• Preventive Nutrition and Food Science
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• v.13 no.3
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• pp.190-195
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• 2008

The $\alpha$-amylase gene, amyL, from Bacillus licheniformis was expressed in Lactobacillus brevis 2.14 and Escherichia coli $DH5{\alpha}$ using two different shuttle vectors, pCW4 and pSJE. E. coli transformants (TFs) harboring either $pCW4T{\alpha}$ or $pSJET{\alpha}$ produced active $\alpha$-amylase but L. brevis TFs did not, as determined by enzyme assays and zymography. But amyL transcripts were synthesized in L. brevis TFs. In terms of plasmid stability, pSJE, a theta-type replicon, was more stable than pCW4, an RCR (rolling circle replication) plasmid, in L. brevis without antibiotic selection.

• Korean Journal of Microbiology
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• v.28 no.3
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• pp.181-187
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• 1990

STA gene coding glucoamylase was introduced into haploid Saccharomyces cerevisiae SHY3 and polyploid Saccharomyces cerevisiae 54. We constructed the recombinant plasmid by substituting the promoter region of alcohol dehydrogenase isoenzyme I gene for that of STA gene to increase the expression of STA gene and found that the activity of glucoamylase was increased in transformants. The plasmid stability was improved remarkably when we got the STA gene into the plasmid which had centromere. The activity of glucoamylase and transformation frequency of it, however, was decreased because of low copy number. Industrial polyploid strain was transformed with the recombinant plasmid having the $2\mu$ origin of replication and STA gene. It produced more alcohol than host when fermented in liquefied starch media. The industrial strain, however, was not transformed with the autonomously replicating plasmid containing centromere.

• Journal of Microbiology
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• v.37 no.1
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• pp.35-40
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• 1999

Sporulation-specific glucoamylase (SGA) gene was isolated from genomic library of Saccharomyces diastaticus 5114-9A by using glucoamylase non-producing mutant of S. diastaticus as a recipient. When the glucoamylase activities of culture supernatant, periplasmic, and intracellular fraction of cells transformed with hybrid plasmid containing SGA gene were measured, the highest activity was detected in culture supernatant. SGA produced by transformant and extracellular glucoamylase produced by S. diastaticus 5114-9A differed in enzyme characteristics such as optimum temperature, thermostability, and resistance to SDS and urea. But the characteristics of SGA produced by sporulating yeast cells and vegetatively growing transformants were identical.

• Journal of Microbiology
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• v.45 no.3
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• pp.213-218
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• 2007

Trametes versicolor has a lignin degrading enzyme system, which is also involved in the degradation of diverse recalcitrant compounds. Manganese-dependent peroxidase (MnP) is one of the lignin degrading enzymes in T. versicolor. In this study, a cDNA clone of a putative MnP-coding gene was cloned and transferred into an expression vector (pBARGPE1) carrying a phosphinothricin resistance gene (bar) as a selectable marker to yield the expression vector, pBARTvMnP2. Transformants were generated through genetic transformation using pBARTvMnP2. The genomic integration of the MnP clone was confirmed by PCR with bar-specific primers. One transformant showed higher enzyme activity than the recipient strain did, and was genetically stable even after 10 consecutive transfers on non-selective medium.

• Journal of Microbiology
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• v.45 no.3
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• pp.241-245
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• 2007

This study was conducted to determine the optimal conditions for introduction of exogenous DNA into Synechocystis sp. PCC 6803. Of the three transformation techniques studied, electroporation, ultrasonic transformation and natural transformation, natural transformation showed the highest efficiency. Additionally, this study demonstrated that the higher plasmid concentration and longer homologous recombining fragments resulted in a greater number of transformants. For successful transformation, the lowest concentration of plasmid was $0.02\;{\mu}g/ml$, and the shortest homologous recombining fragment was 0.2 kb. Use of Synechocystis sp. PCC 6803 in the logarithmic growth phase resulted in two-fold higher transformation rate than that of the same organism when cells in the latent phase or the plateau phase were used for transformation. Pretreatment of the host strain, Synechocystis sp. PCC 6803, with EDTA (2 mM) for two days prior to transformation increased the transformation efficiency by 23%. Additionally, incubation of the cells and DNA for 5 h under light conditions increased the transformation efficiency by two orders of magnitude. Moreover, recovery treatment of the cells before they were plated onto antibiotic medium also increased the transformation efficiency.

