• KSBB Journal
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• 제7권4호
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• pp.284-289
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• 1992

유전자 조작된 Klebsiella pnuemonia를 이용하여 트립토판 생산을 위한 최적 조건 및 플라스미드 안정성에 대한 연구를 수행하였으며, 최적온도는 $37^{\circ}C$, 최적온도에서의 비증식속도는 1.05$h^{-1}$로 측정되었다. 포도당을 기질로 사용하여 첨가회분배양 36시간 후의 트립토판 농도는 0.175g/l 이었으며, 이 결과는 다른 회분배양이나 플라스크 배양에 비해 1.2 및 1.6배 증가한 수치이다. 첨가회분배양에서의 균주안정성은 플라스크에서의 연속 교대 배양에서는 95%가 유지되었으나 첨가 회분배양에서는 50%만이 유지됨을 측정했다.

• 생명과학회지
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• 제9권1호
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• pp.84-91
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• 1999

폐기름 유출지역에서 분리 동정된 미생물 Xl, X2, X3은 폐유나 그 주성분인 난분해성 물질들을 유일 탄소원으로 자랄 수 있었다. Naphthalene과 2-methyl naphthalene은 7일만에 약 80$\%$ 분해되었다. Hexane과 hexadecane은 거의 대부분 분해되며 60$\%$의 분해가 폐유에서 관찰되었다. 합성 계면활성제인 Triton X-100와 Tween 20은 세포의 성장과 분해에 오히려 저해함을 보였다. Xl, X2은 그람 음성을 X3은 그람 양성을 보이며 항생제 ampicillin에 저항성을 가진다. Xl의 30kb plasmid을 E.coli에 transform하여 유전공학적 활용 가능성을 보였다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 1994년도 Proceedings of International Symposium on BIOLOGICAL CONTROL OF PLANT DISEASES Korean Society of Plant Pathology
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• pp.11-26
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• 1994

Crown gall of stonefruit and nut trees is one of the very few plant diseases subject to efficient biological control. The disease is caused by the soil-inhabiting bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes and the original control organism was a non-pathogenic isolate of A. rhizogenes strain K84. Control is achieved by dipping planting material in a cell suspension of strain K84 which specifically inhibits pathogenic strains containing a nopaline Ti plasmid. Because the agrocin 84-encoding plasmid (pAgK84) is conjugative, it can be transmitted from the control strain to pathogenic strains which, as a result, become immune to agrocin 84 and cannot be controlled. To prevent this happening, the transfer genes on pAgK84 were located and then largely eliminated by recombinant DNA technology. The resulting construct, strain K1026, is transfer deficient but controls crown gall just as effectively as does strain K84. Field data from Spain confirm that pAgK84 can transfer to pathogenic recipients from strain K84 but not from strain K1026. The latter has been registered in Australia as a pesticide and is the first genetically engineered organism in the world to be released fro commercial use. It is recommended as a replacement for strain K84 to prevent a breakdown in the effectiveness of biological control of crown gall. Several reports indicate that both strains K84 and K1026 sometimes control crown gall pathogens that are resistant to agrocin 84. A possible reason for this is that both strains produce a second antibiotic called 434 which inhibits growth of nearly all isolates of A. rhizogenes, both pathogens and non-pathogens. Crown gall of grapevine is caused by another species, Agrobacterium vitis. It is resistant to agrocin 84 and cannot be controlled by strains K84 or K1026. It is different from other crown gall pathogens in several characteristics, including the fact that, although a rhizosphere coloniser, its also lives systemically in the vascular tissue of grapevine. Pathogen free propagating material can be obtained from tissue culture or, less surely, by heat therapy of dormant cuttings. A number of laboratories are searching for a biocontrol strain that will prevent, or at least delay, reinfection. A non-pathogenic A. vitis strain F/25 from South Africa looks very promising in this regard.

• Journal of Microbiology and Biotechnology
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• 제33권4호
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• pp.552-558
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• 2023

Levulinic acid (LA) is a valuable chemical used in fuel additives, fragrances, and polymers. In this study, we proposed possible biosynthetic pathways for LA production from lignin and poly(ethylene terephthalate). We also created a genetically encoded biosensor responsive to LA, which can be used for screening and evolving the LA biosynthesis pathway genes, by employing an LvaR transcriptional regulator of Pseudomonas putida KT2440 to express a fluorescent reporter gene. The LvaR regulator senses LA as a cognate ligand. The LA biosensor was first examined in an Escherichia coli strain and was found to be non-functional. When the host of the LA biosensor was switched from E. coli to P. putida KT2440, the LA biosensor showed a linear correlation between fluorescence intensity and LA concentration in the range of 0.156-10 mM LA. In addition, we determined that 0.156 mM LA was the limit of LA detection in P. putida KT2440 harboring an LA-responsive biosensor. The maximal fluorescence increase was 12.3-fold in the presence of 10 mM LA compared to that in the absence of LA. The individual cell responses to LA concentrations reflected the population-averaged responses, which enabled high-throughput screening of enzymes and metabolic pathways involved in LA biosynthesis and sustainable production of LA in engineered microbes.

