Producing renewable sources for a variety of fuels and chemicals is of great importance to a world with increasing demand for such products. While petroleum is a product of decayed plant and other matter that has been incubated beneath the earth's surface for millions of years, some efforts today focus on the direct use of plants and other organisms to generate, e.g., lipids, which can include fatty acids and derivatives thereof, for use in the fuel and chemical industries. Specifically, recent effort has been directed to designing algae to produce lipids for biofuel production because algae can proliferate over a wide range of environmental conditions and because algae do not compete with food crops for arable growth space. See, Hu et al. (2008) Plant J. 54:621-39.
Algal cells are a promising source of biofuels. Wijffels & Barbosa (2010) Science 329:796-99. Their ability to harness solar energy to convert carbon dioxide into carbon-rich lipids already exceeds the abilities of oil-producing agricultural crops, with the added advantage that algae grown for biofuel do not compete with crops for agricultural land (Wijffels & Barbosa, 2010). In order to maximize algal fuel production, new algal strains will need to be engineered for growth and carbon fixation at an industrial scale (Wijffels & Barbosa, 2010).
Triacylglycerol or triglyceride (TAG), a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bonds, is an excellent molecule for high-concentration metabolic-energy storage. TAG is the major form of energy storage in many eukaryotic algae under stress conditions, such as under nutrient limitation or depletion. Nitrogen depletion (where there is essentially no available nitrogen in the culture medium) is particularly effective in increasing TAG production in many eukaryotic algal species. However, culturing algae under nitrogen deficiency simultaneously limits overall lipid productivity of the culture by limiting overall biomass accumulation (Brennan & Owende (2010) Renewable and Sustainable Energy Reviews 14:557-77). Improving the scalability, controllability, and cost-effectiveness of TAG production would be beneficial to the development of renewable energy and chemical sources.
One means of boosting TAG production is to grow algae in a two-step process alternating between nutrient-rich and nutrient-limited conditions. The nutrient-rich growth phase allows the algae to proliferate, while nutrient limitation (e.g., nitrogen depletion) results in the production of storage lipids. See, Rodolfi et al. (2009) Biotechnol. Bioeng. 102:100-12. This process makes TAG production more expensive, because it requires long periods of growth during which the algae are producing little to no TAG.
Another means of boosting TAG production is to grow the algae heterotrophically by supplying extra organic carbon. For example, in various scenarios, organic carbon may be supplied as glycerol, one or more sugars, one or more organic acids, or other reduced carbon sources added to the growth medium. See, Allnutt et al. (WO 2011/026008). This heterotrophic growth technique not only increases the expense of TAG production, it also risks the contamination of the algal cultures with exogenous bacteria or fungi whose growth can be stimulated by the added carbohydrates. See, Scott et al. (2010) Curr. Opin. Biotechnol. 21:277-86.
The biosynthesis pathways leading to the production of TAG have been studied. In the final reaction of the Kennedy pathway, diacylglycerol (DAG), a precursor to both membrane and storage lipids, is covalently linked to a fatty acyl to produce TAG. This reaction is catalyzed by the diacylglycerol acyltransferase (DGAT) enzyme (Kennedy (1961) Fed. Pro. Fed. Am. Soc. Exp. Biol. 20:934-40). There are two distinct gene families in eukaryotic organisms which encode enzymes which catalyze this reaction, DGAT1 and DGAT2, which have little sequence similarity. Evidence from higher plants as well as mammals suggests that the two gene families have different functions, although the exact role of each type of DGAT has not been elucidated, and may differ in different species (Yen et al. (2008) J. Lipid Res. 49:2283-301).
Although the use of recombinant DGAT enzymes to enhance TAG production in oleaginous organisms is known to the art (Xu et al. (2008) Plant Biotechnol. J. 6:799-818), comparatively little attention has been given to the subcellular localization of these recombinant DGAT enzymes.
It has recently been reported that a DGAT1 gene in the diatom species Phaeodactylum tricornutum contains a PH domain-encoding sequence. However, PH domains are not found in known plant DGATs, despite close evolutionary relationships to orthologous algal DGATs. See, FIGS. 1 & 2.
Guiheneuf et al. (WO 2012/059925) reports a PH domain at the amino-terminal end of a DGAT1 in Phaeodactylum tricornutum. 
Further, Liu et al. (CN 102492672) report a DGAT1 sequence from the diatom Thalassiosira pseudonana with a PH domain at the amino-terminal end.
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