• The Plant Pathology Journal
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• v.33 no.3
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• pp.318-328
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• 2017

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was $50^{\circ}C$. However, it was inactivated with time when it was incubated at $40^{\circ}C$ and $50^{\circ}C$. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

• KSBB Journal
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• v.8 no.2
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• pp.133-142
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• 1993

The effects of genetic and environmental factors on productivity of a cloned protein were studied in recombinant Saccharomyces cerevisiae. Plasmid stability and copy level were very high for a $REP^+$ system(at ca. 10 generations, stability: 65-90%, plasmid copy number per cell: 40-200), whereas these were very low for a yep- system(at ca. 10 generations, stability: 30%, plasmid copy number per cell 20). In plasmids containing the $2{\mu}m$ circle genome, a $[cir^o]$ strain was a preferred host cell since the plasmid stability and the copy number in a $[cir^o]$ strain were higher than in a $[cir^+]$strain. Cloned gene expression was dependent on plasmid copy number and stability. The inducer (galactose) level played a very important role in cloned lacZ gene expression, showing that a galactose concentration of 0.8% was sufficient for induction of gene expression. Induction rate was very fast in the case of plasmids exhibiting high stability and copy number by a factor of 4 to 25. The time to reach the peak value of gene expression was longer when galactose was added at the start of fermentation (ca. 26 hours) than at the mid-exponential phase (ca. 6 hours). Glucose repression was reduced by a factor of 2 to 5 as the relative inducer level increased.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.510-515
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• 2012

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

• Molecules and Cells
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• v.28 no.4
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• pp.321-329
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• 2009

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and $H_2O_2$ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.

• Journal of Life Science
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• v.20 no.1
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• pp.97-102
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• 2010

Streptomyces platensis YK-2, newly isolated from forest soil, produces transglutaminase (TGase), which catalyses an acyl transfer reaction between the primary grade amine and protein or $\gamma$-carboxyamide group of peptide bound glutamine residues. For a molecular genetic study of S. platensis, an effective transformation method was established by using a conjugal transfer of DNA from Escherichia coli to spores of actinomycetes. The highest transconjugation frequency of S. platensis was obtained on an MS medium containing 50 mM $MgCl_2$, using $5{\times}10^7\;E$. coli as a DNA donor and $1{\times}10^8$ spores without heat treatment as a host. We also identified that S. platensis contains a single attB site within an ORF encoding a pirin-homolog, and that its attB site sequence shows high homology to that of S. logisporoflavus. In addition, it was confirmed by phenotypic analyses of exconjugants that the introduction of heterologous DNA into the attB site of the S. platensis chromosome does not affect its morphological differentiation and TGase production.

• KSBB Journal
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• v.9 no.5
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• pp.532-540
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• 1994

Secretion efficiency is generally affected by promoter, signal sequence, characteristics of foreign protein and host. Secretion efficiencies of glucoamylase in recombinant plasmid-harboring yeast and chromosome-integrated yeast which had STA signal sequences were 74% and 65% at the 4th day of incubation, respectively. The high secretion efficiencies of the yeasts were obtained due to the fact that the expression levels were not reached at the secretory apparatus capacities of the host yeasts. In both yeasts, most of the intracellular glucoamylase were detected in cytoplasm and small portion (below 10%) of glucoamylase were located in periplasm. The characteristics of secreted heterologous glucoamylase from recombinant Saccharomyces cerevisiaes were investigated by using Western blot analysis. The secreted mature glucoamylase was heterogeneous and its molecular weight was about 200 to 300 kilodalton. The carbohydrate content of mature glucoamylase was higher than 80%, and several bands of about 55 to 65 kilodalton indicate the endoplasmic reticulum forms of intracellular glucoamylase.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.337-346
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• 2016

Three combinations of molecular chaperones from Escherichia coli (i.e., DnaK-DnaJ-GrpE-GroEL-GroES, GroEL-GroES, and DnaK-DnaJ-GrpE) were overproduced in E. coli BL21, and their in vitro stabilizing effects on a nitrile hydratase (NHase) were assessed. The optimal gene combination, E. coli groEL-groES (ecgroEL-ES), was introduced into Rhodococcus ruber TH3. A novel engineered strain, R. ruber TH3G was constructed with the native NHase gene on its chromosome and the heterologous ecgroEL-ES genes in a shuttle plasmid. In R. ruber TH3G, NHase activity was enhanced 37.3% compared with the control, TH3. The in vivo stabilizing effect of ecGroEL-ES on the NHase was assessed using both acrylamide immersion and heat shock experiments. The inactivation behavior of the in vivo NHase after immersion in a solution of dynamically increased concentrations of acrylamide was particularly evident. When the acrylamide concentration was increased to 500 g/l (50%), the remaining NHase activity in TH3G was 38%, but in TH3, activity was reduced to 10%. Reactivation of the in vivo NHases after varying degrees of inactivation was further assessed. The activity of the reactivated NHase was more than 2-fold greater in TH3G than in TH3. The hydration synthesis of acrylamide catalyzed by the in vivo NHase was performed with continuous acrylonitrile feeding. The final concentration of acrylamide was 640 g/l when catalyzed by TH3G, compared with 490 g/l acrylamide by TH3. This study is the first to show that the chaperones ecGroEL-ES work well in Rhodococcus and simultaneously possess protein-folding assistance functions and the ability to stabilize and reactivate the native NHases.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.511-520
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• 2015

