• Korean Journal of Microbiology
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• v.33 no.4
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• pp.274-276
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• 1997

Transformation of Coprinus congregatus with a linearized plasmid has been successfully carried out using phosphinothricin resistance gene as a dominant selectable marker. The transforming frequency was about 500 transformants per microgram of DNA using the protoplast-$CaCl_2$ method. The transforming vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from a transformant by Southern hybridization.

• Journal of agriculture & life science
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• v.44 no.6
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• pp.141-146
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• 2010

In this study, Agrobacterium-mediated transformation was tested to the mycelium culture of Flamulina velutipes which is very popular as an edible mushroom in Korea. Particularly, aluminum oxide particles were used to generate wounds in F. velutipes mycelia via vigorous shaking prior to agro-infiltration. The result showed that transformants resistant to hygromycin could be obtained only from the mycelia with physical wounds. Gene transfer was verified by genomic DNA PCR. This study suggested a convenient tool to improve Agrobacterium-mediated transformation of F. velutipes.

• Plant Resources
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• v.7 no.1
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• pp.39-46
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• 2004

To demonstrate the importance of transformation efficiency in independent event, molecular and cytogenetic analysis were conducted with genomic DNA and chromosome of transgenic plants produced by Agrobacterium tumefeciens LBA4404 (pSBM-PPGN: gusA and bar). Selection ratios of putative transgenic calli were similar in independent experiments, however, transformation efficiencies were critically influenced by the type of regeneration media. MSRK5SS-Pr regeneration mediun, which contains 5 mgL$^{-1}$ kinetin, 2% (w/v) sucrose in combination with 3% (w/v) sorbitol, and 500 mgL$^{-1}$ proline, was efficient to produce transgenic plant of rice from putative transgenic callus in the presence of L-phosphinotricin (PPT). With MSRK5SS-Pr medium, transformation efficincies of Nagdongbyeo were significantly enhanced from 3.7% to 6.3% in independent callus lines arid from 7.3% to 19.7% in plants produced, respectively. Stable integration and expression of bar gene were confirmed by basta herbicide assay, PCR amplification and Southern blotting of bar gene, and fluorescence in situ hybridization (FISH) analysis using pSBM-PPGN as a probe. In Southern blot analysis, diverse band patterns were observed in total 44 transgenic plants regenerated from 20 independent PPT resistant calli showing from one to five copies of T-DNA segments, however, the transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• Journal of Microbiology and Biotechnology
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• v.32 no.2
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• pp.248-255
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• 2022

Phlebia sp. MG-60 is the salt-tolerant, white-rot fungus which was isolated from a mangrove forest. This fungus expresses three kinds of manganese peroxidase (MGMnP) isozymes, MGMnP1, MGMnP2 and MGMnP3 in low nitrogen medium (LNM) or LNM containing NaCl. To date, there have been no reports on the biochemical salt-tolerance of these MnP isozymes due to the difficulty of purification. In present study, we established forced expression transformants of these three types of MnP isozymes. In addition, the fact that this fungus hardly produces native MnP in a high-nitrogen medium (HNM) was used to perform isozyme-selective expression and simple purification in HNM. The resulting MGMnPs showed high tolerance for NaCl compared with the MnP of Phanerochaete chrysosporium. It was worth noting that high concentration of NaCl (over 200 mM to 1200 mM) can enhance the activity of MGMnP1. Additionally, MGMnP1 showed relatively high thermo tolerance compared with other isozymes. MGMnPs may have evolved to adapt to chloride-rich environments, mangrove forest.

• Proceedings of the Botanical Society of Korea Conference
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• 1995.06a
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• pp.67-78
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• 1995

Monoclonal antibodies (MoAbs) are a highly diversified class of proteins with major research and commercial applications such as diagnostics and therapeutics. Currently, the dominant method for producing MoAbs is through the hybridoma technique. However, this technique is slow, tedious, labor intensive, and expensive. The production of MoAbs in cultured transgenic plant cells can offer some advantages over that in the over that in the mammalian systems. The media to cultivate plant cells are well defined and inexpensive. Contamination by bacteria or fungi is easily monitored in plant tissue cultures. Furthermore, these contaminants are usually not potent pathogens to human beings. In our interdisciplinary research efforts, heavy chain monoclonal antibody (HC MAb) was inserted into Ti plasmid vector and transferred into A. tumefaciens for the transformation in tobacco cells. It was found that 76% of the transformants produced HC MAb. The presence of HC MAb in the cell membrane fraction indicated that the signal peptide was functional and efficient. The change of the HC MAb concentration during a batch culture followed a similar trend as dry cell concentration, indicating that the production of HC MAb was growth related. The long-term repeated subcultures of 11 cell lines showed that there was no obvious trend of neither the decrease nor the increase of the productivity with the repeated subcultures.

