• Microbiology and Biotechnology Letters
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• v.45 no.3
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• pp.271-275
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• 2017

The expression of Deinococcus radiodurans dr1558 and pprM genes was examined for enhanced lysine production in recombinant Corynebacterium glutamicum. These genes are known to confer high tolerance to pH and osmotic shock in Escherichia coli. D. radiodurans dr1558 and pprM genes were expressed in C. glutamicum by using 6 synthetic promoters of different strengths, to evaluate the effect of expression efficiency on lysine production. Recombinant C. glutamicum expressing DR1558 under the L26 and I64 promoters showed higher lysine production than that expressing DR1558 under other promoters. Similarly, recombinant C. glutamicum expressing PprM under same promoters (L26 and I64) showed a higher increase in lysine production compared to that expressing PprM under other promoters. In the absence of $CaCO_3$ in the medium, the expression of DR1558 or PprM also increased lysine concentration in C. glutamicum depending on the promoter used. Together, these results suggest that genes involved in radiation tolerance in D. radiodurans can be used to enhance production of amino acids and their derivatives.

• Korean Journal of Plant Tissue Culture
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• v.22 no.6
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• pp.351-355
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• 1995

A truncated Polygalacturonase (PG) cDNA was fused in reverse orientation to the CaMV 35S promoter of the binary vector pCA643, and introduced into tomato cells by Agrobaderium - mediated transformation. Transformed cells were selected using kanamycin as select agent then regenerated into plants. After selfed, one transgenic line (T9), was germinated and grown on MS medium containing 1 mg/mL of kanamycin Genomic Southern analysis of a T9 progeny with labelled PG2 cDNA probe showed a single antisense PC fragment as well as the endogenous PG2 gene, suggesting that PC antisense gene was integrated into tomato genome. Northern blot analysis demonstrated that the antisense RNA was produced from the transgene at much tiger level than the endogenous PG2 gene. Polygalacturonase activity analysis of the fruit from transgenic plants demonstrated that the antisense transgene expression caused 4 to 60% reduction of endogenous PG activity.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.241-247
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• 2016

Natamycin is a widely used antifungal antibiotic. For natamycin biosynthesis, the gene pimE encodes cholesterol oxidase, which acts as a signalling protein. To confirm the positive effect of the gene pimE on natamycin biosynthesis, an additional copy of the gene pimE was inserted into the genome of Streptomyces gilvosporeus 712 under the control of the ermE* promoter (p_ermE*) using intergeneric conjugation. Overexpression of the target protein engendered 72% and 81% increases in the natamycin production and cell productivity, respectively, compared with the control strain. Further improvement in the antibiotic production was achieved in a 1 L fermenter to 7.0 g/l, which was a 153% improvement after 120 h cultivation. Exconjugants highly expressing pimE and pimM were constructed to investigate the effects of both genes on the increase of natamycin production. However, the co-effect of pimE and pimM did not enhance the antibiotic production obviously, compared with the exconjugants highly expressing pimE only. These results suggest not only a new application of cholesterol oxidase but also a useful strategy to genetically engineer natamycin production.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.928-934
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• 2005

ATF2 is a cellular transcription factor which belongs to the CREB/ATF class and it is leucine zipper protein which generally binds to DNA as dimers. This paper presents the procedure for subcloning the ATF2 gene and the results of experiment used the expressed ATF2. The pET expression vector was used since it produced 6xHis fusion protein for easy purification using affinity column. The Nickel chelating chromatography was used for Purifying the expressed ATE2 from E- codi BL2l. Subsequen시y In vitro binding pull-down assay showed the binding specificity of ATF2 with AP-1 family factors such as BATF, c-Fos, c-Jun and ATF2 itselgf. ATF2 forms homodimer as well as strong heterodimer with BATF. It also forms stable dimer with c-Jun but barely binds with c-Fos.

• Journal of Microbiology and Biotechnology
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• v.34 no.9
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• pp.1857-1866
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• 2024

Endophytic fungi have been shown to synthesize bioactive secondary metabolites, some of which promote plant growth through various mechanisms. In our previous study, endophytic fungi were isolated from mango trees (Mangifera indica L.). The present study examined fifty endophytic fungal isolates for mineral solubilization activity, ammonia production, and siderophore production. It was shown that these isolates could produce phytohormones indole-3-acetic acid and gibberellic acid, as well as inhibit plant pathogens, specifically Colletotrichum gloeosporioides and Lasiodiplodia theobromae. The results showed that all the isolated fungal endophytes exhibited various activities. Based on the findings, two fungal endophytes-Aureobasidium pullulans CY.OS 13 and Aspergillus tamarii CY.OS 144-were selected for dual inoculation in chili plants under pot-scale conditions to investigate their potential to improve growth-related traits such as seed germination, shoot and root length, biomass, and chlorophyll content. Seed treated with A. pullulans CY.OS 13 and/or A. tamarii CY.OS 144 showed a significant (p < 0.05) increase in seed germination and growth parameters of chili plants grown under pot-scale conditions. Particularly, chili plants whose seeds were injected with a combination of the two selected endophytic fungi showed the highest plant development traits. Therefore, the selected endophytic fungi have the potential to be used as biofertilizers, especially when combined. They could eventually replace chemical fertilizers because they are environmentally friendly, beneficial to humans, and can even promote sustainable agriculture.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1470-1476
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• 2016

