br Materials and methods br Results
Materials and methods
Discussion The present work demonstrates the functional role of DGAT2 in elevating the neutral lipid accumulation in engineered N. oceanica, which have aroused a great deal of interest in industrial scale biofuel production. Recently, there have been increasing reports on algal lipid enhancement by means of metabolic pathway engineering. Metabolic engineering represents a promising strategy to enhance the algal lipid content by targeted engineering of metabolic pathways (Xue et al., 2015). The Kennedy pathway is considered to be the predominant metabolic pathway in microalgal TAG biosynthesis. DGAT catalyzes the last and committed step in glycerol 3-phosphate pathway. Lipidomic and transcriptomic analyses showed that increased gene doses and elevated transcript abundance of DGAT were responsible for increased lipid content in N. oceanica IMET1 (Li et al., 2014). Heterologous complementation assays also revealed the functional role of algal DGAT2 in TAG accumulation (Hung et al., 2013). In this study, putative DGAT2 was identified and overexpressed in N. oceanica. Glycerolipid content was found to be significantly increased in engineered N. oceanica by 129% as compared with WT Mitomycin C and we also observed the increased oil body volume in engineered cells. Indeed, overexpression of C. reinhardtii DGAT2 in Arabidopsis resulted in elevated leaf TAG content (Sanjaya Miller et al., 2013). Similarly, introduction of Arabidopsis DGAT in tobacco showed seven fold increase of TAG accumulation (Bouvier-Nave et al., 2000). Thus, the results are in accordance with the laser confocal microscopic observation of increased oil body size in the engineered cells. These results suggest that overexpression of DGAT2 could accumulate the TAG via catalyzing the final and committed step in the TAG biosynthetic pathway, which was in agreement with the increased level of DGAT2 transcripts as revealed by qPCR. In addition to neutral lipid accumulation, our study also illuminated that DGAT2 from N. oceanica could alter the composition of fatty acids. The results showed the strong correlation between neutral lipid content and fatty acid, because fatty acid content was considered to be an important parameter in preferred biofuel production. In this study, the content of SFAs was found to be increased while MUFAs and PUFAs decreased significantly in engineered N. oceanica. The elevated SFA content in microalgae was reported to increase oxidative stability and ignition quality of the biofuel, which is a highly desirable trait for high quality biofuel production (Gosch et al., 2012). However, the fatty acid composition of DGAT2-overexpressing algae is still needed for further improvement. There have been multiple types of algal DGAT2 reported which influenced the diversified fatty acid composition (Chen and Smith, 2012, Nguyen et al., 2011). Overexpression of P. tricornutum DGAT2B in yeast increased the content of MUFAs and PUFAs, whereas decreased the SFAs content (Gong et al., 2013). Similarly, Oryza sativa DGAT2B was identified as the most active among three OtDGAT2s and could significantly alter the fatty acid composition (Wagner et al., 2010). From these reports it was proposed that type B of DGAT2 was found to be a more potent isoform. In spite of the annotation of N. oceanica CCMP1779 genome (Vieler et al., 2012), the functional characterization of DGAT2 in N. oceanica has remained unclear. Jia et al. (2015) reported the altered fatty acid composition in Nannochloropsis under nitrogen depletion. Decreased level of long fatty acids such as eicosenoic acid (C20:1) and oleic acid (C18:1) was detected in transgenic Arabidopsis expressing DGAT1 than wild type (Zou et al., 1999). These reports corroborate with our findings that expression of DGAT2 in N. oceanica resulted in altered fatty acid content and elevated neutral lipid accumulation. DGAT2 was reported to play a role in channeling the unusual fatty acids into lipids in plants (Turchetto-Zolet et al., 2011). Thus, these data are in good agreement with the elevated lipid accumulation in engineered N. oceanica.