Enhancement of biomass and lipid biosynthesis in nannochloropsis salina by overexpression of transcription factors

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, Nannochloropsis salina was successfully transformed with particle bomabardment, and the sfCherry fluorescent protein was overexpressed and visualized in N. salina for the first time. Genomic integration was confirmed by RESDA PCR ensuring stable expression of transgenes. We increased transformation efficiency by about 2-fold by using 30% conditioned medium. Using the developed transformation toolbox, NsbHLH2 transcription factor was overexpressed in N. salina, and the transformants showed enhanced growth under the normal and nitrogen limitation conditions. Analyses of NsbHLH2 expression with qRT-PCR revealed that NsbHLH2 may be involved in increased growth mainly under these conditions. The increased growth of NsbHLH2 transformants was linked to increased biomass and FAME productivities, particularly at day 8. However, growth of NsbHLH2 transformants became similar with WT possibly due to nutrient limitation at 12 day, so we conducted continuous cultivation allowing nutrient supplementation at various dilution rates. At high dilution rate ($D=0.5 day^{-1}$), biomass productivity of NsbHLH2 was 378 mg/L/d which was highest compared to WT. Subsequently, we induced maximum FAME productivity by N limitation at high dilution rate, resulting in 83.6 mg/L/d of FAME productivity. In order to understand how NsbHLH2 transformant grew better than WT, we carried out transcriptome analysis. Analyses of differentially expressed genes (DEGs) revealed that translational activity was greatly reduced in the transformant, which may be interpreted as enhanced stress responses. Instead of increased lipid accumulation by enhanced stress response, the transformant showed increased carbon fixation, metabolism of cellulose and acyl-CoA, which may contribute to the improved growth. Taken together, overexpression of NsbHLH2 in N. salina increased of biomass and lipid productivities under batch and continuous cultivation, which may apply to other industrial microalgae.