Abstract
As the field of plant synthetic biology continues to grow, Agrobacterium-mediated transient expression has become an essential method to rapidly test pathway candidate genes in a combinatorial fashion. This is especially important when elucidating and engineering more complex pathways to produce commercially relevant chemicals like many terpenoids, a widely diverse class of natural products of often industrial relevance. Agrobacterium-mediated transient expression has facilitated multiplex expression of recombinant and modified enzymes, including synthetic biology approaches to compartmentalize the biosynthesis of terpenoids subcellularly. Here, we describe methods on how to deploy Agrobacterium-mediated transient expression in Nicotiana benthamiana to rapidly develop terpenoid pathways and compartmentalize terpenoid biosynthesis within plastids, the cytosol, or at the surface of lipid droplets.
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Acknowledgments
This work was supported by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018409. We would also like to acknowledge partial support from the Department of Biochemistry and Molecular Biology startup funding and support from AgBioResearch (MICL02454). AB is supported by the NSF-IMPACTS Training Grant (DGE-1828149). Figures were made using BioRender.com. We wish to thank Davis Mathieu for assistance with the figure for the gas chromatography/mass spectrometry. We collectively acknowledge that Michigan State University occupies the ancestral, traditional, and contemporary Lands of the Anishinaabeg – Three Fires Confederacy of Ojibwe, Odawa, and Potawatomi peoples. In particular, the University resides on Land ceded in the 1819 Treaty of Saginaw. We recognize, support, and advocate for the sovereignty of Michigan’s twelve federally recognized Indian nations, for historic Indigenous communities in Michigan, for Indigenous individuals and communities who live here now, and for those who were forcibly removed from their Homelands. By offering this Land Acknowledgement, we affirm Indigenous sovereignty and will work to hold Michigan State University more accountable to the needs of American Indians and Indigenous peoples.
Competing Interests
The authors declare that there is no conflict of interest.
Author Contributions
JDB and BH conceptualized experiments. JDB performed cloning, expression, extractions, and analytics. JDB wrote the paper with minor critical input from BH and AB. AB helped with last-minute experiments to visualize the vacuum infiltration. All authors read the manuscript and contributed and agreed to its final form.
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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Bibik, J.D., Bryson, A.E., Hamberger, B. (2024). Compartmentalized Terpenoid Production in Plants Using Agrobacterium-Mediated Transient Expression. In: Braman, J.C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 2760. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3658-9_2
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DOI: https://doi.org/10.1007/978-1-0716-3658-9_2
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