• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.1841-1848
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• 2006

Insights into gene expression have the potential for improvement of antibiotic yield and the development of robust production hosts for use in recombinant biomolecule production. $Cubicin^{TM}$ (daptomycin for injection) is a recently approved antibiotic active against many Gram(+) pathogens, including those resistant to methicillin, vancomycin, and fluoroquinolones. Daptomycin is produced as a secondary metabolite by Streptomyces roseosporus. A 128 kb region of DNA including the daptomycin biosynthetic gene cluster (dpt) has been cloned. and sequenced. Using a selected array of nucleic acid probes representing this region, we compared the expression levels of the dpt genes between S. roseosporus wild-type (WT) and derived S. roseosporus high-producer of daptomycin (HP). We observed that the majority of the biosynthetic genes were upregulated in HP compared with WT; a total of 12 genes, including those encoding daptomycin synthetase, showed consistently and significantly higher expression levels, at least 5-fold, in HP compared with WT. In contrast, some genes, flanking the dpt cluster, were expressed at higher levels in the WT strain. The expression of housekeeping genes such as S. roseosporus rpsL, rpsG, and 16S (positive controls) and presumptive intergenic regions in the dpt cluster (negative control) were identical in the two strains. In addition, we compared transcription during the early, mid-log, and early-stationary phases of growth in the HP strain. The same set of genes was upregulated and downregulated under all conditions examined; housekeeping genes showed no relative change in expression level over the periods of growth tested. Analyses of this type would be of value in studies of strain improvement and also for the identification of gene regulation processes that are important for secondary metabolite production.

• Journal of Microbiology and Biotechnology
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• 제13권3호
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• pp.429-436
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• 2003

Pediocin K1 is a bacteriocin produced by Pediococcus sp. KCA 1303-10, isolated from traditionally fermented flatfish in Korea. Pediocin K1-dependent antibiosis and pediocin K1-independent antibiosis against Listeria monocyrogenes were investigated by comparing antibiosis potential of the ped＋ wild-type strain of Pediococcus sp. KCA1303-10 with that of the ped- mutant strain in 3 different media at 3 different temperatures. In the synthetic MRS-APT medium, bacteriocin (pediocin K1)-dependent antibiosis (BDA) acted as the major driving force of overall antibiosis at the initial stage before the pH of the media was not sufficiently lowered, while bacteriocin-independent antibiosis (BIA) took over the major role at the late stage of antibiosis by killing otherwise resistant cells in the modium. The role of BDA increased as the temperature of the system decreased. The antibiosis potential of BDA among the overall antibiosis of Pediococcus against Listeria at $37^{\circ}C$ was calculated as 46%, and as 75% at $25^{\circ}C$. In the skim milk medium, antibiosis of Pediococcus against Listeria was weakened more than 4 log cycles compared to that of the synthetic medium; however, BDA worked as the main antibiosis force regardless of the culturing temperature in the skim milk medium. In the bean soup medium, BDA also worked as the major killing mechanism against Listeria, but BIA played as another suppressing mechanism against otherwise pediocin-resistant Listeria population. These results suggest that a large portion of the inhibitory action of the ped+Pediococcus sp. KCA1303-10 was attributable to the bacteriocin produced by the strain and that viable Pediococcus sp. KCA1303-10 was superior to the purified bacteriocin in suppressing the occurrence of the bacteriocin-resistant Listeria monocytogenes in food systems.

