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Akifumi Sugiyama,
Philip J Linley,
Kanako Sasaki,
Takuto Kumano,
Hideaki Yamamoto,
Nobukazu Shitan,
Kazuaki Ohara,
Kojiro Takanashi,
Emiko Harada,
Hisakazu Hasegawa, Teruhiko Terakawa,
Tomohisa Kuzuyama,
Kazufumi Yazaki
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ABSTRACT: Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.
Metabolic Engineering 08/2011; 13(6):629-37. · 5.61 Impact Factor
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ABSTRACT: Phosphinothricin (PPT) is the active ingredient in bialaphos, which specifically inhibits glutamine synthetase in land plants. We isolated a novel PPT-resistant gene from a soil bacterium, Nocardia sp., and characterized it. The encoded protein, consisting of 177 amino acids, showed significant similarity to bacterial N-acetyltransferases, and we originally designated the gene MAT (methionine sulfone N-acetyltransferase). The recombinant MAT protein exhibited functions as a methionine sulfone and PPT N-acetyltransferase in vitro. The PPT N-acetyltransferase activity reached the maximum at pH 8-8.5, indicating that the protein might optimally function in chloroplasts. We therefore constructed a MAT gene, encoding the enzyme with a chloroplast-localizing signal in its amino-terminus. Plant transformation with the construct resulted in the generation of PPT-resistant rice and Arabidopsis. Furthermore, the transformed Arabidopsis was selectable in a synthetic medium containing PPT. The MAT gene thus facilitated establishment of herbicide-resistant plants, and as a new selectable gene marker.
Bioscience Biotechnology and Biochemistry 06/2009; 73(5):1000-6. · 1.28 Impact Factor
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Kyo Wakasa,
Hisakazu Hasegawa,
Hiroshi Nemoto,
Fumio Matsuda,
Haruna Miyazawa,
Yuzuru Tozawa,
Keiko Morino,
Akira Komatsu,
Tetsuya Yamada, Teruhiko Terakawa,
Hisashi Miyagawa
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ABSTRACT: Metabolic manipulation of plants to improve their nutritional quality is an important goal of plant biotechnology. Expression in rice (Oryza sativa L.) of a transgene (OASA1D) encoding a feedback-insensitive alpha subunit of rice anthranilate synthase results in the accumulation of tryptophan (Trp) in calli and leaves. It is shown here that the amount of free Trp in the seeds of such plants is increased by about two orders of magnitude compared with that in the seeds of wild-type plants. The total Trp content in the seeds of the transgenic plants was also increased. Two homozygous lines, HW1 and HW5, of OASA1D transgenic rice were generated for characterization of agronomic traits and aromatic metabolite profiling of seeds. The marked overproduction of Trp was stable in these lines under field conditions, although spikelet fertility and yield, as well as seed germination ability, were reduced compared with the wild type. These differences in agronomic traits were small, however, in HW5. In spite of the high Trp content in the seeds of the HW lines, metabolic profiling revealed no substantial changes in the amounts of other phenolic compounds. The amount of indole acetic acid was increased about 2-fold in the seeds of the transgenic lines. The establishment and characterization of these OASA1D transgenic lines have thus demonstrated the feasibility of increasing the Trp content in the seeds of rice (or of other crops) as a means of improving its nutritional value for human consumption or animal feed.
Journal of Experimental Botany 02/2006; 57(12):3069-78. · 5.36 Impact Factor
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ABSTRACT: A selection system based on a mutant rice gene for a feedback-insensitive subunit of anthranilate synthase (OASA1D) was developed for the transformation of rice and potato. Expression of OASA1D conferred resistance to the tryptophan analog 5-methyltryptophan (5MT) in transformed cells of rice and potato. The selection system based on OASA1D and 5MT was associated with a high transformation efficiency, a short time frame for the generation of transgenic plants, simple culture procedures, and it was as effective as hygromycin B selection in rice (monocotyledon) and kanamycin selection in potato (dicotyledon). Transgenic rice and potato plants established by 5MT selection had normal morphology and accumulated tryptophan when OASA1D was expressed under the control of a constitutive promoter. These results demonstrate the efficacy of OASA1D as a selectable marker and they suggest that the 5MT selection system based on this gene will prove applicable to a wide range of plant species and culture procedures.
Molecular Breeding 12/2004; 14(4):363-373. · 2.85 Impact Factor
