The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Hiroshi Ezura - One of the best experts on this subject based on the ideXlab platform.
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the accumulation of recombinant Miraculin is independent of fruit size in tomato
Plant Biotechnology, 2021Co-Authors: Azusa Ono, Kyoko Hiwasatanase, Satoko Nonaka, Hiroshi EzuraAbstract:The taste-modifying protein Miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of Miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest Miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.
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comparison of the n glycosylation on recombinant Miraculin expressed in tomato plants with native Miraculin
Plant Biotechnology, 2018Co-Authors: Hiroshi Ezura, Kyoko Hiwasatanase, Hiroyuki Kajiura, Kazuhito FujiyamaAbstract:Miraculin is a promising protein with taste-modifying properties. Focusing on the unique function and potential of Miraculin, recombinant Miraculin production has been explored with the use of heterologous expression systems, but the activities of recombinant Miraculins were much lower than those of native Miraculin, probably due to the difference in post-translational modification, especially N-glycosylation. For practical use therefore, the differences between N-glycan of recombinant Miraculin compared to that of native Miraculin should be minimized. Here, to establish the platform for functional Miraculin production, we expressed Miraculin in tomato plants with the same taste-modifying activity as native Miraculin purified from miracle fruit, and we compared the N-glycan structures with those of native Miraculin. Our N-glycan structural analysis using purified Miraculin, followed by hydrazynolysis, 2-pyridylamine (PA)-labeling, high-performance liquid chromatography, and a liquid chromatography tandem-mass spectrometry analysis revealed that both the native and recombinant Miraculins carried an M3 structure as a predominant structure and that most of the N-glycan structures on the Miraculins were pauci-mannosidic structures with a smaller amount of plant-specific α1,3-fucosylated and/or β1,2-xylosylated N-glycans and without a Lewis a epitope. These results indicate that the N-glycoform of native Miraculin from miracle fruit and recombinant Miraculin expressed in tomato plants are almost identical to each other with similar ratios and that, therefore, plant-specific N-glycans are essential for showing the full taste-modifying activity of Miraculin.
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an e8 promoter hsp terminator cassette promotes the high level accumulation of recombinant protein predominantly in transgenic tomato fruits a case study of Miraculin
Plant Cell Reports, 2013Co-Authors: Natsuko Kurokawa, Kyoko Hiwasatanase, Tadayoshi Hirai, Mariko Takayama, Hiroshi EzuraAbstract:The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant Miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the Miraculin concentration was the highest in the ripening fruits, representing 30–630 μg Miraculin of the gram fresh weight. The highest level of Miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.
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From miracle fruit to transgenic tomato: mass production of the taste-modifying protein Miraculin in transgenic plants
Plant Cell Reports, 2012Co-Authors: Kyoko Hiwasa-tanase, Kazuhisa Kato, Tadayoshi Hirai, Narendra Duhita, Hiroshi EzuraAbstract:The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, Miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, Miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of Miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying Miraculin and future advances required to achieve industrial use.
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cultivation under salt stress increases the concentration of recombinant Miraculin in transgenic tomato fruit resulting in an increase in purification efficiency
Plant Biotechnology, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, Narendra Duhita, Hiroshi EzuraAbstract:Abstract High target protein concentrations in source materials are important for achieving high purification efficiency. We sought to produce the taste-modifying protein, Miraculin, in transgenic tomato fruits and then to extract and purify this protein. In order to improve the efficiency of recombinant Miraculin purification from transgenic tomatoes, we tested a salt-stress cultivation technique aimed at increasing Miraculin concentration in the tomatoes. Two lines of transgenic tomatoes, 56B and 5B, were grown under salt-stress conditions. There was a higher Miraculin content per gram of fresh weight in transgenic tomatoes cultivated under salt stress than in tomatoes grown under non-stressed conditions. The observed increase in Miraculin concentration was due to an enrichment effect caused by the miniaturization of tomatoes cultivated under salt stress. When the Miraculin-enriched tomatoes were used for Miraculin purification, the recovery rate of Miraculin was higher.
Tadayoshi Hirai - One of the best experts on this subject based on the ideXlab platform.
