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Title: Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts

item KUMAR, SHASHI - Yulex Corporation
item Hahn, Frederick
item BAIDOO, EDWARD - Lawrence Berkeley National Laboratory
item Kahlon, Talwinder
item Wood, Delilah - De
item McMahan, Colleen
item CORNISH, KATRINA - Yulex Corporation
item KEASLING, JAY - Lawrence Berkeley National Laboratory
item DANIELL, HENRY - University Of Central Florida
item Whalen, Maureen

Submitted to: Metabolic Engineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2011
Publication Date: 11/21/2011
Citation: Kumar, S., Hahn, F.M., Baidoo, E., Kahlon, T.S., Wood, D.F., Mcmahan, C.M., Cornish, K., Keasling, J., Daniell, H., Whalen, M.C. 2011. Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts. Metabolic Engineering. 14(1):19-28.

Interpretive Summary: Sustainable production of natural rubber in the USA requires high yielding rubber-producing crops. The tools of biotechnology offer an unprecedented opportunity to increase yields of rubber, produced in plants via the isoprenoid biochemical pathway, by metabolic engineering. Rubber is biosynthesized only in the presence of excess IPP (isopentenyl pyrophosphate), so approaches to metabolic engineering usually aim to increase the amount of IPP in cells. Plants contain chloroplasts, subcellular organelles that produce compounds including chlorophyll (an IPP derivative), so IPP can be produced both in the cytosol of the cell and in the chloroplasts. Engineering of chloroplasts has advantages including 1) high production level of foreign proteins, 2) precise and predictable insertions of multiple genes, and 3) no transgene flow in pollen. In this study, chloroplasts of tobacco were engineering to produce high levels of IPP derivatives.

Technical Abstract: Metabolic engineering to enhance production of isoprenoid metabolites for industrial and medical purposes is an important goal. The substrate for isoprenoid synthesis in plants is produced by the mevalonate pathway (MEV) in the cytosol and by the 2 C methyl-D-erythritol 4 phosphate (MEP) pathway in plastids. A multi-gene approach was employed to insert the entire cytosolic MEV pathway into tobacco chloroplasts. Molecular analysis confirmed the site-specific insertion of seven transgenes and homoplasmy. Functionality was demonstrated by unimpeded growth on fosmidomycin, which specifically inhibits the MEP pathway. Transplastomic plants containing the MEV pathway genes accumulated higher levels of mevalonate, chlorophyll, carotenoids, squalene, sterols and triacyglycerols than control plants. This is the first time an entire pathway with six enzymes has been transplastomically expressed in plants. Thus, we have generated an important tool for use in isoprenoid metabolic engineering to redirect metabolic fluxes, and have demonstrated a viable multigene strategy for engineering metabolism in plants.