S96: Engineering microalgae for production systems: metabolic manipulation without GMO classification

Metabolic engineering of microalgae for production of fuels or chemicals currently relies on heterologous selectable markers (such as antibiotics), complementation of metabolic deficiencies, or mutagenesis and screening.  Complementation approaches are time-consuming, mutagenesis prohibits specific gene or pathway targeting, and heterologous markers classify the resultant strain as a genetically-modified organism (GMO).  To date, no GMO algae have completed the stringent regulation process to be grown in outdoor pond systems, and some regions of the U.S. outright ban outdoor GMO cultivation.  We present here a method to metabolically engineer specific genes in microalgae using only native sequences of the target organism, thus resulting in strains not classified as GMOs.  We have previously shown that knock-down of the lipase Thaps3_264297 in the diatom Thalassiosira pseudonana using antisense RNA results in strains with increased TAG accumulation and cell viability compared to wild-type after nutrient starvation – phenotypes that can be used as selectable markers.  Co-transformation of Thaps3_264297 antisense RNA with other native DNA constructs (e.g. to manipulate other pathways) would enable the selection of transgenic strains by screening for the lipase knock-down phenotype.  Multiple rounds of nutrient limitation, fluorescence-activated cell sorting and nutrient replenishment will be used to select against wild-type and enrich for transgenic cells. This method, which can be applied to other microalgal strains, allows for the targeted manipulation of metabolic pathways combined with a simple phenotypic screening process to produce engineered strains not classified as GMOs that can be used in outdoor production systems.