Stable nuclear transformation of the oleaginous microalga Neochloris oleoabundans by electroporation

Abstract

Biodiesel from microalgae is technically feasible, but not yet economically viable. A potential approach to improve microalgae as an economically viable biodiesel feedstock is to increase microalgal lipid content via genetic engineering. Genetic manipulation of microalgae requires the accessibility to stable nuclear transformation. In this study, we describe a strategy for developing a stable nuclear transformation system of the oleaginous microalga Neochloris oleoabundans using electroporation. The hygromycin B-resistant gene Hyg3, which was used as a positively selectable marker, consisted of aph7” gene encoding aminoglycoside phosphotransferase of Streptomyces hygroscopicus and intron1 of Chlamydomonas reinhardtii rbcS2 gene, under the control of C. reinhardtii HSP70A-RBCS2 hybrid promoter. The transformation frequency was 5.2 × 10⁻⁴ transformants mg⁻¹ DNA. The transformants showed stable hygromycin B-resistant phenotype for at least 6 months in the absence of the antibiotic selection. Co-transformation frequency of unselectable green fluorescent protein gene (Gfp) adapted to C. reinhardtii codon usage (ChGfp) and selectable Hyg3 gene was 2.6 × 10⁻⁴ transformants mg⁻¹ DNA; up to 90 % of the transformants exhibited green fluorescent protein (GFP) activity. The ChGfp and Hyg3 gene were integrated into the nuclear genome of N. oleoabundans. The GFP fluorescence signal of the transformants under confocal laser scanning microscope was visible. The successful stable nuclear transformation system not only provides a basis for molecular genetics study, but also enables subsequent genetic engineering in the microalga to increase lipid content for biodiesel production. The strategy for developing the stable nuclear transformation system presented in this study may be applicable to other microalgal species without sequenced genome.