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an e8 promoter hsp terminator cassette promotes the high level accumulation of recombinant protein predominantly in transgenic tomato fruits a case study of Miraculin
Plant Cell Reports, 2013Co-Authors: Natsuko Kurokawa, Kyoko Hiwasatanase, Tadayoshi Hirai, Mariko Takayama, Hiroshi EzuraAbstract:The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant Miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the Miraculin concentration was the highest in the ripening fruits, representing 30–630 μg Miraculin of the gram fresh weight. The highest level of Miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.
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From miracle fruit to transgenic tomato: mass production of the taste-modifying protein Miraculin in transgenic plants
Plant Cell Reports, 2012Co-Authors: Kyoko Hiwasa-tanase, Kazuhisa Kato, Tadayoshi Hirai, Narendra Duhita, Hiroshi EzuraAbstract:The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, Miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, Miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of Miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying Miraculin and future advances required to achieve industrial use.
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cultivation under salt stress increases the concentration of recombinant Miraculin in transgenic tomato fruit resulting in an increase in purification efficiency
Plant Biotechnology, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, Narendra Duhita, Hiroshi EzuraAbstract:Abstract High target protein concentrations in source materials are important for achieving high purification efficiency. We sought to produce the taste-modifying protein, Miraculin, in transgenic tomato fruits and then to extract and purify this protein. In order to improve the efficiency of recombinant Miraculin purification from transgenic tomatoes, we tested a salt-stress cultivation technique aimed at increasing Miraculin concentration in the tomatoes. Two lines of transgenic tomatoes, 56B and 5B, were grown under salt-stress conditions. There was a higher Miraculin content per gram of fresh weight in transgenic tomatoes cultivated under salt stress than in tomatoes grown under non-stressed conditions. The observed increase in Miraculin concentration was due to an enrichment effect caused by the miniaturization of tomatoes cultivated under salt stress. When the Miraculin-enriched tomatoes were used for Miraculin purification, the recovery rate of Miraculin was higher.
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the hsp terminator of arabidopsis thaliana induces a high level of Miraculin accumulation in transgenic tomatoes
Journal of Agricultural and Food Chemistry, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, Kazuhisa Kato, Narendra Duhita, Natsuko Kurokawa, Ko Kato, Hiroshi EzuraAbstract:High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the Miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the Miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the Miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of Miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.
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ubiquitin promoter terminator cassette promotes genetically stable expression of the taste modifying protein Miraculin in transgenic lettuce
Plant Cell Reports, 2011Co-Authors: Tadayoshi Hirai, Megumu Yano, You Wang Kim, Abdullah Mohammad Shohael, Hiroshi EzuraAbstract:Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant Miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein Miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with Miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and Miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the Miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the Miraculin protein in lettuce and hence this platform is of benefit for recombinant Miraculin production on a commercial scale.
Kyoko Hiwasatanase - One of the best experts on this subject based on the ideXlab platform.
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the accumulation of recombinant Miraculin is independent of fruit size in tomato
Plant Biotechnology, 2021Co-Authors: Azusa Ono, Kyoko Hiwasatanase, Satoko Nonaka, Hiroshi EzuraAbstract:The taste-modifying protein Miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of Miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest Miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.
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comparison of the n glycosylation on recombinant Miraculin expressed in tomato plants with native Miraculin
Plant Biotechnology, 2018Co-Authors: Hiroshi Ezura, Kyoko Hiwasatanase, Hiroyuki Kajiura, Kazuhito FujiyamaAbstract:Miraculin is a promising protein with taste-modifying properties. Focusing on the unique function and potential of Miraculin, recombinant Miraculin production has been explored with the use of heterologous expression systems, but the activities of recombinant Miraculins were much lower than those of native Miraculin, probably due to the difference in post-translational modification, especially N-glycosylation. For practical use therefore, the differences between N-glycan of recombinant Miraculin compared to that of native Miraculin should be minimized. Here, to establish the platform for functional Miraculin production, we expressed Miraculin in tomato plants with the same taste-modifying activity as native Miraculin purified from miracle fruit, and we compared the N-glycan structures with those of native Miraculin. Our N-glycan structural analysis using purified Miraculin, followed by hydrazynolysis, 2-pyridylamine (PA)-labeling, high-performance liquid chromatography, and a liquid chromatography tandem-mass spectrometry analysis revealed that both the native and recombinant Miraculins carried an M3 structure as a predominant structure and that most of the N-glycan structures on the Miraculins were pauci-mannosidic structures with a smaller amount of plant-specific α1,3-fucosylated and/or β1,2-xylosylated N-glycans and without a Lewis a epitope. These results indicate that the N-glycoform of native Miraculin from miracle fruit and recombinant Miraculin expressed in tomato plants are almost identical to each other with similar ratios and that, therefore, plant-specific N-glycans are essential for showing the full taste-modifying activity of Miraculin.
