• Fisheries and Aquatic Sciences
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• 제26권9호
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• pp.558-568
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• 2023

Vibrio vulnificus is an aquatic bacterium causing septicemia and wound infection in humans. To understand this pathogen at the genomic level, it was performed whole genome sequencing of a cefoxitin-resistant strain, V. vulnificus 1908-10 possessing virulence-related genes (vvhA, viuB, and vcgC) isolated from Gadeok island coastal seawater in South Korea. The genome of V. vulnificus 1908-10 consisted of two circular contigs and no plasmid. The total genome size was estimated to be 5,018,425 bp with a guanine-cytosine (GC) content of 46.9%. We found 119 tRNA and 34 rRNA genes respectively in the genome, along with 4,352 predicted protein sequences. Virulence factor (VF) analysis further revealed that V. vulnificus 1908-10 possess various virulence genes in classes of adherence, antiphagocytosis, chemotaxis and motility, iron uptake, quorum sensing, secretion system, and toxin. In the comparison of the presence/absence of virulence genes, V. vulnificus 1908-10 had fur, hlyU, luxS, ompU, pilA, pilF, rtxA, rtxC, and vvhA. Of the 30 V. vulnificus comparative strains, 80% of the C-genotype strains have all of these genes, whereas 40% of the E-genotype strains have all of them. In particular, pilA were identified in 80% of the C-type strains and 40% of the E-type strains, showing more difference than other genes. Therefore, V. vulnificus 1908-10 had similar VF characteristics to those of type C strains. Multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin of V. vulnificus 1908-10 contained 8 A-type repeats (GXXGXXXXXG), 25 B.1-type repeats (TXVGXGXX), 18 B2-type repeats (GGXGXDXXX), and 7 C-type repeats (GGXGXDXXX). The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) showed that the RtxA protein of V. vulnificus 1908-10 had the effector domain in the order of cross-liking domain (ACD)-C58_PaToxP-like domain- α/β hydrolase-C58_PaToxP-like domain.

• 한국수정란이식학회지
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• 제9권3호
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• pp.243-247
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• 1994

최근 의약적으로 유용한 단백질을 대량 생산키 위한 실현 가능한 방법이 유전자변환 가축의 이용과 관련되어 발전되어 왔다. 이러한 유전자 변환동물은 이종의 단백질을 유즙속으로 분비시키는 생체반응기로서 이용되고 있다. 이러한 전략적 목적을 위해 현재 유전자 변환동물의 생산을 위한 이용에 있어 여러 가지 방법들이 보고되고 있다. 그러나 ES 세포의 사용이 이러한 방법들 사이에서 가장 실질적인 것으로 추정되고 있다. 본 실험에서는 유전자 구축을 위해 사람 황체 호르몬(human luteinizing hormone; hLH)의 전사를 유도하기 위해 각각 2.2 및 0.5 kb의 토끼 $\beta$-casein pronoter 단편을 이용하여 생쥐의 유선에 hLH를 발현시키도록 조절하고 발현이 thynidine kinase(TK) pronoter에 의해 좌우되는 neo 유전자를 selectable marker로서 plasnid속에 삽입하였다. 그 결과 생긴 구축 유전자는 각각 pCas 2.2와 pCas 0.5로 명명하였다. 구축된 유전자로 2$\times$107의 TT-2 ES세포를 170V, 550$\mu$F로 100$\mu$g의 선상 plasmid에 의해 electroporation 시켰다. 감염된 colony들은 250$\mu$g/$m\ell$ G418을 함유하는 ESM 배양액에서 선별 7일 이후에 회수하여 성공적으로 감염된 ES세포는 PCR 및 Southern blot에 의해 확인되었고 그들 중 나머지는 trypsin 처리 후 각각 미세조작과 공배양 기술을 사용하여 ICR 생쥐의 8세포기 수정란 속에 도입하였다. 결국 24시간 동안 37$^{\circ}C$, 5% $CO_2$에서 배양된 배반포를 chimera의 생산을 위해 위임신 유기된 G418 선발처리 이후 400 및 275개의 ES 세포 colony가 생존하였으며, 3개의 ES 세포으 colony 의 genome 속에 임의적으로 plamid가 삽입된 것을 Southern blot에 의해 확인되었다. 총 13 chimera 생쥐가 3 colony로부터 생산되었으나 germ-line chimera는 현재 조사중이다. chimera 생산빈도는 공배양 기술보다 주입방법에서 현저히 높았다.

