Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes
Artemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants <i>Artemisia annua</i> L. Due to the limitation of this source, considerable efforts are now directed to the develo...
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MDPI AG
2020-04-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/9/4/537 |
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| author | Aleksey Firsov Tatiana Mitiouchkina Lyubov Shaloiko Alexander Pushin Alexander Vainstein Sergey Dolgov |
| author_facet | Aleksey Firsov Tatiana Mitiouchkina Lyubov Shaloiko Alexander Pushin Alexander Vainstein Sergey Dolgov |
| author_sort | Aleksey Firsov |
| collection | DOAJ |
| description | Artemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants <i>Artemisia annua</i> L. Due to the limitation of this source, considerable efforts are now directed to the development of methods for artemisinin production using heterologous expression systems. Artemisinin is a sesquiterpene lactone, synthesized through the cyclization of farnesyl diphosphate involved in other sesquiterpene biosynthetic systems. <i>Chrysanthemum</i> species as well as <i>A. annua</i>, belong to <i>Asteraceae</i> family, and had been characterized by containing highly content of sesquiterpenes and their precursors. This makes chrysanthemum a promising target for the production of artemisinin in heterologous host plants. Chrysanthemum (<i>C. morifolium</i> Ramat.) was transformed by <i>Agrobacterium tumefaciens</i> carrying with the binary vectors p1240 and p1250, bearing artemisinin biosynthesis genes coding: amorpha-4,11-diene synthase, artemisinic aldehyde Δ11(13) reductase, amorpha-4,11-diene monooxygenase (p1240 was targeted to the mitochondria and p1250 was targeted to the cytosol), cytochrome P450 reductase from <i>A. annua</i>, as well as yeast truncated 3-hydroxy-3-methylglutarylcoenzyme A reductase. This study obtained 8 kanamycin-resistant lines after transformation with the p1240 and 2 lines from p1250. All target genes were detected in 2 and 1 transgenic lines of the 2 vectors. The transformation frequency of all target genes were 0.33% and 0.17% for p1240 and p1250, relative to the total transformed explant numbers. RT-PCR analysis revealed the transcription of all transferred genes in two lines obtained after transformation with the p1240 vector, confirming the possibility of transferring genetic modules encoding entire biochemical pathways into the chrysanthemum genome. This holds promise for the development of a chrysanthemum-based expression system to produce non-protein substances, such as artemisinin. |
| first_indexed | 2024-03-10T20:20:25Z |
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| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
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| spelling | doaj.art-83e3c6ae5c7b4f1990184c1112145a522023-11-19T22:14:35ZengMDPI AGPlants2223-77472020-04-019453710.3390/plants9040537Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway GenesAleksey Firsov0Tatiana Mitiouchkina1Lyubov Shaloiko2Alexander Pushin3Alexander Vainstein4Sergey Dolgov5Branch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Moscow Region, 142290 Pushchino, RussiaBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Moscow Region, 142290 Pushchino, RussiaBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Moscow Region, 142290 Pushchino, RussiaBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Moscow Region, 142290 Pushchino, RussiaThe Hebrew University of Jerusalem, Robert H. Smith Faculty of Agriculture, Food and Environment, POB 12, Rehovot 76100, IsraelBranch of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Moscow Region, 142290 Pushchino, RussiaArtemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants <i>Artemisia annua</i> L. Due to the limitation of this source, considerable efforts are now directed to the development of methods for artemisinin production using heterologous expression systems. Artemisinin is a sesquiterpene lactone, synthesized through the cyclization of farnesyl diphosphate involved in other sesquiterpene biosynthetic systems. <i>Chrysanthemum</i> species as well as <i>A. annua</i>, belong to <i>Asteraceae</i> family, and had been characterized by containing highly content of sesquiterpenes and their precursors. This makes chrysanthemum a promising target for the production of artemisinin in heterologous host plants. Chrysanthemum (<i>C. morifolium</i> Ramat.) was transformed by <i>Agrobacterium tumefaciens</i> carrying with the binary vectors p1240 and p1250, bearing artemisinin biosynthesis genes coding: amorpha-4,11-diene synthase, artemisinic aldehyde Δ11(13) reductase, amorpha-4,11-diene monooxygenase (p1240 was targeted to the mitochondria and p1250 was targeted to the cytosol), cytochrome P450 reductase from <i>A. annua</i>, as well as yeast truncated 3-hydroxy-3-methylglutarylcoenzyme A reductase. This study obtained 8 kanamycin-resistant lines after transformation with the p1240 and 2 lines from p1250. All target genes were detected in 2 and 1 transgenic lines of the 2 vectors. The transformation frequency of all target genes were 0.33% and 0.17% for p1240 and p1250, relative to the total transformed explant numbers. RT-PCR analysis revealed the transcription of all transferred genes in two lines obtained after transformation with the p1240 vector, confirming the possibility of transferring genetic modules encoding entire biochemical pathways into the chrysanthemum genome. This holds promise for the development of a chrysanthemum-based expression system to produce non-protein substances, such as artemisinin.https://www.mdpi.com/2223-7747/9/4/537transgenic plantschrysanthemumartemisininmetabolic engineering |
| spellingShingle | Aleksey Firsov Tatiana Mitiouchkina Lyubov Shaloiko Alexander Pushin Alexander Vainstein Sergey Dolgov Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes Plants transgenic plants chrysanthemum artemisinin metabolic engineering |
| title | Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes |
| title_full | Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes |
| title_fullStr | Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes |
| title_full_unstemmed | Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes |
| title_short | Agrobacterium-Mediated Transformation of Chrysanthemum with Artemisinin Biosynthesis Pathway Genes |
| title_sort | agrobacterium mediated transformation of chrysanthemum with artemisinin biosynthesis pathway genes |
| topic | transgenic plants chrysanthemum artemisinin metabolic engineering |
| url | https://www.mdpi.com/2223-7747/9/4/537 |
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