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

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 the tobacco chloroplast genome. 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, carotenoids, squalene, sterols, and triacyglycerols than control plants. This is the first time an entire eukaryotic pathway with six enzymes has been transplastomically expressed in plants. Thus, we have developed an important tool to redirect metabolic fluxes in the isoprenoid biosynthesis pathway and a viable multigene strategy for engineering metabolism in plants.

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

“…18 Multigene engineering is especially relevant for studies with FVIII because all domains may be simultaneously expressed in a single transformation cassette. 47 Further optimization of the system and clinical implications…”

The complete chloroplast genome of Origanum vulgare L. (Lamiaceae)

“…In case of commercial taxa chloroplast sequence information may also find practical applications in further disciplines such as e.g. DNA-barcoding (Boccacci et al, 2012;Khan and Azim, 2011), plant breeding (Wang et al, 2012), conservation genetics (Dzialuk et al, 2009;Shanjani et al, 2010) or plant biotechnology (Kumar et al, 2012).…”

Transplastomic plants for innovations in agriculture. A review

“…Carotenoid biosynthesis normally makes use of precursors from the non-mevalonate pathway. Recently, Kumar et al (2012) successfully introduced the entire cytoplasmic mevalonate pathway (six genes from yeast on a synthetic operon) into the tobacco chloroplast genome (Fig. 2).…”

“…Similarly to Kumar et al (2012), a general increment in isoprenoid content (chlorophyll a, phytol, β-carotene, lutein, antheraxanthin, solanesol, and β-sitosterol) was observed when the enzyme responsible for the first committed step of the plastidial nonmevalonate pathway, i.e., 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) from Synechocystis was overexpressed in transplastomic tobacco plants (Hasunuma et al 2008a). …”