• Molecules and Cells
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• 제37권10호
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• pp.727-733
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• 2014

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-Omethylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong $ermE^*$ promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.278-281
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• 2004

The $\alpha$1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 plays a key role for the outer chain initiation of the N-linked oligosaccharides. A search for Hansenula polymorpha genes homologous to S. cerevisiae OCHI (ScOCH1) has revealed seven open reading frames (ORF100, ORF142, ORF168, ORF288, ORF379, ORF576, ORF580). All of the seven ORFs are predicted to be a type II integral membrane protein containing a transmembrane domain near the amino-terminal region and has a DXD motif, which has been found in the active site of many glycosyltransferases. Among this seven-membered OCH1 gene family of H. polymorpha, we have carried out a functional analysis of H. polymorpha ORF168 (HpOCH2) showing the highest identity to ScOCH1. Inactivation of this protein by disruption of corresponding gene resulted in several phenotypes suggestive of cell wall defects, including hypersensitivity to hygromycin B and sodium deoxycholate. The structural analysis of N-glycans synthesized in HpOCH2-disrupted strain (Hpoch2Δ) and the in vitro $\alpha$1,6-mannosyltransferase activity assay strongly indicate that HpOch2p is a key enzyme adding the first $\alpha$1,6-mannose residue on the core glycan Man$_{8}$GlcNAc$_2$. The Hpoch2Δ was further genetically engineered to synthesize a recombinant glycoprotein with the human compatible N-linked oligosaccharide, Man$_{5}$GlcNAc$_2$, by overexpression of the Aspergillus saitoi $\alpha$1,2-mannosidase with the 'HDEL” ER retention signal.gnal.

• 한국연초학회지
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• 제20권1호
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• pp.57-65
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• 1998

This study was conducted to develop a resistant tobarro against Potato virus Y (PVY) by transformation of the plants with genetically engineered viral genes. The complimentary DNAs (cDNAS) of potato virus Y-necrosis strain (PVY-Vn) replicase mutant genes (3'-deleted, 5'-deleted and ADD-mutant Nlbs) were synthesized through RT-PCR by using purified PVY-VN RNA and synthesized primers, and cloned in the sense orientation into a plant expression vector (pMBPI), The cDNAS of the genes were transferred into Agrobacterium tumefaciens LBA 4404, and then transformed into tobacco (Nicotiana tabacum cv. Burley 21) plants. Regenerated plants were tested for PVY resistance by inoculation test; 13 transgenic plants including 7 for 3'-deleted Nlb, 3 for 5'-deleted Nlb, and 3 for ADD-mutant Nlb appeared to be resistant at 4 weeks after inoculation with PVY-VN. Among the 13 transgenic tobacco plants, 8 plants had no symptom up to 14 weeks after inoculation. The progenies ($T_1$) from self-fertilization of the transgenic lines varied 0.0% to 81.2% in their resistance (% of resistant plants). The analysis of Nlb-31deleted, -5'deleted and -ADD mutant in the $T_1$ plants by polymerase chain reaction (PCR) showed that Nlb-3'deleted, -5'deleted and -ADD mutants were detected in all of the resistant plants. These results suggest that the PVY resistance was inherited in the $T_1$ generation.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.88-94
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• 2008

Two sugar biosynthetic cassette plasm ids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003-OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ $Na^+$] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12-membered ring aglycon (10-deoxymethynolide, 1) and a 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.

• Journal of Microbiology and Biotechnology
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• 제3권4호
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• pp.232-237
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• 1993

A strain of yeast which can convert starch directly to ethanol was developed by the intergeneric protoplast fusion between Schwanniomyces alluvius possessing $\alpha$ amylase as well as glucoamylase with debranching activity and FSC-14-75 which previously had been formed from a heterologous transformation and subsequent intergeneric protoplast fusion. Fusants were selected on minimal medium after protoplasts of auxotrophic mutant of S. alluvius fused with heat-treated protoplasts of FSC-14-75 in the presence of 30%(w/v) PEG and 20 mM $CaCl_2$. The fusion frequency was in the range of $10^{-6}$ order. All fusants tested were intermediate types of parental strains for carbon compound assimilation, and their cell volumes were approximately 1.1 times larger than FSC-14-75 and 1.8 times larger than S. alluvius. The fusants were unable to sporulate like FSC-14-75, while S. alluvius could sporulate. In flask scale the most promising fusant, FSCSa-R10-6, produced 7.83%(v/v) and 10.17%(v/v) ethanol from 15% and 20% of liquefied potato starch, respectively, indicating that the fermetation efficiency of each case increased 1.2 times and 1.6 times than that of FSC-14-75. The elution pattern on DEAE-cellulose chromatography showed that FSCSa-R10-6 has four distinct amylase peaks of which two peaks originated from S. alluvius and the other two from FSC-14-75. These results suggest that the enhanced fermentation efficiency of the fusant might be due to almost-complemented parental amylases.

• Journal of Periodontal and Implant Science
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• 제38권1호
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• pp.41-50
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• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.