Nuclear transformation of the versatile microalga Euglena gracilis

Abstract Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium -mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium- mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.