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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/14/11425 |
| Summary: | 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. |
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| ISSN: | 1661-6596 1422-0067 |