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an e8 promoter hsp terminator cassette promotes the high level accumulation of recombinant protein predominantly in transgenic tomato fruits a case study of Miraculin
Plant Cell Reports, 2013Co-Authors: Natsuko Kurokawa, Kyoko Hiwasatanase, Tadayoshi Hirai, Mariko Takayama, Hiroshi EzuraAbstract:The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant Miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the Miraculin concentration was the highest in the ripening fruits, representing 30–630 μg Miraculin of the gram fresh weight. The highest level of Miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.
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cultivation under salt stress increases the concentration of recombinant Miraculin in transgenic tomato fruit resulting in an increase in purification efficiency
Plant Biotechnology, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, Narendra Duhita, Hiroshi EzuraAbstract:Abstract High target protein concentrations in source materials are important for achieving high purification efficiency. We sought to produce the taste-modifying protein, Miraculin, in transgenic tomato fruits and then to extract and purify this protein. In order to improve the efficiency of recombinant Miraculin purification from transgenic tomatoes, we tested a salt-stress cultivation technique aimed at increasing Miraculin concentration in the tomatoes. Two lines of transgenic tomatoes, 56B and 5B, were grown under salt-stress conditions. There was a higher Miraculin content per gram of fresh weight in transgenic tomatoes cultivated under salt stress than in tomatoes grown under non-stressed conditions. The observed increase in Miraculin concentration was due to an enrichment effect caused by the miniaturization of tomatoes cultivated under salt stress. When the Miraculin-enriched tomatoes were used for Miraculin purification, the recovery rate of Miraculin was higher.
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the hsp terminator of arabidopsis thaliana induces a high level of Miraculin accumulation in transgenic tomatoes
Journal of Agricultural and Food Chemistry, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, Kazuhisa Kato, Narendra Duhita, Natsuko Kurokawa, Ko Kato, Hiroshi EzuraAbstract:High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the Miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the Miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the Miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of Miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.
Hyeon-jin Sun - One of the best experts on this subject based on the ideXlab platform.
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article biochemistry functional expression of Miraculin a taste modifying protein in transgenic miyagawa wase satsuma mandarin citrus unshiu marc
한국응용생명화학회지(구 한국농화학회지), 2011Co-Authors: Hyo Yeon Lee, Jeong Won Park, Hyeon-jin Sun, Jaehoon Kim, Key Zung Riu, Yongwoo Kim, Seong Beom Jin, Kyung Hwan Boo, Mohammad Adnan BachchuAbstract:Miraculin is a taste modifying plant protein, which displays a peculiar property of being able to modify sour taste into sweet taste. Thus, there has been an increasing interest in Miraculin due to this amazing property. In the present study, Miraculin gene was introduced into the Miyagawa Wase Satsuma mandarin (Citrus unshiu Marc.) callus by Agrobacterum-mediated transformation to produce a citrus transgenic plant and express the recombinant Miraculin protein under the control of 35S promoter. Satsuma mandarin is one of the choicest citrus varieties grown widely and commercially in Korea, especially in Jeju Island, and Japan. Expression of this protein in the transgenic plant resulted in the accumulation of a significant amount of the Miraculin protein in the leaves. To investigate whether the expressed protein was correctly modified, the dimerization and N-glycosylation of recombinant Miraculin in the transgenic plants were analyzed. The recombinant protein also showed a sufficient biological activity. These results open up a new way of expression system in woody plants such as citrus and can provide a suitable alternative for producing recombinant Miraculin.
