DXS expression in Morinda citrifolia cell suspension cultures for increasing anthraquinones production

QUEVEDO, CV; PERASSOLO, M; GIULIETTI, AM; RODRÍGUEZ TALOU, JULIÁN

Dalian, China

Simposio; 13th International Biotechnology Symposium and Meeting; 2008

Plant cell suspension cultures are attractive alternatives for large scale production of plant-derived natural products, in particular of secondary metabolites. Morinda citrifolia, which belongs to Rubiaceae family, is an example of this, since it is able to produce Anthraquinones (AQs) that have potential therapeutic use, including antitumor, immunomodulatory and antioxidative properties [1]. AQs are anthracene derivatives which have two keto groups, usually in positions 9 and 10. The basal compound, anthraquinone (9, 10-dioxoanthracene), can be substituted in various ways, resulting in a great diversity of structures. The A and B rings of AQs are synthetized from the carbon skeleton of shikimic and ¦Á-ketoglutaric acids via the ischorismate/o-succinylbenzoate pathway. The C ring is originated from a terpenoid block synthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway (MEP), [2]. 1-deoxy-D-Xylulose-5-phosphate synthase (DXS), the first enzyme in the (MEP) pathway catalyzes the transketolase reaction converting pyruvate and glyceraldehyde 3-phosphate into 1-deoxy-D-Xylulose-5-phosphate [2]. In order to increase the AQs production, genetic strategies have been applied, including the overexpression of key enzymes. The aim of this work was to overexpress the DXS in M. citrifolia suspension cultures. For this purpose, it was constructed an expression vector with DXS. The dxs cDNA obtained from Catharantus roseus cloned in a pGemTeasy vector, was digested and ligated between the Cauliflower mosaic virus 35S-promoter (CaMV-35S) and the potato proteinase inhibitor terminator (PIt) of pMOG843 (MOGEN, Leiden, The Netherlands). Subsequently, the cassette expression containing dxs was inserted into the binary vector pMOG22-GUS, which contains hygromycin resistance gene, GUS reporter gene and the left and right T-DNA borders from Agrobacterium tumefaciens.  M citrifolia cell cultures were transformed by biolistic methods and using A. tumefaciens strain LBA4404 containing the compatible plasmid pBRIMCS carrying a constitutive virG gene. Transformation was confirmed by analysis of the GUS reporter gene. The DXS activity was determined using a fluorometric end-point assay based on 1-deoxyfructose and others 1-deoxysugars reaction with 3,5-diaminobenzoic acid in an acidic medium to form highly fluorescent quinaldine derivatives [4]. The transgenic cells lines obtained presented DXS higher activity compared to the wild type cell lines. These results are promising for AQs production by M citrifolia suspension cultures.                               References. [1] Komaraiah, P., Kavi Kishor, P.B., Carlsson, M., Magnusson, K. and Mandenius, C.F. 2005 Enhancement of antrhaquinone accumulation in Morinda citrifolia suspension cultures. Plant Science 168: 1337-1344. [2] Han, Y. S., van der Haijden, R. and Verpoorte, R. (2001) Bioshyntesis of antrhaquinone in cell cultures of the Rubiaceae. Plant Cell, Tissue and Organ Culture 67: 201-220. [3] Est¨¦vez JM, Cantero A, Reindl A, Reichler S, Le¨®n P. (2001) 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants.  J Biol Chem. 276(25): 22901-9 [4] Querol, J., Besumbes, O., Lois, M.L., Boronat, A. and Imperial, S. (2001) A fluorometric Assay for the Determination of 1-Deoxy-D-xylulose 5-Phosphate Synthase Activity. Analytical Biochemistry 296: 101-105.