• 한국미생물·생명공학회지
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• 제34권1호
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• pp.35-39
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• 2006

높은 C4 대사활성을 보이는 반추위미생물이 가지는 포도당 발효대사 조절양식의 한가지를 대장균에서 모사하였다. 대장균은 glycolytic condition에서는 phosphoenolpyruvate(PEP) ${\leftrightarrow}$ oxaloacetate(OAA)간 반응을 phosphenolpyruvate carboxylase(PPC)에 의해, gluconeogenetic condition에서는 phosphoenolpyruvate carboxykinase(PCK)에 의해 촉매하도록 조절한다. 반면 반추위미생물은 glycolytic condition에서 PCK를 통하여 반응이 촉매된다. 이러한 조절양식의 차이점이 C4 대사활성에 미치는 영향을 조사하기 위하며 ppc가 돌연변이되고 대신 인위적으로 PCK를 발현할 수 있는 대장균을 제조하였다. 이렇게 PEP-OAA간 대사조절이 변이된 대장균 K12 ppc-/pck+는 야생형 K12보다 2.5배의 높은 C4대사활성을 보였다. 대장균에서의 C4 대사생리를 증가시키는 연구는 대사공학을 이용한 여러가지 유용물질(i.e. 숙신산, ALA)생산에 응용하기 위한 기초자료로 활용될 수 있을 것으로 기대된다.

• 한국미생물·생명공학회지
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• 제48권3호
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• pp.296-302
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• 2020

Rapamycin, derived from Streptomyces rapamycinicus, is an important bioactive compound having a therapeutic value in managing Parkinson's disease, rheumatoid arthritis, cancer, and AIDS. Because of its pharmaceutical activity, studies over the past decade have focused on the biosynthesis of rapamycin to enhance its yield. In this study, the effect of rapG on rapamycin production was investigated. The rapG expression vector was constructed by utilizing the integration vector pSET152 under the control of the erythromycin resistance gene (ermE^⁎), a strong constitutive promoter. The rapamycin yield of wild type (WT) and WT/rapG overexpression mutant strains, under fermentation conditions, was analyzed by high-performance liquid chromatography (HPLC). Our results revealed that overexpression of rapG increased rapamycin production by approximately 4.9-fold (211.4 mg/l) in MD1 containing 15 g/l of glycerol, compared to that of the WT strain. It was also found that Illicium verum powder (10 g/l), containing shikimic acid, enhanced rapamycin production in both WT and WT/rapG strains. Moreover, the amount of rapamycin produced by the WT/rapG strain was statistically higher than that produced by the WT strain. In conclusion, the addition 15 g/l glycerol and 15 g/l I. verum powder produced the optimal conditions for rapamycin production by WT and WT/rapG strains.

• The Plant Pathology Journal
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• 제15권3호
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• pp.137-143
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• 1999

A novel chromosomal gene tagging technique using a specific fragment of the fatty acid desaturase-like open reading frame (des-like ORF) from the tox-argK gene cluster of Pseudomonas syringae pv. phaseolicola was developed to identify Xanthomonas spp.released into the environment as biocontrol agents. X. campestris pv. convolvuli FB-635, a pathogen of Convolvulus arvensis L., (bindweed), was chosen as the organism in which to develop and test the system. A 0.52 kb DES fragment amplified from P. syringae pv. phaseolicola C-199 was inserted into pGX15, a cosmid clone containing a 10.3 kb Eco RI-HindIII fragment derived from the xanthomonadin biosynthetic gene cluster contained in plasmid pIG102, to create a pigG::DES insertion. The 10.8 kb EcoRI-BamHI fragment carrying the pigG:: DES insertion was cloned into pLAFR3 to generate pLXP22. pLXP22 was then conjugated into X. campestris pv. convolvuli FB-635 and the pigG::DES insertion integrated into the bacterial chromosome by marker exchange. Rifampicin resistant, tetracycline sensitive, starch hydrolyzing, white colonies were used to differentiate the marked strain from yellow pigmented wild-type ones. PCR primers specific for the unique DES fragment were used for direct detection of the marked strain. Result showed the marked strain could be detected at very low levels even in the presence of high levels of other closely related or competitive bacteria. This PCR-based DES-tagging system provides a rapid and specific tool for directly monitoring the dispersal and persistence of Xanthomonas spp.released into the environment.