• 생명과학회지
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• 제20권11호
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• pp.1577-1581
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• 2010

조혈촉진 cytokine인 Granulocyte colony stimulating factor (G-CSF)는 골수세포를 자극하여 granulocyte로 증식, 분화시키는 기능을 가지며, 현재 아주 고가의 치료제로 사용되고 있다. 인간 G-CSF (hG-CSF)를 아직 시도되지 않은 누에 유래 세포주인 BM5 세포에서 발현시키고 생산 효율을 높이기 위해 hG-CSF cDNA를 변형하였다. hG-CSF의 cDNA의 endoplasmic reticulum (ER) signal sequence 부분을 누에의 소포체에서 분비되는 단백질인 prophenoloxidase (PPAE), protein disulfide isomerase (PDI)와 bombyxin (BX)에서 유래한 누에특이 ER signal sequence로 대체한 hG-CSF의 cDNA 함유 벡터를 구축하였다. 이들 벡터를 사용하여 형질전환한 BM5 세포의 배양액에 분비된 G-CSF 단백질을 western blot으로 분석하여 발현을 확인하였다. 누에특이 ER signal sequence들로 대체된 hG-CSF cDNA를 포함하는 벡터에 의한 hG-CSF 단백질 생산이 인간 G-CSF cDNA가 든 벡터에 의한 hG-CSF의 생산보다 월등히 효율적이었다. 또한, PPAE-signal sequence를 포함하는 hG-CSF 단백질은 배양배지에서 형질전환 4일 후에 최고에 달하였고, 7 일째까지 비슷한 양이 배지 내에서 검출되었다. 이상의 결과는 인간유래 유전자가 곤충세포 내에서 발현 될 때 인간유래 유전자 보다는 곤충 유전자발현 시스템에 맞게 변형했을 경우 더 효율적인 단백질 발현을 얻을 수 있음을 보여 준다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권1호
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• pp.43-51
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• 2002

The cloning and expression of $\beta-glucosidase$ II, encoded by the gene ${\beta}glu2$, from thermotolerant yeast Pichia etchellsii into Escherichia coli is described. Cloning of the 7.3 kb BamHI/SalI yeast insert containing ${\beta}glu2$ in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid into E. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At $50^{\circ}C$, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of $0.14\;h^{-l}$. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2002년도 학술발표대회
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• 한국미생물·생명공학회지
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• 제47권3호
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• pp.343-349
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• 2019

Gul jeotgals (GJs) were prepared using solar salt aged for 3 years. One sample was fermented using starters, such as Bacillus subtilis JS2 and Tetragenococcus halophilus BS2-36 (each $10^6CFU/g$), and another sample was fermented without starters for 49 days at $10^{\circ}C$. Initial counts of bacilli and lactic acid bacteria (LAB) in non-starter GJ were found to be $3.20{\times}10^2$ and $7.67{\times}10^1CFU/g$ on day 0, and increased to $1.37{\times}10^3$ and $1.64{\times}10^6CFU/g$ on day 49. Those of starter GJ were found to be $2.10{\times}10^5$ and $3.30{\times}10^7CFU/g$ on day 49, indicating the growth of starters. The pH values of GJ were $5.93{\pm}0.01$ (non-starter) and $5.92{\pm}0.01$ (starter) on day 0 and decreased to $5.78{\pm}0.01$ (non-starter) and $5.75{\pm}0.01$ (starter) on day 49. Amino-type nitrogen (ANN) production increased continuously during fermentation, and $407.19{\pm}15.85$ (non-starter) and $398.04{\pm}13.73$ (starter) mg% on day 49. Clone libraries of 16S rRNA genes were constructed from total DNA extracted from non-starter GJ on days 7, 21, and 42. Nucleotide sequences of Escherichia coli transformants harboring recombinant pGEM-T easy plasmid containing 16S rRNA gene inserts from different bacterial species were analyzed using BLAST. Uncultured bacterium was the most dominant group and Gram - bacteria such as Acidovorax sp., Afipia sp., and Variovorax sp. were the second dominant group. Bacillus amyloliquefaciens (day 7), Bacillus velezensis (day 21 and 42), and Bacillus subtilis (day 42) were observed, but no lactic acid bacteria were detected. Acidovorax and Variovorax species might play some role in GJ fermentation. Further studies on these bacteria are necessary.

• 한국동물위생학회지
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• 제29권3호
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• pp.277-285
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• 2006

Plasmids are covalently closed circular molecules of DNA that are stably inherited and replicate somewhat independently of the bacterial chromosome. Genes carried on plasmids can mediate a wide variety of important functions, including antibiotics (R plasmids) and heavy metals resistance, toxins production, cell penetration, iron chelation, complement resistance, and metabolic characteristics such as sucrose and lactose fermentation. Fifty strains of lactobacilli were isolated from 26 staters and 29 fermented milk products. They were classified 27 strains as Lactobacillus paracasei subsp paracasei, 11 stains as Lactococcus lactis subsp cremoris, 6 strains as L delbrueckii subsp lactis, 4 strains as L acidophius, and 2 strains as L delbrueckii subsp bulgaricus. All of these strains were examined for drug resistance and transferability of R plasmids. All of the isolates were sensitive to Am, C, CF, E, NB, P, T, and Te. But resistant to SXT 94% (47 strains), K 66% (33 strains), S 56% (28 strains), ENR 50% (25 strains), NOR 38% (19 strains) CIP 38% (19 strains), GM 16% (8 strains), and N 14% (7 strains), in order. And 32 different resistant patterns were found. The most frequently encountered patterns were CIP-ENR-K-NOR-S-SXT (5 strains). In vitro R plasmids transfer experiment, 57 antibiotic resistant strains which were not transfer to the recipient 2 Escherichia coli strains by conjugation, These results indicate that Lactobacillus in internal trade market' stater recognize R factor but transmissible R plasmid is not existed.