Triclosan, the widely used biocide, specifically targets enoyl-acyl carrier protein reductase (ENR) in the bacterial fatty acid synthesis system. Although the fish pathogen Aeromonas salmonicida subsp. salmonicida exhibits triclosan resistance, the nature of this resistance has not been elucidated. Here, we aimed to characterize the triclosan resistance of A. salmonicida subsp. salmonicida causing furunculosis. The fosmid library of triclosan-resistant A. salmonicida subsp. salmonicida was constructed to select a fosmid clone showing triclosan resistance. With the fosmid clone showing triclosan resistance, a subsequent secondary library search resulted in the selection of subclone pTSR-1. DNA sequence analysis of pTSR-1 revealed the presence of a chromosomal-borne fabV-encoding ENR homolog. The ENR of A. salmonicida (FabVas) exhibited significant homology with previously known FabV, including the catalytic domain YX₍₈₎K. fabVas introduction into E. coli dramatically increased its resistance to triclosan. Heterologous expression of FabVas might functionally replace the triclosan-sensitive FabI in vivo to confer E. coli with triclosan resistance. A genome-wide search for fabVas homologs revealed the presence of an additional fabV gene (fabVas2) paralog in A. salmonicida strains and the fabVas orthologs from other gram-negative fish pathogens. Both of the potential FabV ENRs expressed similarly with or without triclosan supplement. This is the first report about the presence of two potential FabV ENRs in a single pathogenic bacterium. Our result suggests that triclosan-resistant ENRs are widely distributed in various bacteria in nature, and the wide use of this biocide can spread these triclosan-tolerant ENRs among fish pathogens and other pathogenic bacteria.

• Journal of Microbiology
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• v.42 no.2
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• pp.111-116
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• 2004

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli $tRNA_{1}$$^{Gln}$ with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-$tRNA_{1}$$^{Gln}$ formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also amelio-rated growth inhibition, presumably by competitively preventing $tRNA_{1}$$^{Gln}$ misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of $tRNA_{1}$$^{Gln}$, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-$tRNA^{Gln}$ amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mis-charging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli Glu-$tRNA_{1}$$^{Gln}$, and converts it to the cognate Gln-$tRNA_{1}$$^{Gln}$ species. B. subtilis GluRS-dependent Glu-$tRNA_{1}$$^{Gln}$ formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.6
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• pp.510-515
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• 2006

The putative EPA synthesis gene cluster was mined from the entire genome sequence of Shewanella oneidensis MR-1. The gene cluster encodes a PKS-like pathway that consists of six open reading frames (ORFs): ORFSO1602 (multi-domain beta-ketoacyl synthase, KS-MAT-4ACPs-KR), ORFSO1600 (acyl transferase, AT), ORFSO1599 (multi-domain beta-ketoacyl synthase, KS-CLF-DH-DH), ORFSO1597 (enoyl reductase, ER), ORFSO1604 (phosphopentetheine transferase, PPT), and ORFSO1603 (transcriptional regulator). In order to prove involvement of the PKS-like machinery in EPA synthesis, a 20.195-kb DNA fragment containing the genes was amplified from S. oneidensis MR-1 by the long-PCR method. Its identity was confirmed by the methods of restriction enzyme site mapping and nested PCR of internal genes orfSO1597 and orfSO1604. The DNA fragment was cloned into Escherichia coli using cosmid vector SuperCos1 to form pCosEPA. Synthesis of EPA was observed in four E. coli clones harboring pCosEPA, of which the maximum yield was 0.689% of the total fatty acids in a clone designated 9704-23. The production yield of EPA in the E. coli clone was affected by cultivation temperature, showing maximum yield at $20^{\circ}C$ and no production at $30^{\circ}C$ or higher. In addition, production yield was inversely proportional to glucose concentration of the cultivation medium. From the above results, it was concluded that the PKS-like modules catalyze the synthesis of EPA. The synthetic process appears to be subject to regulatory mechanisms triggered by various environmental factors. This most likely occurs via the control of gene expression, protein stability, or enzyme activity.