Stable transformation of the green algae Acutodesmus obliquus and Neochloris oleoabundans based on E. coli conjugation

Abstract

Microalgae are an ideal platform for the production of high-value chemicals, nutritional products and biofuels. Genetic engineering could speed up the development of microalgae derived products and reduce the overall production costs. Genetic methods such as particle bombardment, electroporation, Agrobacterium tumefaciens mediated transformation (ATMT), and agitation with glass beads and silicon carbide whiskers have been developed for the genetic transformation of microalgae. However, the transformation efficiency is species dependent, so a variety of transformation methods are required to engineer a wide range of microalgae species. The oleaginous microalgae Acutodesmus obliquus and Neochloris oleoabundans have a great potential as production platforms due to their ability to produce large amounts of triacylglycerol (TAG). Genetic modification techniques however are required to increase TAG levels further or to modify the fatty acid composition. Recently, a conjugation-based method for the delivery of episomes from bacteria to diatom microalgae has been reported. In this study, we have achieved the successful transformation of green oleaginous microalgal strains by transferring an expression vector via conjugation from E. coli. Since delivery of exogenous DNA into the microalgae cells is only the first step in obtaining transgenic microalgae, we further analyzed transformation efficiencies by PCR and expression of the Clover fluorescent protein in the targeted species.