• 미생물학회지
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• 제30권1호
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• pp.47-52
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• 1992

본 연구에서는 토양 내에서 비선택적으로 작용하는 제초제인 phosphinothricin(PPT) 을 분해할 수 있는 세균을 분리. 동정하고 돌연변이 유도 및 세포융합의 기법을 통해 그 능력을 개량하였으며, 아울러 다른 제초제인 glyphosate 저항성 균주 (Pseudomonas cepacia) 와의 종간 세포 융함을 이용하여 두가지 제초제에 동시에 작용 할 수 있는 균주의 개발 가능성을 알아보았다. 이때, 분리된 PPT 분해균주는 Pseudomonas paucimobilis 로 동정되었고, ethylmethansulfate 를 처리하여 영양 요구성 돌연변이를 얻은뒤, 이를 종내 세포융합을 위한 균주로 사용하였다. Lysozyme 과 EDTA 를 이용하여 원형질체를 형성시켰을때, 원형질체 재생율은 P. paucimoblis 의 경우 6.5%, P. cepacia 의 경우 8.8% 로 나타났다. 세포융합의 fusogen 으로 polyethylenglycol 6,000 을 사용하여, 종내 융합을 통한 융합체 F1, F2, 종간 융합을 통한 융합체 F3, F4 를 얻었다. 종내 융합의 결과, 융합체 F1 위 경우 야생형에 비해 PPT 분해능이 약 11% 정도 향상되었으며, 종간융합을 통하여 얻은 융합체의 경우, PPT 분해능 및 glyphosate 저항성 등의 모균주 특성을 모두 지니고 있다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.457-460
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• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.

• Mycobiology
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• 제30권1호
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• pp.22-26
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• 2002

This study was carried out to investigate the possibility for seeds germination of Gastrodia elata using symbiotic fungi. Since seeds of G. elata are very small and lack an endosperm and other nutrients, their germination is difficult without requirement for external nutrients. Out of twenty six isolates collected from protocorms of G. elata and roots of native orchids inhabited in wild, two strains(H-2 and H-21) were observed to stimulate the seed germination of G. elata. The seed germination of G. elates was excellent on oak tree leaves medium. The optimal conditions for mycelial growth of symbiotic fungi were $25^{\circ}C$ and pH 6.0, respectively. The mycelial growth of H-2 strain was excellent on YMA medium, while H-21 was poor on PDA medium. In case of carbon sources, the mycelial growth of H-2 and H-21 was good on media supplemented with glucose and dextrin, respectively. Calcium nitrate was good for mycelial growth of H-2 strain as a nitrogen sources, whereas urea was effective to H-21 strain.

• Journal of Microbiology and Biotechnology
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• 제23권3호
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• pp.304-312
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase ${\delta}$ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'${\rightarrow}$5' exonuclease activity. The resulting $HpPOL3^*$ gene encoding the error-prone proofreading-deficient DNA polymerase ${\delta}$ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a $URA3^-$ mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of $HpPOL3^*$. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

• 한국미생물·생명공학회지
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• 제7권4호
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• pp.217-223
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• 1979

자연계에서 분리한 곰팡이 중 전분을 잘 당화하며 산생성력이 강한 한 균주를 선별하여 현재 막걸리 제국용 균종으로 많이 사용되고 있는 백국균(Aspergillus Kawachii)과 비교하여 각종 성분을 경시적으르 분석한 결과는 다음과 같다. 1) 분리균중 당화력 및 액화력이 비교적 강한 M-80을 공시균으로 선별하였다. 2) 백국균과 분리균의 $\alpha$-amylase 역가는 140W. V.로 비슷하였으나 $\beta$-amylase 역가는 54 A. U. 로 분리균이 높았다. 3) acid protease 역가는 분리균이, alkaline pro-tease 역가는 백국균이 높았다. 4) 총산, ethanol, fusel oil의 생성량은 분리균이 약간 높았으며 methanol 함량은 비슷하였다. 5) 술덧중의 유리 아미노산의 함량은 분리균이 백국균보다 약 10%정도 높았으며, 15종의 유리아미노산이 확인되었다.