Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research
The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is e...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Association for the Advancement of Science (AAAS)
2022-01-01
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| Series: | BioDesign Research |
| Online Access: | http://dx.doi.org/10.34133/2022/9834989 |
| _version_ | 1797287400270987264 |
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| author | Chenfei Tian Yixin Zhang Jianhua Li Yong Wang |
| author_facet | Chenfei Tian Yixin Zhang Jianhua Li Yong Wang |
| author_sort | Chenfei Tian |
| collection | DOAJ |
| description | The emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N. benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator–promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0–362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering. |
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| format | Article |
| id | doaj.art-ed2af7764fb0403798a71673f0c55ca9 |
| institution | Directory Open Access Journal |
| issn | 2693-1257 |
| language | English |
| last_indexed | 2024-03-07T18:32:35Z |
| publishDate | 2022-01-01 |
| publisher | American Association for the Advancement of Science (AAAS) |
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| series | BioDesign Research |
| spelling | doaj.art-ed2af7764fb0403798a71673f0c55ca92024-03-02T05:59:44ZengAmerican Association for the Advancement of Science (AAAS)BioDesign Research2693-12572022-01-01202210.34133/2022/9834989Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology ResearchChenfei Tian0Yixin Zhang1Jianhua Li2Yong Wang3CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China; University of Chinese Academy of Sciences, Beijing 100039, ChinaCollege of Life Science, Jilin Agricultural University, Changchun 130118, ChinaCAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, ChinaCAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, ChinaThe emerging plant synthetic metabolic engineering has been exhibiting great promise to produce either value-added metabolites or therapeutic proteins. However, promoters for plant pathway engineering are generally selected empirically. The quantitative characterization of plant-based promoters is essential for optimal control of gene expression in plant chassis. Here, we used N. benthamiana leaves and BY2 suspension cells to quantitatively characterize a library of plant promoters by transient expression of firefly/Renilla luciferase. We validated the dual-luciferase reporter system by examining the correlation between reporter protein and mRNA levels. In addition, we investigated the effects of terminator–promoter combinations on gene expression and found that the combinations of promoters and terminators resulted in a 326-fold difference between the strongest and weakest performance, as reflected in reporter gene expression. As a proof of concept, we used the quantitatively characterized promoters to engineer the betalain pathway in N. benthamiana. Seven selected plant promoters with different expression strengths were used orthogonally to express CYP76AD1 and DODA, resulting in a final betalain production range of 6.0–362.4 μg/g fresh weight. Our systematic approach not only demonstrates the various intensities of multiple promoter sequences in N. benthamiana and BY2 cells but also adds to the toolbox of plant promoters for plant engineering.http://dx.doi.org/10.34133/2022/9834989 |
| spellingShingle | Chenfei Tian Yixin Zhang Jianhua Li Yong Wang Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research BioDesign Research |
| title | Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research |
| title_full | Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research |
| title_fullStr | Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research |
| title_full_unstemmed | Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research |
| title_short | Benchmarking Intrinsic Promoters and Terminators for Plant Synthetic Biology Research |
| title_sort | benchmarking intrinsic promoters and terminators for plant synthetic biology research |
| url | http://dx.doi.org/10.34133/2022/9834989 |
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