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Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells
Journal of Plant Physiology, 2009Co-Authors: Tadayoshi Hirai, Megumu Yano, Mayuko Sato, Kiminari Toyooka, Hyeon-jin Sun, Hiroshi EzuraAbstract:A taste-modifying protein, Miraculin, is highly accumulated in ripe fruit of miracle fruit (Richadella dulcifica) and the content can reach up to 10% of the total soluble protein in these fruits. Although speculated for decades that Miraculin is secreted into intercellular spaces in miracle fruit, no evidence exists of its cellular localization. To study the cellular localization of Miraculin in plant cells, using miracle fruit and transgenic tomato that constitutively express Miraculin, immunoelectron microscopy, imaging GFP fusion proteins, and immunological detection of secreted proteins in culture medium of transgenic tomato were carried out. Immunoelectron microscopy showed the specific accumulation of Miraculin in the intercellular layers of both miracle fruit and transgenic tomato. Imaging GFP fusion protein demonstrated that the Miraculin-GFP fusion protein was accumulated in the intercellular spaces of tomato epidermal cells. Immunological detection of secreted proteins in culture medium of transgenic tomato indicated that Miraculin was secreted from the roots of transgenic tomato expressing Miraculin. This study firstly showed the evidences of the intercellular localization of Miraculin, and provided a new insight of biological roles of Miraculin in plants.
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transgenic strawberry expressing the taste modifying protein Miraculin
Plant Biotechnology, 2008Co-Authors: Toshiyuki Sugaya, Megumu Yano, Tadayoshi Hirai, Hyeon-jin Sun, Hiroshi EzuraAbstract:The gene encoding the taste-modifying protein Miraculin was introduced under the control of the 35S or El2 promoter into strawberry (Fragaria x ananassa) by Agrobacterium-mediated transformation to produce transgenic plants. Although Miraculin was detected in the leaves and fruits of the transgenic plants, the level of accumulation among the transgenic lines, which ranged from 0.5 to 2.0 μg g−1 fresh fruit, was not significantly different and was lower than that in miracle fruits (145 μg g−1 fresh fruit). High levels of Miraculin accumulation were detected in the mature fruits. The transgenic lines were subsequently propagated via the runners for three vegetative generations, and Miraculin was detected at equal levels in the leaves and fruits of the plants from each generation. In conclusion, although the level of accumulation was not high, Miraculin was stably expressed and accumulated in the vegetative progeny of the transgenic strawberry plants.
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Genetically stable expression of functional Miraculin, a new type of alternative sweetener, in transgenic tomato plants
Plant biotechnology journal, 2007Co-Authors: Hyeon-jin Sun, Megumu Yano, Hiroshi Kataoka, Hiroshi EzuraAbstract:Miraculin is a taste-modifying protein isolated from the red berries of Richadella dulcifica, a shrub native to West Africa. Miraculin by itself is not sweet, but it is able to turn a sour taste into a sweet taste. This unique property has led to increasing interest in this protein. In this article, we report the high-yield production of Miraculin in transgenic tomato plants. High and genetically stable expression of Miraculin was confirmed by Western blot analysis and enzyme-linked immunosorbent assay. Recombinant Miraculin accumulated to high levels in leaves and fruits, up to 102.5 and 90.7 microg/g fresh weight, respectively. Purified recombinant Miraculin expressed in transgenic tomato plants showed strong sweetness-inducing activity, similar to that of native Miraculin. These results demonstrate that recombinant Miraculin was correctly processed in transgenic tomato plants, and that this production system could be a good alternative to production from the native plant.
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functional expression of the taste modifying protein Miraculin in transgenic lettuce
FEBS Letters, 2006Co-Authors: Hyeon-jin Sun, Minlong Cui, Hiroshi EzuraAbstract:Taste-modifying proteins are a natural alternative to artificial sweeteners and flavor enhancers and have been used in some cultures for centuries. The taste-modifying protein, Miraculin, has the unusual property of being able to modify a sour taste into a sweet taste. Here, we report the use of a plant expression system for the production of Miraculin. A synthetic gene encoding Miraculin was placed under the control of constitutive promoters and transferred to lettuce. Expression of this gene in transgenic lettuce resulted in the accumulation of significant amounts of Miraculin protein in the leaves. The Miraculin expressed in transgenic lettuce possessed sweetness-inducing activity. These results demonstrate that the production of Miraculin in edible plants can be a good alternative strategy to enhance the availability of this protein.
Kazuhisa Kato - One of the best experts on this subject based on the ideXlab platform.
