br Experimental Procedures br Acknowledgments br Introductio

Experimental Procedures
Acknowledgments
Introduction Incessant consumption of fossil fuels brings on global energy crisis and serious environmental concerns. Hence, the researchers have paid considerable attention to alternative renewable bioenergy. Microalgae has been considered as potential biofuel feedstock over terrestrial crops, because of their higher growth rate, CO2 fixation, cultivable in wide range of water without encroaching farm land (Subramaniam et al., 2010) and capability of accumulating higher triacylglycerols (TAG) and oils that can be readily converted into biodiesel by transesterification (Chisti, 2007). However, lack of microalgal strains with higher TAG content and 5,7-Dichlorokynurenic acid is found to be one of the major obstacles in the economically viable fuel industry (Chisti, 2007). Metabolic engineering represents a promising strategy to enhance the microalgal TAG content by targeted engineering of metabolic pathways. Identification and manipulation of key genes that influence TAG biosynthesis are the critical determinants for engineering the microalgae for enhanced TAG production. The assembly of TAG occurs in the ER where TAG can be synthesized by sequential acylation of glycerol starting with glycerol 3-phosphate. Diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) catalyzes the last and committed step in the TAG production, by transferring an acyl group from acyl-CoA to diacylglycerol (DAG) to form triacylglycerol. Previous studies showed that DGAT overexpression in plants resulted in elevated TAG accumulation (Andrianov et al., 2010, Bouvier-Nave et al., 2000). Overexpression of Arabidopsis DGAT in Saccharomyces cerevisiae resulted in increased DGAT activity, thereby increasing the TAG content (Jako et al., 2001). In addition, overexpression of DGAT2 in green microalga Chlamydomonas reinhardtii apparently increased the TAG content up to 9-fold (Hung et al., 2013). In algae, three types of DGAT have been characterized, and among them DGAT2 has been identified as the potent enzyme in TAG biosynthesis (Chungjatupornchai and Watcharawipas, 2015, Hung et al., 2013). Algal DGAT2s diverge from those of other eukaryotes by possessing multiple paralogs (Chen and Smith, 2012). The genome sequencing of Nannochloropsis revealed the existence of one form of DGAT1 and eleven forms of DGAT2s (Wang et al., 2014). DGAT1 and DGAT2 have been characterized in many different algal species, such as marine diatom Phaeodactylum tricornutum (Guiheneuf et al., 2011, Niu et al., 2013) and green microalga C. reinhardtii (La Russa et al., 2012). Especially, DGAT2 was more potent in TAG biosynthesis than DGAT1 in mice (Stone et al., 2004) and plants (Kroon et al., 2006), possibly due to its conserved orthologs in nature (Shockey et al., 2006) and its higher affinity towards the substrates (Chen and Smith, 2012). However, identification of DGAT2 in oleaginous microalga Nannochloropsis oceanica and elucidation of its functional role in microalgal TAG synthesis remain unclear. Nannochloropsis species are unicellular photosynthetic heterokonts distributed in wide range of waters and ranging in size from 2 to 5μm. Nannochloropsis has been of commercial interest because of its promising characteristics such as high TAG content, synthesis of economically valuable by-products and higher growth rate (Doan and Obbard, 2010, Wang et al., 2014). Our burgeoning knowledge of the microalgal genomes and metabolic pathways open up new opportunities to study and improve microalgae. Genetic manipulation to enhance lipid accumulation has been conducted in the model microalgal organisms such as C. reinhardtii (Mussgnug et al., 2007) and P. tricornutum (Xue et al., 2015). However, genetic engineering of oleaginous non-model microalgal species for enhanced lipid accumulation has been rarely achieved yet (Muto et al., 2015). It is of great interest to develop genetically improved strains of such microalga with high industrial potential. In this study, we demonstrate the identification, cloning and overexpression of a putative DGAT2 from N. oceanica. This report showed the promising role of DGAT2 in microalgal TAG biosynthesis and would also pave the way for metabolic engineering in photosynthetically driven cell factories for commercialization.