 and GC3S were shown to have no meaningful relationship. Further research revealed that natural selection primarily shaped the codon usage in A. belladonna and other nightshade genomes for translational efficiency. The sequencing properties of these chloroplast genomes were also investigated by investing the occurrences of palindromes and inverted repeats, which would be useful for future research on medicinal plants.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.5
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• pp.432-443
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• 2005

Microarray technology has contributed Significantly to the understanding of bacterial genetics and transcriptional regulation. One neglected aspect of this technology has been optimization of microarray-generated signals and quality of generated information. Full genome microarrays were developed for Clostridium acetobutylicum through spotting of PCR products that were designed with minimal homology with all other genes within the genome. Using statistical analyses it is demonstrated that Signal quality is significantly improved by increasing the hybridization volume. possibly increasing the effective number of transcripts available to bind to a given spot, while changes in labeled probe amounts were found to be less sensitive to improving signal quality. In addition to Q-RT-PCR, array validation was tested by examining the transcriptional program of a mutant (M5) strain lacking the pSOL1 178-gene megaplasmid relative to the wildtype (WT) strain. Under optimal conditions, it is demonstrated that the fraction of false positive genes is 1% when considering differentially expressed genes and 7% when considering all genes with signal above background. To enhance genomic-scale understanding of organismal physiology, using data from these microarrays we estimated that $40{\sim}55%$ of the C. acetobutylicum genome is expressed at any time during batch culture, similar to estimates made for Bacillus subtilis.

• The Journal of Korean Society of Virology
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• v.26 no.1
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• pp.121-129
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• 1996

Multiplication of Herpes simplex virus type-1 was observed by electronmicroscopy, a gene library of the genome was constructed and thymidine kinase gene was cloned. Vero cells infected with the virus were lysed 48 h p.j. and multinucleated giant cells were observed approximately at 72 h p.i. The nucleocapsids were observed in nuclei and cytoplasm, and the assembled nucleocapsids were budded out through the vacuole and cytoplasmic membranes, and then virions were released from the cells. HSV-1 genome DNA was digested with BamHI and BglII enzymes and then the gene library of the genome fragments were constructed. The BamHI cleaved the genome DNA into twenty-seven fragments in the range of 1.1 - 14 kb, and BglII cleaved the genome DNA into sixteen fragments in the range of $4.5{\sim}20.1\;kb$. The pHLA-12 and pHLB-4 recombinant plasmids were contained TK gene by Southern blot analysis. The molecular sizes of the fragments which contained the TK gene were 3.74 in pHLA-12 and 6.41kb in pHLB-4 recombinant plasmid, respectively.

• Microbiology and Biotechnology Letters
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• v.48 no.3
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• pp.373-382
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• 2020

Candidatus Carsonella ruddii is an endosymbiont that resides in specialized cells within the body cavity of plant sap-feeding insects called psyllids. The establishment of symbiotic associations is considered one of the key factors for the evolutionary success of psyllids, as it may have helped them adapt to imbalanced food resources like plant sap. Although C. ruddii is defined as a psyllid primary symbiont, the genes for some essential amino acid pathways are absent. Complete genome sequences of several C. ruddii strains have been published. However, in-depth intra-species comparison of C. ruddii strains has not yet been done. This study therefore aimed to perform a comparative genome analysis of six C. ruddii strains, allowing the interrogation of phylogenetic group, functional category of genes, and biosynthetic pathway analysis. Accordingly, overall genome size, number of genes, and GC content of C. ruddii strains were reduced. Phylogenetic analysis based on the whole genome proteomes of 30 related bacterial strains revealed that the six C. ruddii strains form a cluster in same clade. Biosynthetic pathway analysis showed that complete sets of genes for biosynthesis of essential amino acids, except tryptophan, are absent in six C. ruddii strains. All genes for tryptophan biosynthesis are present in three C. ruddii strains (BC, BT, and YCCR). It is likely that the host may depend on a secondary symbiont to complement its deficient diet. Overall, it is therefore possible that C. ruddii is being driven to extinction and replacement by new symbionts.