• Microbiology and Biotechnology Letters
• /
• v.17 no.2
• /
• pp.160-164
• /
• 1989

A 5.7Kb EcoRI fragment containing alkaline amylase gene of Bacillus sp. AL-8 obtained in the previons experiment (10) was transformed in B. subtilis via plasmid pUB110. The enzymatic proper-ties of the amylase produced by the transformants were Identical to those of the donor strain. Thus, the alkaline amylase activity from the transformant was maximum at pH 10 and 5$0^{\circ}C$. And the enzyme was very stable over the ranges of alkaline pH. In order to determine the location of the alkaline amylase gene within the 5.7Kb DNA fragment, the fragment was subcloned in E. coli. It was found that the alkaline amylase gene was located k EcoRI fragment of 3.7Kb.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1542-1546
• /
• 2009

Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. Here, we report cloning of the crtX gene responsible for zeaxanthin diglucoside biosynthesis from Paracoccus haeundaensis and the production of the corresponding carotenoids in transformed cells carrying this gene. An expression plasmid containing the crtX gene (pSTCRT-X) was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 46 kDa. Biosynthesis of zeaxanthin diglucoside was obtained when the plasmid pSTCRT-X was co-transformed into E. coli containing the pET-44a(+)-CrtEBIYZ carrying crtE, crtB, crtI, crtY, and crtZ genes required for zeaxanthin $\beta$-D-diglucoside biosynthesis.

• Journal of Pharmaceutical Investigation
• /
• v.33 no.3
• /
• pp.209-213
• /
• 2003

The objective of this study was to construct the pegylated liposomes containing plasmid DNA with optimal encapsulation efficiency. Plasmid DNA $(pGL2\;clone\;753,\;{\sim}6\;kb)$ was encapsulated by the freeze/thawing method into liposomes composed of 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphocholine (POPC), didodecyl dimethyl ammonium bromide (DDAB), distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG 2000) and DSPE-PEG 2000-male-imide. The liposomes containing plasmid DNA were then extruded through two stacked polycarbonate filters with series of different pore sizes to control the liposome size. The plasmid DNA entrapped in the liposomes was separated from free plasmid DNA by Sephadex CL-4B column chromatography. The decreased pore size of polycarbonate filters resulted in the decreased size of liposomes. The encapsulation efficiency was markedly affected by the cationic lipid (DDAB) concentration, but to a low degree by the size of liposomes and by the amount of plasmid DNA.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1994.04a
• /
• pp.178-178
• /
• 1994

Hepatitis B Virus (HBV) is a DNA virus with a 3.2kb partially double-stranded genome. The life cycle of the virus involves a reverse transcription of the greater than genome length 3.5kb mRNA. This pegenomic RNA contains all the genetic information encoded by the virus and functions as an intermediate in viral replication. Tumor suppressor p53 has previously been shown to interact with the X-gene product of the HBV, which led us to hypothesize that p53 may act as a negative regulator of HBV replication and the role of the X-gene product is to overcome the p53-mediated restriction. As a first step to prove the above hypothesis, we tested whether p53 represses the propagation of HBV in in vitro replication system. By transient cotransfection of the plasmid containing a complete copy of the HBV genome and/or the plasmid encoding p53, we found that the replication of HBV is specifically blocked by wild-type p53. The levels of HBV DNA, HBs Ag and HBc/e Ag secreted in cell culture media were dramatically reduced upon coexpresion of wild-type p53 but not by the coexpression of the mutants of p53 (G154V and R273L). Furthermore, levels of RNAs originated from HBV genome were repressed more than 10 fold by the cotransfection of the p53 encoding plasmid. These results clearly states that p53 is a nesative regulator of the HBV replication. Next, to addresss the mechanism by which p53 represses the HBV replication, we performed the transient transfection experiments employing the pregenomic/core promoter-CAT(Chloramphenicol Acetyl Transferase) construct as a reporter. Cotransfection of wild-type p53 but not the mutant p53 expression plasmids repressed the CAT activity more than 8 fold. Integrating the above results, we propose that p53 represses the replication of HBV specifically by the down-regulation of the pregenomic/core promoter, which results in the reduced DNA synthesis of HBV. Currently, the mechanism by which HBV overcomes the observed p53-mediated restriction of replication is tinder investigation.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.11
• /
• pp.1801-1809
• /
• 2015

A phytoene synthase gene, crtB, was isolated from Kocuria gwangalliensis. The crtB with 1,092 bp full-length has a coding sequence of 948 bp and encodes a 316-amino-acids protein. The deduced amino acid sequence showed a 70.9% identity with a putative phytoene synthase from K. rhizophila. An expression plasmid, pCcrtB, containing the crtB gene was constructed, and E. coli cells containing this plasmid produced the recombinant protein of approximately 34kDa , corresponding to the molecular mass of phytoene synthase. Biosynthesis of lycopene was confirmed when the plasmid pCcrtB was co-transformed into E. coli containing pRScrtEI carrying the crtE and crtI genes encoding lycopene biosynthetic pathway enzymes. The results obtained from this study will provide a base of knowledge about the phytoene synthase of K. gwangalliensis and can be applied to the production of carotenoids in a non-carotenoidproducing host.

