Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway

• Published in: Journal of biotechnology 2014-04, Vol.176, p.20-28
• Main Authors: Ritala, Anneli, Dong, Lemeng, Imseng, Nicole, Seppänen-Laakso, Tuulikki, Vasilev, Nikolay, van der Krol, Sander, Rischer, Heiko, Maaheimo, Hannu, Virkki, Arho, Brändli, Johanna, Schillberg, Stefan, Eibl, Regine, Bouwmeester, Harro, Oksman-Caldentey, Kirsi-Marja
• Format: Article
• Language: English
• Subjects: Acyclic Monoterpenes; Alkaloids; Biomass; Bioreactors; catharanthus-roseus; cells; Cosmetics; cultures; Derivatives; DNA, Plant; essential oils; Fragrances; Geraniol; Hairy root; Indoles; isoprenoid biosynthesis; key enzyme; monoterpene; Nicotiana - enzymology; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Roots - enzymology; Plant Roots - genetics; Plant Roots - metabolism; Plants, Genetically Modified - enzymology; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; plastidial pathways; Roots; Secologanin Tryptamine Alkaloids - metabolism; Secondary Metabolism; synthase; Terpenes - metabolism; Terpenoid indole alkaloid; Tobacco; Transgenic; Valerian - genetics; Valeriana officinalis
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2014.01.031

•Tobacco hairy roots were engineered to express geraniol synthase.•A detailed metabolite analysis of transgenic hairy root cultures was performed.•Generated hairy roots contained geraniol derivatives in six major glycoside forms.•Total geraniol accumulation was as high as 204.3μg/g DW in engineered hairy roots.•A scale-up to a 20L wave bioreactor was developed containing mg amounts of geraniol. The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastid-targeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC–MS screening tool was developed for the rapid evaluation of production clones. The GC–MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7μg/g dry weight (DW) and a maximum of 31.3μg/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3μg/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag.