br Conclusions In conclusion we identified
Conclusions In conclusion, we identified and overexpressed DGAT2 in fast growing, oleaginous N. oceanica for the first time. The engineered N. oceanica overexpressing DGAT2 resulted in enhanced neutral lipid accumulation and altered fatty BMS-754807 molecular composition. Altogether, our findings demonstrate the potential role of DGAT2 in algal TAG biosynthesis. This report may have great utility in profitable and sustainable biofuel production from oleaginous microalgae.
Acknowledgments This work was supported by the Natural Science Foundation of China (41576132), the Guangdong Natural Science Foundation (2014A030308010) and the Department of Education of Guangdong (2014KTSCX022).
Introduction Oilseeds are the main source of lipids used in food and biofuels and are increasingly important renewable chemical feedstocks (Napier and Graham, 2010, Wilson and Hildebrand, 2010). Most storage lipids in oil-accumulating seeds are composed entirely of triacylglycerols (TAGs), molecules that consist of three fatty acid (FA) chains esterified to a glycerol backbone. Unlike major commercial oilseeds containing oil comprised predominantly of just five main FAs, namely palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids, many exotic plant species have been found to contain high levels of unusual fatty acids (UFAs) such as hydroxy, epoxy, acetylenic and conjugated fatty acids in their seeds (van de Loo et al., 1993). Many of these UFAs have unique properties that can make them valuable as renewable raw materials for the chemical industry, in making dyes, paints, coatings, adhesives, composites, plastics and a variety of other bio-based products (Jaworski and Cahoon, 2003). However, commercial production of UFAs through traditional farming of these wild species is badly hampered because of their poor agronomic properties such as small seeds, low yields, and limited geographical growing areas. Alternatively, expanding efforts are focusing on the production of these UFAs in seeds of agronomically suitable plants, particularly oil crops by metabolic engineering (Damude and Kinney, 2008, Dyer and Mullen, 2008, Napier and Graham, 2010). A number of variant FAD2s (or Δ12 desaturases) have been identified to be the main enzymes for synthesis of UFAs in high-accumulating plants (Cahoon and Ohlrogge, 1994, Cahoon et al., 2006, Cahoon et al., 2002, Cahoon et al., 2007, Hitz, 1998, Lee et al., 1998). These divergent FAD2s exhibit functions such as epoxidation, hydroxylation, acetylation and conjugation rather than the function of the typical FAD2 that catalyzes the introduction of a cis-Δ12 double bond in oleic acid (C18:1) to form linoleic acid (C18:2). Transgenic expression of variant FAD2 genes alone led to the synthesis of the UFAs at levels significantly lower than those found in native sources from which the genes were isolated (Burgal et al., 2008, Cahoon et al., 2007, Jaworski and Cahoon, 2003, Singh et al., 2005, Thelen and Ohlrogge, 2002), indicating that additional enzymes are required for the metabolism and accumulation of UFAs in seeds of transgenic plants. Diacylglycerol acyltransferases (DGAT; EC 18.104.22.168) that can catalyze the final acylation of TAG have been proposed to be one of the rate-limiting steps in plant storage lipid accumulation and play an essential role in controlling both the quantitative and qualitative flux of fatty acids into storage TAGs (Abe et al., 2006, He et al., 2004, Ichihara et al., 1988, Jako et al., 2001, Lung and Weselake, 2006, Perry et al., 1999, Sørensena et al., 2005, Sovero, 1996). There are two classes of membrane-bound DGATs designated as DGAT1 and DGAT2 in plants (Adelsberger et al., 2004, Cases et al., 2001, Kroon et al., 2006, Shockey et al., 2006, Zou et al., 1999), with DGAT2 showing particular propensity for UFA accumulation (Li et al., 2010a, Shockey et al., 2006). Shockey et al. (2006) presented good evidence that DGAT2 may be involved in the selective accumulation of the unusual fatty acid, eleostearic acid (a conjugated FA), in tung (Vernicia fordii) oil. Castor DGAT1 (He et al., 2004) and DGAT2 (Kroon et al., 2006) are reported to play a dominant role in the production of the hydroxy fatty acid (HFA) ricinoleate in castor (Ricinus communis) seed oil. Castor DGAT2 can nearly double ricinoleate accumulation in Arabidopsis seeds (from ∼17 to ∼30%) by coexpression of this gene along with the castor hydroxylase compared to the hydroxylase gene alone (Browse et al., 2008, Burgal et al., 2008). In addition to DGATs, phospholipid: diacylglycerol acyltransferases (PDATs; E.C.22.214.171.124), another acyltransferase acting in the final acylation step in TAG synthesis, can directly transfer the sn-2 acyl chain from phosphatidylcholine (PC) to sn-1, 2-diacylglycerol (DAG) and form TAG and lyso-PC (Dahlqvist et al., 2000, Mhaske et al., 2005). PDAT has overlapping functions with DGAT for both TAG synthesis in seed and pollen and the development of the two organs in Arabidopsis (Zhang et al., 2009), and are also suggested to contribute to the incorporation of UFAs into TAGs (Mhaske et al., 2005, Ståhl et al., 2004). Moreover, castor PDAT1A can increase HFA accumulation up to 27% when co-expressed with castor hydroxylase in Arabidopsis seed oil compared with 16.9% of HFA in the transgenic Arabidopsis expressing hydroxylase alone (van Erp et al., 2011). Taken together, coexpression of divergent FAD2 with UFA-specific TAG synthesis enzymes are promising for UFA production in oilseeds by metabolic engineering, although most of these studies have been done using Arabidopsis.