• Journal of Life Science
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• v.9 no.2
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• pp.153-159
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• 1999

To express constitutively the inulinase gene (INUl) of Kluyveromyces marxianus in Saccharomyces cerevisiae, three yeast promoters such as GAPDH, ADH1 and ENO1 were connected upstream of INUl. The resulting plasmids, pYIGP, pADHl-INU, and pENO-INU were introduced to S. cerevisiae SEY2102 host strain, respectively, and then each transformants were selected by staining of colonies on sucrose-agar plate. When the yeast transformants were cultivated on 2$\%$ dextrose media, the total expression levels of inulinase reached to 1.11 unit/mL, 0.88 unit/mL, and 0.69 unit/mL for respective GAPDH, ADH1, and ENO1 promoter systems. On 4% dextrose media, however, the inulinase activities were observed at 2.00 unit/mL for pYIGP, 0.71 unit/mL for pADH1-INU, and 1.40 unit/mL for pENO-INU. This result indicates that the constitutive expression of INUl was significantly affected by the initial concentration of dextrose and the promoter strength was in the order GAPDH, ENO1, and ADH1 promoter at high dextrose concentration. Taking into account the plasmid stability, however, it is suggested that the ENO1 promoter system is more suitable for the INU1 expression on high dextrose medium or in the fed-batch cultivation accumulating ethanol at high level.

• Korean Journal of Microbiology
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• v.20 no.1
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• pp.11-20
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• 1982

A study was undertaken to examine the effect of curing agents on the stability, curing and segregation of R plasmid pSL100. And also the stability, transfer frequency, and recombination of its segregants obtained from curing agent treatment were studied. Ethidium bromide, acridine orange, and mitomycin-C were used as curing agent. The results obtained were as follows ; 1. The curing agent ethidium bromide, acridine orange, and mitomycin-C were not effective for curing the multiple antibiotic resistant determinant of pSL100 in Salmonella typhimurium and Escherichia coli. However, they induced plasmid segregation with high frequency in S.typhimuruim LT-2strains. TcApSmCm, TcSmCmKm, TcApCm, TcAp, TcKm, Tc segregants were obtained. 2. The resistant markers of the segregents were transferred to S.typhimurium LT-2 strains with high frequencies whereas they were transferred to E.coli K-12 only with low frequencies. 3. The transconjugants obtained from conjugation between two different S.typhimurium segregants were similar to the phenotype of the original R factor pSL100 and the resistant markers were transferred to the S.typhimurium LT-2 or E.coli strain with equal frequencies, indicating that they are recombinants. 4. The transconjugants obtained from conjugation between pSL100 segrgants and pKM101, or pBR322 possessed the resistant markers of the two parental plasmids and they were transferred to both S.typhimurium and E.coli K-12 strains with the same frequencies and maintained stably, suggesting that they are also recombinants. 5. The recombinant pSL100 could be also obtained in rec A-strains of E.coli, suggesting that the gene function of rec A is required for the recombination of pSL100 segregants in E.coli.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.274-280
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• 1991

Effects of IPTG induction on cell growth and production of ${\beta}$-galactosidase-preS2 fusion protein (${\beta}$gal-preS2) were studied in a defined medium using a recombinant Escherichia coli JM109/pCMHB30. IPTG was added (0.2 mM) to induce the cloned-gene expression in the early-, mid-, and late-log growth phases. The most serious decreases in growth rate and plasmid stability were observed for the induction in the early-log growth phase. The expression level of ${\beta}$gal-preS2 attained by the induction in the mid-log phase was about 0.51 mg fusion protein/mg total cellular protein, which was 2- and 5-fold improvement over the levels obtained with the inductions in the early- and late-log phases. Formation of acidic byproducts including acetate and pyruvate showed different profiles during the fermentation period for each cases of induction; pyruvate was the major byproduct for the induction in the early-log phase while acetate production became more significant for the cases of inductions in the mid- and late-log phases.

• Korean Journal of Microbiology
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• v.27 no.4
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• pp.323-333
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• 1989

Plasmid DNA molecules containing strong promoter upstream from IS50L or IS50R, the two insertion sequences that flank Tn5, were constructed to amplify the synthesis of Tn5-encoded polypeptides. When proteins made by cells that contain these plasmids were analyzed on polyacrylamide gels, enhanced synthesis of IS50R polypeptides could be detected. Synthesis of this polypeptide apparently is initiated within the large open reading frame of this element. In addition, the stability of IS50L-and IS50R-encoded polypeptides was analyzed. It was found that IS50L polypeptides are relatively unstable in vivo. This instability could account for the observed inability of this element to promote transposition.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.551-553
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• 2000

A strong constitutive $P_{JH}$ promoter from Bacillus was applied to overexpress the endoxylanase gene in B. subtilis. The expression plasmid, pHJKJ4, was designed to contain the $P_{JH}$ promoter and endoxylanase promoter ($P_B$), and introduced into B. subtilis DB104. Through batch fermentation of the trasformant cell on a maltose medium, endoxylanase was produced in a growth-associated manner as the predominant protein. The total activity reached about 600 unit/ml at the end of the cultivation, which corresponded to 698 mg endoxylanase protein/l with a specific activity of 860 unit/mg protein. It was also found that the segregational plasmid instability was less than 30% and most of the endoxylanase activity was detected in the culture medium. This result suggests that the secretory production of endoxylanase can be significantly enhanced with the use of the $P_{JH}$ promoter and high-cell density culture techniques, quantitatively as well as qualitatively.