Octacosanol, which has prominent physiological activities and functions, has been recognized as a potential growth promoter in animals. A total of 392 1-d-old male Arbor Acres broiler chicks with similar body weight were randomly distributed into four dietary groups of seven replicates with 14 birds each supplemented with 0, 12, 24, or 36 mg octacosanol (extracted from rice bran, purity >92%)/kg feed. The feeding trial lasted for six weeks and was divided into the starter (day 1 to 21) and the grower (day 22 to 42) phases. The results showed that the feed conversion ratio (FCR) was significantly improved in broilers fed a diet containing 24 mg/kg octacosanol compared with those fed the control diet in the overall phase (day 1 to 42, p = 0.042). The average daily gain and FCR both showed linear effects in response to dietary supplementation of octacosanol during the overall phase (p = 0.031 and 0.018, respectively). Broilers fed with 24 or 36 mg/kg octacosanol diet showed a higher eviscerated yield, which increased by 5.88% and 4.26% respectively, than those fed the control diet (p = 0.030). The breast muscle yield of broilers fed with 24 mg/kg octacosanol diet increased significantly by 12.15% compared with those fed the control diet (p = 0.047). Eviscerated and breast muscle yield increased linearly with the increase in dietary octacosanol supplementation (p = 0.013 and 0.021, respectively). Broilers fed with 24 or 36 mg/kg octacosanol diet had a greater (p = 0.021) $pH_{45min}$ value in the breast muscle, which was maintained linearly in response to dietary octacosanol supplementation (p = 0.003). There was a significant decrease (p = 0.007) in drip loss value between the octacosanol-added and the control groups. The drip loss showed linear (p = 0.004) and quadratic (p = 0.041) responses with dietary supplementation of octacosanol. These studies indicate that octacosanol is a potentially effective and safe feed additive which may improve feed efficiency and meat quality, and increase eviscerated and breast muscle yield, in broiler chicks. Dietary supplementation of octacosanol at 24 mg/kg diet is regarded as the recommended dosage in the broilers' diet.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1519-1528
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• 2013

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2-9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.15-19
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• 2004

The expression of a chimeric transgene (nos-npt II) has been examined in 9 years old transgenic poplars (Populus koreana x P. nigra) growing in a nursery. The expression of the gene in twenty six independentely transformed plants were examined by 1) enzyme (NPT II) assay, 2) RT-PCR, and 3) resistance to kanamycin. High NPT II activities in young leaves of all the transformed plants were found even without a selection pressure for antibiotics for 9 years. However, the activity varied with the positions of leaves in the stem in that young leaves showed higher activity than did mature tissues. When leaf segments were cultured in the presence of 150 mg/l kanamycin, only those from young leaves produced vigorously growing callus. However, as in the case of NPTII assay, the leaf segments from mature leaves did not form callus well on the media. RT-PCR with nptII specific primers also showed that amplification products were observed only when RNAs from young tissues were used. The total RNA gel showed that while RNA in young leaves are relatively stable and in a large quantity, those in old leaves were mostly degraded. All the above results suggest that the gene is transcriptionally active only in young tissue even though it is attached to a constituitive promoter. Therefore, the expression of foreign gene in poplar plants seemed to be affected by the metabolic state of the cells and thus vary greatly with the developmental stages and the age of tissue.

• Journal of Microbiology and Biotechnology
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• v.18 no.12
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• pp.1938-1944
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• 2008

The procpb gene encoding human procarboxypeptidase B (proCPB, GeneBank access code AJ224866) was cloned and its Pichia expression plasmid, $pPIC9{\alpha}$/hproCPB (9.2 kb), was constructed, in which procpb was under the control of the AOXl promoter and connected to the downstream of the mating factor ${\alpha}$-1 ($MF{\alpha}1$) signal sequence. The plasmid was linearized by digestion with Sacl, and integrated into the genome of P. pastoris strain GS115. By culturing of Pichia transformant on methanol medium, the human proCPB was successfully expressed and secreted into the culture supernatant. Moreover, Western blot analysis of the extracellular proteins showed proCPB bands clearly at a molecular mass of 45 kDa, confirming the expression of proCPB with its right size. The CPB activity reached about 3.5 U/ml and 12.7 U/ml in the flask and fermentor batch cultures of Pichia transformant, respectively. No CPB enzyme activity was found in the intracellular fraction. When the fed-batch cultivation was performed with methanol and glycerol mixture as a feeding medium, the extracellular CPB activity was increased to 42.0 U/ml, which corresponds to a 3.3-fold higher level of CPB activity than that of batch culture. The $K_m$ and $k_{cat}$ values of recombinant human CPB enzyme for hippuryl-$_L$-Arg as a substrate were estimated to be 0.16 mM and $11.93\;sec^{-1}$, respectively.

• Journal of Microbiology and Biotechnology
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• v.24 no.11
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• pp.1516-1524
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• 2014

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and $55^{\circ}C$. In addition, it displayed very high thermal stability, with a half-life of 82 min at $60^{\circ}C$. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.