Towards metabolic engineering of triterpene pathway in green microalgae

Microalgae are able to synthesis a large arsenal of bio-molecules and so far have been discovered 100000 different chemical compounds (Sasso et al 2012, FEMS Microbiol Rev 36: 761-785). These compounds mainly belong to the chemical family of isoprenoids, alkaloids, polyunsaturated fatty acid. Among them, triterpenes molecules have received a major scientific interest due to their use for human health, pharmacological properties and in synthetic chemistry. In addition, these molecules can be remarkable for the production of biofuel, as reported for botryococcene detected in Botryococcus braunii (Tom et al 2011. PNAS 108:12260-12265). However, the pathway of triterpenoids is not completely understood which reduce to apply an metabolic engineering approach. To overcome this limitation a heterologous overexpression of a gene involved in the biosynthesis of these molecules might improve their content and modify the profile. For this purpose, we have evaluated the effects of the ectopic expression of a oxidosqualene cyclase (AsOXA1) isolated from Aster sedifolius (Cammareri et al 2008, Plant Sci 175:255-261) in several species of green microalgae. The AsOXA1 gene was cloned in to a binary expression vector driven by the constitutive promoter CaMV35S. Indirect nuclear genetic transformation was performed in 19 different species belongs to Ankistrodesmus, Scenedesums, Chlamydomonas, Chlorella genera. The microalgae colonies were detected after 14 days of co-cultivation on selective medium in 8 different accession. The highest frequency of transformation was obtained in the accession #103 and only 4 colonies for the accession # 101. In order to reduce the escape, 96 putative transgenic colonies from each accession were inoculated into liquid selective medium. The accession #124 and #110 shown a lower frequency of escape compared to the accession#112. PCR analysis were carried out on 70 colonies from 9 microalgae accession that were grown in selective liquid medium. The genome integration of AsOXA1 gene was detected in 63 out of 70 colonies assayed. The transgenic algae expressing AsOXA1 will be analysed by the appropriate techniques that allow to determine a modification in content and profiles of triterpenes.