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From miracle fruit to transgenic tomato: mass production of the taste-modifying protein Miraculin in transgenic plants
Plant Cell Reports, 2012Co-Authors: Kyoko Hiwasa-tanase, Kazuhisa Kato, Tadayoshi Hirai, Narendra Duhita, Hiroshi EzuraAbstract:The utility of plants as biofactories has progressed in recent years. Some recombinant plant-derived pharmaceutical products have already reached the marketplace. However, with the exception of drugs and vaccines, a strong effort has not yet been made to bring recombinant products to market, as cost-effectiveness is critically important for commercialization. Sweet-tasting proteins and taste-modifying proteins have a great deal of potential in industry as substitutes for sugars and as artificial sweeteners. The taste-modifying protein, Miraculin, functions to change the perception of a sour taste to a sweet one. This taste-modifying function can potentially be used not only as a low-calorie sweetener but also as a new seasoning that could be the basis of a new dietary lifestyle. However, Miraculin is far from inexpensive, and its potential as a marketable product has not yet been fully developed. For the last several years, biotechnological production of this taste-modifying protein has progressed extensively. In this review, the characteristics of Miraculin and recent advances in its production using transgenic plants are summarized, focusing on such topics as the suitability of plant species as expression hosts, the cultivation method for transgenic plants, the method of purifying Miraculin and future advances required to achieve industrial use.
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the hsp terminator of arabidopsis thaliana induces a high level of Miraculin accumulation in transgenic tomatoes
Journal of Agricultural and Food Chemistry, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, Kazuhisa Kato, Narendra Duhita, Natsuko Kurokawa, Ko Kato, Hiroshi EzuraAbstract:High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the Miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the Miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the Miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of Miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.
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a trial of production of the plant derived high value protein in a plant factory photosynthetic photon fluxes affect the accumulation of recombinant Miraculin in transgenic tomato fruits
Plant Signaling & Behavior, 2011Co-Authors: Kazuhisa Kato, Tadayoshi Hirai, Kyoko Hiwasatanase, Tsuyoshi Mizoguchi, Shinichiro Maruyama, Eiji Goto, Hiroshi EzuraAbstract:One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories. To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant Miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m(-2) s(-)1) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although Miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant Miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.
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uniform accumulation of recombinant Miraculin protein in transgenic tomato fruit using a fruit ripening specific e8 promoter
Transgenic Research, 2011Co-Authors: Tadayoshi Hirai, Kyoko Hiwasatanase, You Wang Kim, Kazuhisa Kato, Hiroshi EzuraAbstract:The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the Miraculin gene encoding the taste-modifying protein in tomato. The accumulation of Miraculin protein and mRNA was compared among transgenic tomatoes expressing the Miraculin gene driven by these promoters. Recombinant Miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, Miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the Miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.
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high level accumulation of recombinant Miraculin protein in transgenic tomatoes expressing a synthetic Miraculin gene with optimized codon usage terminated by the native Miraculin terminator
Plant Cell Reports, 2011Co-Authors: Kyoko Hiwasatanase, Tadayoshi Hirai, Kazuhisa Kato, Mpanja Nyarubona, Takanari Ichikawa, Hiroshi EzuraAbstract:In our previous study, a transgenic tomato line that expressed the MIR gene under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator (tNOS) produced the taste-modifying protein Miraculin (MIR). However, the concentration of MIR in the tomatoes was lower than that in the MIR gene’s native miracle fruit. To increase MIR production, the native MIR terminator (tMIR) was used and a synthetic gene encoding MIR protein (sMIR) was designed to optimize its codon usage for tomato. Four different combinations of these genes and terminators (MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR) were constructed and used for transformation. The average MIR concentrations in MIR-tNOS, MIR-tMIR, sMIR-tNOS and sMIR-tMIR fruits were 131, 197, 128 and 287 μg/g fresh weight, respectively. The MIR concentrations using tMIR were higher than those using tNOS. The highest MIR accumulation was detected in sMIR-tMIR fruits. On the other hand, the MIR concentration was largely unaffected by sMIR-tNOS. The expression levels of both MIR and sMIR mRNAs terminated by tMIR tended to be higher than those terminated by tNOS. Read-through mRNA transcripts terminated by tNOS were much longer than those terminated by tMIR. These results suggest that tMIR enhances mRNA expression and permits the multiplier effect of optimized codon usage.