Abstract
Terpenes constitute a distinct class of natural products1 that attract insects2, defend against phytopathogenic microbes3 and combat human diseases4. However, like most natural products, they are usually made by plants and microbes in small amounts and as complex mixtures. Chemical synthesis is often costly and inefficient, and may not yield enantiomerically pure terpenes, whereas large-scale microbial production requires expensive feedstocks. We engineered high-level terpene production in tobacco plants by diverting carbon flow from cytosolic or plastidic isopentenyl diphosphate through overexpression in either compartment of an avian farnesyl diphosphate synthase and an appropriate terpene synthase. Isotopic labeling studies suggest little, if any, metabolite exchange between these two subcellular compartments. The strategy increased synthesis of the sesquiterpenes patchoulol and amorpha-4,11-diene more than 1,000-fold, as well as the monoterpene limonene 10–30 fold, and seems equally suited to generating higher levels of other terpenes for research, industrial production or therapeutic applications.
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Acknowledgements
We thank Dale Poulter, University of Utah, for the avian farnesyl diphosphate synthase gene, Peter Brodelius, Kalmar University, for the amorpha-4,11-diene synthase gene and Randy Dinkins, University of Kentucky, for providing the Arabidopsis RUBISCO transit peptide sequence DNA. Special thanks to Nihar Nayak for his excellent advice concerning plant transformation, Scott Kinison for logistical and technical support, and Suphata Kaewpraparn for assistance with the insect bioassay experiment, all associated with the University of Kentucky. This work was supported by grants from Firmenich, Geneva (to J.C.) and from the US National Institutes of Health (GM 13956 to R.M.C).
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Shuiqin Wu conceived and performed the experimental work; Michel Schalk and Anthony Clark made conceptual contributions to the work; Brandon Miles carried out the NMR experimental work; Robert Coates helped with interpretation of NMR spectra and data; and Joe Chappell conceived the work, helped to interpret the data and prepared the manuscript.
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M.S. and A.C. are current employees of Firmenich, which provided financial support for this work.
Supplementary information
Supplementary Fig. 1
Expression levels and processing of the patchoulol synthase (PTS) and farnesyl diphosphate synthase (FPS) proteins in transgenic plants were measured by immunodetection. (PDF 562 kb)
Supplementary Fig. 2
Head-space gas analysis of volatile emissions from control and PTS transgenic plants. (PDF 1281 kb)
Supplementary Fig. 3
A Ti vector system compatible for recombination cloning was developed to facilitate vector construction (pBDON). (PDF 80 kb)
Supplementary Table 1
Reports of genetic engineering terpene synthases in plants. (PDF 66 kb)
Supplementary Data
Supplementary Data for GC and NMR analyses performed at the University of Illinois. (PDF 69 kb)
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Wu, S., Schalk, M., Clark, A. et al. Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotechnol 24, 1441–1447 (2006). https://doi.org/10.1038/nbt1251
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DOI: https://doi.org/10.1038/nbt1251
