Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina

Biotechnol Rep (Amst). 2015 Aug 22:8:10-15. doi: 10.1016/j.btre.2015.08.004. eCollection 2015 Dec.

Abstract

Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA) PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals.

Keywords: EPA, eicosapentaenoic acid; Heterologous protein expression; MARs, matrix attachment regions; Particle bombardment; RESDA PCR, restriction enzyme site-directed amplification polymerase chain reaction; Restriction enzyme site-directed amplification (RESDA) PCR; TAIL PCR, thermal asymmetric interlaced PCR; sfCherry fluorescent protein.