• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.713-713
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• 2003

• Journal of Plant Biotechnology
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• v.31 no.3
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• pp.231-237
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• 2004

Oxycarotenoids are oxygenated carotenoids that perform critical roles in plants. $\beta$-Carotene hydroxylase adds hydroxyl groups to the $\beta$-rings of carotenes and has been cloned from several bacteria and plants including Arabidopsis. This study was carried out to investigate the effect of $\beta$-carotene hydroxylase gene (Chyb) on the oxycarotenoids biosynthesis in the transgenic Arabidopsis. Construct of pGCHYB containing Chyb was established onto Gateway vector system (pENTR3C gateway vector and pH2GW7 destination vector). Arabidopsis thaliana (cv. Columbia) was transformed with Agrobacterium tumerfacience GV3101 harboring pGCHYB construct driven by 35S promoter and hygromycin resistant gene. Seven hundred bases paired PCR products, indicating the presence of Chyb gene, were found in the transformants by PCR analysis using Chyb primers. Hygromycin resistance assay showed that transgenes were stably inherited to next generation. The overexpression of the Chyb gene resulted in the decrease carotenoid content. Especially, astaxanthin unusual oxycarotenoid in wild type Arabidopsis was detected in the transgenic plants. This means that decreased carotenoids might be converted into astaxanthin metabolism with the aid of silent gene in the host.

• Fisheries and Aquatic Sciences
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• v.12 no.1
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• pp.54-59
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• 2009

Carotenoids such as $\beta$-carotene and astaxanthin are used as food colorants, animal feed supplements and for nutritional and cosmetic purposes. In a previous study, an astaxanthin biosynthesis gene cluster was isolated from the marine bacterium, Paracoccus haeundaensis. Geranylgeranyl pyrophosphate (GGPP) synthase (CrtE), encoded by the ortE gene, catalyzes the formation of GGPP from farnesyl pyrophosphate (FPP), which is an essential enzyme for the biosynthesis of carotenoids in early steps. In order to study the biochemical and enzymatic characteristics of this important enzyme, a large quantity of purified GGPP synthase is required. To overproduce GGPP synthase, the crtE gene was subcloned into a pET-44a(+) expression vector and transformed into the Escherichia coli BL21(DE3) codon plus cell. Transformants harboring the crtE gene were cultured and the crtE gene was over-expressed. The expressed protein was purified to homogeneity by affinity chromatography and applied to study its biochemical properties and molecular characteristics.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1542-1546
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• 2009

Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. Here, we report cloning of the crtX gene responsible for zeaxanthin diglucoside biosynthesis from Paracoccus haeundaensis and the production of the corresponding carotenoids in transformed cells carrying this gene. An expression plasmid containing the crtX gene (pSTCRT-X) was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 46 kDa. Biosynthesis of zeaxanthin diglucoside was obtained when the plasmid pSTCRT-X was co-transformed into E. coli containing the pET-44a(+)-CrtEBIYZ carrying crtE, crtB, crtI, crtY, and crtZ genes required for zeaxanthin $\beta$-D-diglucoside biosynthesis.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.570-575
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• 2004

Three different strains of carotenoid accumulating XantlwphyUomyces dendrorhous mutants, JH1, JH2, and JH3, were isolated by NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis, which might potentially be useful for animal feed as well as for studies on the regulation and biosynthesis of astaxanthin. Mutants were selected based on the capability of growth and carotenoid production on the YM agar plate containing chemical inhibitor, $\beta$-ionone. Astaxanthin-overproducing mutant JH1 produced 4.032 mg astaxanthinlg dry cell weight, and this value was about 15-folds higher than that of wild-type. $\beta$-Carotene-overproducing mutant JH2 produced 0.273 mg $\beta$-carotene/g dry cell weight, and this was 4-folds increase from that of wild-type. In contrast, JH3 was a white-colored mutant that was unable to produce carotenoid pigment.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1060-1066
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• 2005

