Limonene production in tobacco with Perilla limonene synthase cDNA.

Limonene synthase (LS) catalyses the stereo-specific cyclization of geranyl diphosphate (GPP) to form a monocyclic monoterpene, limonene. In an attempt to engineer monoterpene biosynthesis, three expression constructs of LS cDNA of Perilla frutescens, which were designed to be localized in either the plastid, the cytosol or the endoplasmic reticulum (ER), were introduced into tobacco in order to examine differences in enzyme activity and the productivity of limonene. High and moderate enzyme activity, respectively, was observed for plastid- and cytosol-localized LS, whereas no enzyme activity was seen for ER-localized LS, suggesting that the plastid is the preferred compartment for LS, while LS may also have an active form in the cytosol. The formation of limonene in vivo was confirmed by gas chromatography-mass spectrometry (GC-MS) in leaf extracts of both plastid- and cytosol-localized LS transgenic plants. The amount of limonene in plastid-localized LS transgenic plants was 143 ng g-1 fresh wt, whereas that in the cytosol-type was 40 ng g-1 fresh wt, and these limonene contents increased by 2.7-fold and 3.0-fold, respectively, with the addition of methyl jasmonate. The headspace analyses showed that the plastid- and the cytosol-localized LS transgenic plants (12 cm high) emitted 390 ng and 515 ng limonene per month, respectively. The possibility of genetically engineering monoterpene production is discussed.