An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i>
Plants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene...
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/14/11425 |
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| author | Edith C. F. Forestier Amy C. Cording Gary J. Loake Ian A. Graham |
| author_facet | Edith C. F. Forestier Amy C. Cording Gary J. Loake Ian A. Graham |
| author_sort | Edith C. F. Forestier |
| collection | DOAJ |
| description | Plants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene casbene in <i>Nicotiana benthamiana</i>, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from <i>Arabidopsis thaliana</i> Hsp101 or <i>Glycine max</i> GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV <i>35S</i>, and the 5′UTR from the tobacco mosaic virus. The resulting four chimeric promoters fused to a green fluorescent protein (GFP) reporter showed that the variant Ara2 had the strongest fluorescent signal after heat shock. We next created a 4-gene cassette driven by the <i>Ara2</i> promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into <i>Nicotiana benthamiana</i>. Metabolic analysis on the transgenic lines revealed that continuous heat outperforms heat shock, with up to 1 μg/mg DW of casbene detected after 32 h of uninterrupted 40 °C heat. These results demonstrate the potential of heat-inducible promoters as synthetic biology tools for metabolite production in plants. |
| first_indexed | 2024-03-11T01:00:26Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T01:00:26Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-2f9a1b958da84d41adf299d5b34e197a2023-11-18T19:39:12ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124141142510.3390/ijms241411425An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i>Edith C. F. Forestier0Amy C. Cording1Gary J. Loake2Ian A. Graham3Centre for Novel Agricultural Products (CNAP), Department of Biology, University of York, Wentworth Way, York YO10 5DD, UKCentre for Novel Agricultural Products (CNAP), Department of Biology, University of York, Wentworth Way, York YO10 5DD, UKInstitute of Molecular Plant Sciences, Daniel Rutherford Building, School of Biological Sciences, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh EH9 3JH, UKCentre for Novel Agricultural Products (CNAP), Department of Biology, University of York, Wentworth Way, York YO10 5DD, UKPlants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene casbene in <i>Nicotiana benthamiana</i>, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from <i>Arabidopsis thaliana</i> Hsp101 or <i>Glycine max</i> GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV <i>35S</i>, and the 5′UTR from the tobacco mosaic virus. The resulting four chimeric promoters fused to a green fluorescent protein (GFP) reporter showed that the variant Ara2 had the strongest fluorescent signal after heat shock. We next created a 4-gene cassette driven by the <i>Ara2</i> promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into <i>Nicotiana benthamiana</i>. Metabolic analysis on the transgenic lines revealed that continuous heat outperforms heat shock, with up to 1 μg/mg DW of casbene detected after 32 h of uninterrupted 40 °C heat. These results demonstrate the potential of heat-inducible promoters as synthetic biology tools for metabolite production in plants.https://www.mdpi.com/1422-0067/24/14/11425<i>Agrobacterium tumefaciens</i>casbenediterpenesheat-shockMoClo<i>Nicotiana benthamiana</i> |
| spellingShingle | Edith C. F. Forestier Amy C. Cording Gary J. Loake Ian A. Graham An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i> International Journal of Molecular Sciences <i>Agrobacterium tumefaciens</i> casbene diterpenes heat-shock MoClo <i>Nicotiana benthamiana</i> |
| title | An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i> |
| title_full | An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i> |
| title_fullStr | An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i> |
| title_full_unstemmed | An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i> |
| title_short | An Engineered Heat-Inducible Expression System for the Production of Casbene in <i>Nicotiana benthamiana</i> |
| title_sort | engineered heat inducible expression system for the production of casbene in i nicotiana benthamiana i |
| topic | <i>Agrobacterium tumefaciens</i> casbene diterpenes heat-shock MoClo <i>Nicotiana benthamiana</i> |
| url | https://www.mdpi.com/1422-0067/24/14/11425 |
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