• Journal of Life Science
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• v.10 no.2
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• pp.125-130
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• 2000

To overproduce endoxylanase from a recombinant Bacillus subtilis harboring the pJHKJ4 plasmid, the effects of carbon and nitrogen sources on the cell growth and expression level of endoxylanase were investigated in the flask cultures. Among the various carbon and nitrogen sources tested, glucose and maltose as carbon source and yeast extract as nitrogen source were found to be the most effective for the cell growth and the endoxylanase expression. When the concentration of glucose was increased from 0.5% to 5%, the highest activity of extracellular endoxylanse, 166 unit/$m\ell$, was observed at 2% glucose. In case of maltose, the endoxylanase was stably produced at the level of 180 unit/$m\ell$, regardless of the concentration of maltose. The higher the concentration of yeast extract, the greater cell growth and endoxylanase expression were obtained. However, the highest endoxylanase activity per unit cell mass was observed with 1% yeast extract. With the optimized medium (2% glucose, 1% yeast extract, etc), about 630 unit/$m\ell$ of endoxylanse was expressed through the batch fermentation in a fermentor, which expression level corresponded to about 0.7 g-endoxylanase protein /$\ell$. It was also found that the plasmid was stably maintained above 70% level, and more than 90% of endoxylanase activity was detected in the extracellular medium.

• Korean Journal of Microbiology
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• v.28 no.1
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• pp.55-64
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• 1990

Cell volume and DNA contents of the fusants were similar to those of parents. Genetic stability of the fusants was increased when they were cultured on minimal medium (MM) rather than on complete medium (CM), and the fusants were stabilized by subculturing 7 generations each 7 day on MM agar. The finally selected fusants after being cultured for 6 months on CM were stable without segregation. The fusants could also form nuclein and ascospores, and show red and pink colors by the test of TTC colorization. Assimilability and fermentability of carbon sources of the fusants were similarto those of parents. The tolerance of KCl, NaCl, sodium propionate and cycloheximide showed the traits of one strain of parents. When the fusants were cultured for 72 hr and 60 hr in the medium containing 20% glucose and sucrose, respectively, the yield of ethanol for FWKS 260 was reached to 9.6 v/v% and 9.8v/v%, respectively. The sensitive strain Kyokai 7 was found to be killed entirely after cultivation of 48 hr by the killer toxin from the fusants. The recipient S 29 and Kyokai 7 were found to have neither L nor M dsRNA plasmid. However, K 52 and fusants had both L and M dsRNA plasmid of 4.7 kb and 2.5 kb, respectively. The curants treated by heat and cycloheximide did not contain M dsRNA plasmid, but had large amounts of L dsRNA plasmid of those of killer yeasts.

• Journal of Life Science
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• v.14 no.3
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• pp.391-395
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• 2004

To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.128-134
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• 2008

In order to examine efficient L-threo-2,3-Dihydroxyphenylserine (L-threo-DOPS) synthesis process using whole cell biocatalyst, a thermostable L-threonine aldolase (L-TA), which cloned from Streptomyces coelicolor A3(2) and improved for stability, was expressed in a Corynebacterium glutamicum R strain. The constructed Corynebacterium expression vector, pCG-H44(1) successfully expressed L-TA in C. glutamicum R strain, but showed very low expression level. In order to improve the expression level, the expression vector named pCG-H44(2) was reconstructed by eliminating 1 nucleotide between SD sequence and start codon of L-TA. The pCG-H44(2) vector plasmid was able to overexpress L-TA approximately 3.2 times higher than pCG-H44(1) in C. glutamicum R strain (CGH-2). When the whole cell of CGH-2 was examined in a repeated batch system, L-threo-DOPS was successfully synthesized with a yield of 4.0 mg/ml and maintain synthesis rate constantly after 30 repeated batch reactions for 130 h.

• Microbiology and Biotechnology Letters
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• v.13 no.4
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• pp.349-354
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• 1985

A 4.7 kb Hind III fragment containing $\alpha$-amylase gene of Bacillus stearothermophilus IAM 11062 was cloned in Escherichia coil HB101, using plasmid pBR322 and runaway plasmid pSY343 as a vector. The cloned gene was stably maintained and expressed In E.coli. The constructed strain of E. coli have at least 3 times higher amylase activity than the donor strain, of B. stearothermophilus. About 75％ of the $\alpha$-amylase produced by the constructed strain of E. coli was localized in the periplasm and it was found that the enzymes can be released by an osmotic shock using EDTA. The enzymatic properties of L-amylase produced in E. coli were very similar to those produced by B. stearothermophilus in terms of optimum temperature, heat stability and molecular weight.