• Preventive Nutrition and Food Science
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• v.15 no.4
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• pp.360-363
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• 2010

To enhance the expression and secretion of pediocin PA-1 from heterologous bacterial hosts, the promoter and deduced signal sequence (PS) of an $\alpha$-amylase gene from a Bifidobacterium adolescentis strain was fused with pediocin PA-1 structural and immunity genes (AB) and the resulting functions were evaluated in Escherichia coli. Two recombinant PCR products were created-one with just the deduced signal sequence and one with the sequence plus the Ser and Thr sequences that are the next two amino acids of the signal sequence. These two products, the PSAB (---AQA::KYY---) and PSABST (---AQA$\underline{ST}$::KYY---), respectively, were inserted into a TA cloning vector (yT&A) and named pPSAB, which was previously reported, and pPSABST. The two recombinant plasmid DNAs were transferred into E. coli JM109 and the transformants displayed antimicrobial activity, where the activity of E. coli JM109 (pPSAB) was stronger than that of E. coli JM109 (pPSABST), indicating that the ST amino acid residues were not necessary for secretion and might have even decreased the antimicrobial activity of recombinant pediocin PA-1.

• Journal of Microbiology and Biotechnology
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• v.7 no.1
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• pp.8-12
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• 1997

In order to overproduce D-xylose isomerase, the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene (xylA) was fused to ${\lambda}P_{L}$ promoter. The promoterless xylA gene containing the ribosome binding site and coding region for D-xylose isomerase was cloned into a site 0.3 kb downstream from the ${\lambda}P_{L}$ promoter on a high copy number plasmid. An octameric XbaI linker containing TAG amber codon was inserted between 33rd codon of ${\lambda}N$ and the promoterless xylA gene. The resulting recombinant plasmid (designated as pPX152) was transformed into E. coli M5248 carrying a single copy of the temperature sensitive ${\lambda}cI857$ gene on its chromosomal DNA. When temperature-induced, the transformants produced 15 times as much D-xylose isomerase as that of D-xylose-induced parent strain. The amount of overproduced D-xylose isomerase was found to be about 60% of total protein in cell-free extracts.

• Journal of Sericultural and Entomological Science
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• v.35 no.1
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• pp.36-42
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• 1993

Expression of insecticidal protein by fusion product of truncated HD-1[CryIA(a)] N-terminal and HD-73[CryIA(c)] C-Terminal fragment of Bacillus thruingiensis subsp. kurstaki was investigate. Immunological analysis of transformants by using polyclonal antisera raised against the whole-crystal protein of HD-1 revealed that SK4 and SK5 were observed cross-reaction with polypeptides of 77-kDa and 105-kDa, respectively. Bioassay of the transformant pSK5 to Plutella maculipennis and Heliothis assulta were 96% and 97%, respectively.

• Journal of Microbiology
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• v.43 no.4
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• pp.361-365
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• 2005

In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 ${\mu}g$/ ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

• Korean Journal of Microbiology
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• v.38 no.3
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• pp.162-167
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• 2002

To analyse proteins and gene related to antifungal activity, SAR01 strain was isolated from a brown seaweed and identified as Streptomyces sp. by FAME(fatty acid methyl ester) analysis. Antifungal activity deficient mutant(SAR535) of Streptomyces sp. SAR01 was induced by gamma radiation$({60}^Co)$. It was found that 6 specific protein spots appeared only in SAR01 by 2-D electrophoresis analysis. Among them, a protein of 10 kDa had homology of 96% with 10 kD chaperonin cpn 10 (GroES) by Basic Local Alignment Search Tool(BLAST, NCBI) analysis. SAR535 transformants into which groES was transferred by electroporation revealed antifungal activity newly similar with SAR01 It suggested that groES be supposed to be related to the antifungal activity of Streptomyces sp. SAR01.