• KSBB Journal
• /
• v.25 no.2
• /
• pp.167-172
• /
• 2010

Streptomyces scabiei producing phytotoxin called thaxtomin, which cause scab disease on economically important crops such as potato. For molecular genetics study of S. scabiei an effective transformation method was established based on conjugal transfer from Escherichia coli ET12567 (pUZ8002) using a phiC31-derived integration vector, pSET152, containing oriT and attP fragments. The high frequency was obtained on MS medium containing 50 mM $MgCl_2$. In addition, the sequence and location of the chromosomal integration attB site of S. scabiei was identified for the first time in the strains producing thaxtomin by the southern blot analysis of exconjugants and the sequencing of plasmid containing DNA flanking the insertion sites from exconjugant chromosome. Similar to the case of Streptomyces species, a single phiC31 attB site of S. scabiei is present within an ORF encoding a pirin-homolog.

• Microbiology and Biotechnology Letters
• /
• v.47 no.4
• /
• pp.546-554
• /
• 2019

This study, for the first time, reports the functional expression of lipase B derived from the yeast Candida antarctica (CALB) in Corynebacterium strain using the Escherichia coli plasmid PK18. The CALB gene fragment encoding a 317-amino-acid protein was successfully obtained from the total RNA of C. antarctica. CALB was readily produced in the Corynebacterium strain without the use of induction methods described in previous studies. This demonstrated the extracellular production of CALB in the Corynebacterium strain. CALB produced in the Corynebacterium MB001 strain transformed with pEC-CALB recombinant plasmid exhibited maximum extracellular enzymatic activity and high substrate affinity. The optimal pH and temperature for the hydrolysis of 4-nitrophenyl laurate by CALB were 9.0 and 40℃, respectively. The enzyme was stable at pH 10.7 in the glycine-KOH buffer and functioned as an alkaline lipase. The CALB activity was inhibited in the presence of high concentration of Mg²⁺, which indicated that CALB is not a metalloenzyme. These properties are key for the industrial application of the enzyme.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.2
• /
• pp.211-218
• /
• 2023

A cryptic plasmid (pTH32) was characterized from Tetragenococcus halophilus 32, an isolate from jeotgal, Korean traditional fermented seafood. pTH32 is 3,198 bp in size with G+C content of 35.84%, and contains 4 open reading frames (ORFs). orf1 and orf2 are 456 bp and 273 bp in size, respectively, and their translation products showed 65.16% and 69.35% similarities with RepB family plasmid replication initiators, respectively, suggesting the rolling-circle replication (RCR) mode of pTH32. orf3 and orf4 encodes putative hypothetical protein of 186 and 76 amino acids, respectively. A novel Tetragenococcus-Escherichia coli shuttle vector, pMJ32E (7.3 kb, Em^r), was constructed by ligation of pTH32 with pBluescript II KS(+) and an erythromycin resistance gene (ErmC). pMJ32E successfully replicated in Enterococcus faecalis 29212 and T. halophilus 31 but not in other LAB species. A pepA gene, encoding aminopeptidase A (PepA) from T. halophilus CY54, was successfully expressed in T. halophilus 31 using pMJ32E. The transformant (TF) showed higher PepA activity (49.8 U/mg protein) than T. halophilus 31 cell (control). When T. halophilus 31 TF was subculturd in MRS broth without antibiotic at 48 h intervals, 53.8% of cells retained pMJ32E after 96 h, and only 2.4% of cells retained pMJ32E after 14 days, supporting the RCR mode of pTH32. pMJ32E could be useful for the genetic engineering of Tetragenococcus and Enterococcus species.

• Mycobiology
• /
• v.38 no.4
• /
• pp.331-335
• /
• 2010

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/${\mu}g$ of DNA in $1{\times}10^7$ protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

• Korean Journal of Microbiology
• /
• v.30 no.4
• /
• pp.322-331
• /
• 1992

In order to understand the transfer and behavior of R gene in water environments. the Kmr gene in the genetically modified microorganisms(GMMs) w,is studied by conjugation. The plasmid variously rearranged in the conjugants were comparatively analyzied by agarosc gel electrophoresis and the specific Km' genes in the gel were tletected with DNA probe. The Kmr genes of the GMM strains(DKC600 and DKC601) were transferred at higher rate than those of natural isola~e(DKI)b, ut the ratc was a little diflurent depending upon the recipient strains. Rearrangement of the plasmids appeared morc drastic in GMM strains than in IIKI as donor. The transfer frequencies of the Km' genes in LR broth were remarkably higher than in the water of AW and FW without regards to the strains. In LA breth. the frequencies of Kmr genes were higher at 25'C-30$^{\circ}$C than at 10$^{\circ}$C and at pH - 7 than pH 9, but temperature and pH of the FW did n,,t affect to the frequency. And the conjugants from GMM strains in FW did not showed any plasmids. except tor 43 kb plasmiil. As results of Southern analysis of the plasmid, variously rearranged in eonjugant cells obtained in LB broth, the Kmr genes were detected at the same position of Km' plasrnids of the donor cell(DK1 and GMM strains). But Km' plasmid disappeared in the conjugants obtained in F'W and their chronlosomes showed strong signal of hybridization. The Kmr plasmid of DKl in the conjugants obtained in FW water was transferred and maintained its size, but the Kmr plasinids of the GMM strains were all integrated into chromosome. Therefore, the Kmr plasmids of DKI anit GMM strains in LH were intactly transferred and other plasmitls were variously rearranged. but Km' gene of DKC600 in FW water was integrated into the chromosorn: without regards to the temperature and pH of the water.