• Journal of Microbiology and Biotechnology
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• 제17권9호
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• pp.1498-1503
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• 2007

To identify novel crystal proteins, Bacillus thuringiensis 2385-1 was isolated from Korean soil samples and characterized. The H-serotype of 2385-1 was identical to that of subsp. kenyae (H4a4c), and its crystal toxin was bipyramidal-shaped. However, 2385-1 showed a much higher toxicity towards Plutella xylostella and Spodoptera exigua larvae than subsp. kenyae. In addition, the crystal protein profile and plasmid DNA pattern of 2385-1 differed from those of subsp. kenyae. To verify the crystal protein gene types of 2385-1, a PCR-RFLP analysis was performed, and the results revealed that 2385-1 contained two novel cry1-type crystal protein genes, cryl-5 and cry1-12, in addition to the crylJal gene. The deduced amino acid sequences of cryl-5 and cry1-12 showed a 97.9% and 75.7% sequence similarity with the CrylAb and CrylJa crystal proteins, respectively. Among the novel crystal proteins, Cry1-5 showed a high toxicity towards P. xylostella and S. exigua larvae. In conclusion, B. thuringiensis 2385-1 is a new isolate in terms of its gene types, and should be a promising source for an insecticide to control lepidopteran larvae.

• 한국축산식품학회지
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• 제39권4호
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• pp.601-609
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• 2019

Bifidobacterium longum KACC 91563 secretes family 5 extracellular solute-binding protein via extracellular vesicle. In our previous work, it was demonstrated that the protein effectively alleviated food allergy symptoms via mast cell specific apoptosis, and it has revealed a therapeutic potential of this protein in allergy treatment. In the present study, we cloned the gene encoding extracellular solute-binding protein of the strain into the histidine-tagged pET-28a(+) vector and transformed the resulting plasmid into the Escherichia coli strain BL21 (DE3). The histidine-tagged extracellular solute-binding protein expressed in the transformed cells was purified using Ni-NTA affinity column. To enhance the efficiency of the protein purification, three parameters were optimized; the host bacterial strain, the culturing and induction temperature, and the purification protocol. After the process, two liters of transformed culture produced 7.15 mg of the recombinant proteins. This is the first study describing the production of extracellular solute-binding protein of probiotic bacteria. Establishment of large-scale production strategy for the protein will further contribute to the development of functional foods and potential alternative treatments for allergies.

• Journal of Plant Biotechnology
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• 제31권3호
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• pp.231-237
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• 2004

Oxycarotenoids는 녹색식물, 곰팡이, 효모, 버섯 및 세균 등이 만들어 내는 황색, 적색 또는 자색의 polyene계 색소로 분자내에 산소를 함유하며 생체내에서 중요한 역할을 담당하고 있다. 본 실험에서는 Oxycarotenoids의 생합성 경로상에 존재하는 $\beta$-carotene hydroxylase 유전자 (Chyb)가 재조합된 Ti-plasmid (pGCHYB)를 A. tumerfacience GV3101에 의해 Arabidopsis thaliana (cv. Columbia)에 형질전환하였다. 50 mg/L hygromycin 함유한 MS 배지에서 선발된 개체를 이용하여 Chyb 유전자의 도입여부를 PCR로 분석한 결과, 대조구에서는 Chyb 유전자의 증폭 되지 않았으나 형질전환체에서는 증폭 산물을 확인 할 수 있었다. 또한 형질전환체의 발현여부를 RT-PCR분석한 결과 도입된 Chyb 유전자가 안정적으로 발현되었다. 형질전환체의 carotenoids를 HPLC 분석한 결과 xanthophyll cycle carotenoids (violaxanthin과 zeaxanthin)의 함량 및 $\beta$-carotene 함량은 감소되었으며, 대조구 Arabidopsis에는 생합성되지 않는 astaxanthin이 생합성되었다. 따라서 본 실험에서 육성된 형질전환체를 이용하여 oxycarotenoids 생합성 과정상의 중간대사물질의 표지, 관여된 transcript 및 metabolite 분석 등을 통해 carotenoids 대사계의 연구소재로 활용 할 수 있을 것으로 기대한다.