Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco

• Published in: The Plant journal : for cell and molecular biology 2004-07, Vol.39 (1), p.135-145
• Main Authors: Lucker, J, Schwab, W, Franssen, M.C.R, Plas, L.H.W. van der, Bouwmeester, H.J, Verhoeven, H.A
• Format: Article
• Language: English
• Subjects: (+)‐trans‐isopiperitenol; Amino Acid Sequence; amino acid sequences; biochemical pathways; Biological and medical sciences; biosynthesis; Citrus - enzymology; Citrus - genetics; Citrus limon; complementary DNA; cytochrome P-450; Cytochrome P-450 Enzyme System - chemical synthesis; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; Flowers - enzymology; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; gene expression regulation; Gene Silencing; genetic transformation; Genetic Vectors; isomerases; limonene; limonene-3-hydroxylase; Mentha spicata; Mentha spicata - genetics; Mentha spicata - metabolism; metabolic engineering; Metabolism; Metabolism. Physicochemical requirements; Mixed Function Oxygenases - chemical synthesis; Mixed Function Oxygenases - genetics; Models, Chemical; Molecular Sequence Data; monoterpene biosynthesis; monoterpene synthase; Monoterpenes - chemistry; Monoterpenes - metabolism; monoterpenoids; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; nucleotide sequences; oxygenases; Plant physiology and development; plant proteins; Plants, Genetically Modified; sequence alignment; Terpenes - chemistry; Terpenes - metabolism; tobacco; Transformation, Genetic; transgenes; transgenic plants; Volatilization
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313X.2004.02113.x

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, γ-terpinene and( -)-β-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.