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.43-49
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• 2000

An efficient and reliable Agrobacterium transformation procedure based on TDZ (thidiazuron)-induced organogenesis was established and applied to six Brazilian eggp1ant varieties. Optimum transgenic plants recovery was achieved upon the study of the following parameters affecting transformation efficiency, using F-100 variety as a model: i) explant source; ii) pre-culture period; iii) physical state of the pre-culture medium and iv) coculture conditions. The highest frequency of kanamycin-resistant calli derived from leaf explants (5%) was obtained without a pre-culture period and co-cultivation for 24 h in liquid medium followed by five days on solid RM (regeneration medium). For cotyledon explants, best results were achieved upon a pre-culture of 24 h in liquid RM and a co-cultivation period of 24 h in liquid RM followed by three days in solid RM, resulting in a transformation Sequency of 22.7%. Kanamycin-resistant organogenic calli were also obtained from cultivars Emb, Preta Comprida, Round nose Shaded, Campineira and Florida Market. The expression pattern of an epidermis-specific promoter was studied using transformants expressing a chimaeric construct comprised by the promoter Atgrp-5 transcriptionally fused to the coding region of the gus gene. The expression pattern was similar to that previously observed in tobacco and Arabidopsis thaliana, with preferential expression at the epidermis and the stem phloem. These results support the idea that the Atgrp-5 promoter can be used to drive defense genes in these tissues, which are sites of pathogen interaction and spread, in programs for the genetic improvement of eggplant.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1308-1315
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• 2007

Lipase B from Candida antarctica (CalB) displayed on the cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB 10 and CalB 14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB 14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of $Leu^{278}Pro$ in CalB10 and $Leu^{278}Pro/Leu^{219}Gln$ in CalB14. The substituted $Pro^{278}$ in both mutants was located near the proline site of the ${\alpha}$10 helix. This mutation was assumed to induce a conformational change in the ${\alpha}$10 helix and increased the $k_{cat}$ value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ ($Leu^{219}Gln$) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.

• Journal of Microbiology and Biotechnology
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• v.6 no.5
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• pp.331-335
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• 1996

The second $\beta$-Xylosidase gene (xylB) from Bacillus stearothermophilus was isolated from the genomic library, cloned into pBR322, and subsequently transferred into Escherichia coli HB101. Six out of 10, 000 transformants were selected from the selective LB medium supplemented with p-nitrophenyl-$\alpha$-L-arabinofuranoside (pNPAf) and ampicillin ($50\mu g$/ml) based on their ability to form a yellow ring around the colony. One of the clones was found to harbor the recombinant plasmid with 5.0 kb foreign DNA, which was identical to the $\alpha$-L-arabinofuranosidase gene (arfI) previously cloned in this lab, while the other five had 3.5 kb of the foreign DNA. Southern blotting experiments confirmed that the 3.5 kb insert DNA was from B. stearothermophilus chromosomal DNA. A zymogram with 4-methylumbelliferyl-$\alpha$-L-arabinofuranoside as the enzyme substrate revealed that the cloned gene product was one of the mutiple $\alpha$-L-arabinofuranosidases produced by B. stearothermophilus. Unlike the arfI gene product, the product of the gene on the insert DNA (xylB) showed an activity not only on pNPAf but also on oNPX suggesting that the cloned gene product could be a bifunctional enzyme having both $\alpha$-L-arabinofuranosidase and $\beta$-xylosidase activities.

• Plant Biotechnology Reports
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• v.5 no.2
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• pp.147-156
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• 2011

Efficient Agrobacterium-mediated genetic transformation of Scoparia dulcis L. was developed using Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pCAMBIA1301 with ${\beta}$-glucuronidase (GUS) (uidA) and hygromycin phosphotransferase (hpt) genes. Two-day precultured leaf segments of in vitro shoot culture were found to be suitable for cocultivation with the Agrobacterium strain, and acetosyringone was able to promote the transformation process. After selection on shoot organogenesis medium with appropriate concentrations of hygromycin and carbenicillin, adventitious shoots were developed on elongation medium by twice subculturing under the same selection scheme. The elongated hygromycin-resistant shoots were subsequently rooted on the MS medium supplemented with $1mg\;l^{-1}$ indole-3-butyric acid and $15mg\;l^{-1}$ hygromycin. Successful transformation was confirmed by PCR analysis using uidA- and hpt-specific primers and monitored by histochemical assay for ${\beta}$-GUS activity during shoot organogenesis. Integration of hpt gene into the genome of transgenic plants was also verified by Southern blot analysis. High transformation efficiency at a rate of 54.6% with an average of $3.9{\pm}0.39$ transgenic plantlets per explant was achieved in the present transformation system. It took only 2-3 months from seed germination to positive transformants transplanted to soil. Therefore, an efficient and fast genetic transformation system was developed for S. dulcis using an Agrobacterium-mediated approach and plant regeneration via shoot organogenesis, which provides a useful platform for future genetic engineering studies in this medicinally important plant.

• Journal of Microbiology and Biotechnology
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• v.27 no.10
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• pp.1867-1876
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• 2017

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.