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- Title
An Engineered Heat-Inducible Expression System for the Production of Casbene in Nicotiana benthamiana.
- Authors
Forestier, Edith C. F.; Cording, Amy C.; Loake, Gary J.; Graham, Ian A.
- Abstract
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 Nicotiana benthamiana, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from Arabidopsis thaliana Hsp101 or Glycine max GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV 35S, 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 Ara2 promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into Nicotiana benthamiana. 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.
- Subjects
NICOTIANA benthamiana; GREEN fluorescent protein; TOBACCO mosaic virus; SYNTHETIC biology; SOYBEAN; CUCUMBER mosaic virus; ARABIDOPSIS thaliana; PROTEIN expression
- Publication
International Journal of Molecular Sciences, 2023, Vol 24, Issue 14, p11425
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms241411425