Under environmental stress, such as strong irradiance or nitrogen deficiency, unicellular green algae of the genus Haematococcus accumulate secondary carotenoids, i.e. astaxanthin, in the cytosol. The induction and regulation of astaxanthin biosynthesis in microalgae has recently received considerable attention owing to the increasing use of secondary carotenoids as a source of pigmentation for fish aquacultures, and as a potential drug in cancer prevention as a free-radical quencher. Accordingly, this study generated expressed sequence tags (ESTs) from a library constructed from astaxanthin-induced Haematococcus pluvialis. Partial sequences were obtained from the 5' ends of 1,858 individual cDNAs, and then grouped into 1,025 non-overlapping sequences, among which 708 sequences were singletons, while the remainder fell into 317 clusters. Approximately $63\%$ of the EST sequences showed similarity to previously described sequences in public databases. H. pluvialis was found to consist of a relatively high percentage of genes involved in genetic information processing ($15\%$) and metabolism ($11\%$), whereas a relatively low percentage of sequences was involved in the signal transduction ($3\%$), structure ($2\%$), and environmental information process ($3\%$). In addition, a relatively large fraction of H. pluvialis sequences was classified as genes involved in photosynthesis ($9\%$) and cellular process ($9\%$). Based on this EST analysis, the full-length cDNA sequence for superoxide dismutase (SOD) of H. pluvialis was cloned, and the expression of this gene was investigated. The abundance of SOD changed substantially in response to different culture conditions, indicating the possible regulation of this gene in H. pluvialis.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.224-224
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• 2005

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.269-280
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• 2010

Lilium ${\times}$ formolongi was genetically engineered by Agrobacterium-mediated transformation with the plasmid pCrtZW-N8idi-crtEBIY, which contains seven enzyme genes under the regulation of the CaMV 35S promoter. In the transformants, ketocarotenoids were detected in both calli and leaves, which showed a strong orange color. In transgenic calli, the total amount of carotenoids [133.3 ${\mu}g/g$ fresh weight (FW)] was 26.1-fold higher than in wild-type calli. The chlorophyll content and photosynthetic efficiency in transgenic orange plantlets were significantly lowered; however, after several months of subculture, they had turned into plantlets with green leaves that showed significant increases in chlorophyll and photosynthetic efficiency. The total carotenoid contents in leaves of transgenic orange and green plantlets were quantified at 102.9 and 135.2 ${\mu}g/g$ FW, respectively, corresponding to 5.6- and 7.4-fold increases over the levels in the wild-type. Ketocarotenoids such as echinenone, canthaxanthin, 3'-hydroxyechinenone, 3-hydroxyechinenone, and astaxanthin were detected in both transgenic calli and orange leaves. A significant change in the type and composition of ketocarotenoids was observed during the transition from orange transgenic plantlets to green plantlets. Although 3'-hydroxyechinenone, 3-hydroxyechinenone, astaxanthin, and adonirubin were absent, and echinenone and canthaxanthin were present at lower levels, interestingly, the upregulation of carotenoid biosynthesis led to an increase in the total carotenoid concentration (+31.4%) in leaves of the transgenic green plantlets.

• Horticultural Science & Technology
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• v.29 no.3
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• pp.267-272
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• 2011

Several reports indicated that astaxanthin and zeaxanthin have more active anticancer activity than pro-vitamin A carotenes. ${\beta}$-carotene hydroxylase is a key enzyme to synthesize zeaxanthin and astaxanthin in carotenoids biosynthesis pathway. We isolated the ChyB gene encoding ${\beta}$-carotene hydroxylase from tomato leaves. The ChyB gene (1.5Kbp) fragment was cloned into the binary vector and designated to pIG121-ChyB-tom. Agrobacterium-mediated infiltration was used for transient assay in Nicotiana benthamiana. Leaf samples were collected 0, 1, 2, 3 days after infiltration (DAI). RT-PCR result showed that the expression of ${\beta}$-carotene hydroxylase transcripts was not detected in control (0DAI), but its expression was detected after 1 DPI and increased later on. When the activity of ${\beta}$-carotene hydroxylase was measured, the 1,1-diphenyl-pricryl hydrazyl (DPPH) radical scavenging activity (27%) at 2 DAI was significantly higher than that (21%) at 0 DAI. These results indicated that anti-oxidant activity dramatically increased at 2 DAI in tobacco leaves was due to over expression of tomato ${\beta}$-carotene hydroxylase. These results can be the foundation to develop tomato cultivars with high oxy-carotenoids content using the ChyB gene transformation.