Abstract
BackgroundPhotosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. The marine microalga, Nannochloropsis oceanica, has been attracting ever-increasing interest because of its fast growth, high triacylglycerol (TAG) content, and available genome sequence and genetic tools. Diacylglycerol acyltransferase (DGAT) catalyzes the last and committed step of TAG biosynthesis in the acyl-CoA-dependent pathway. Previous studies have identified 13 putative DGAT-encoding genes in the genome of N. oceanica, but the functional role of DGAT genes, especially type-I DGAT (DGAT1), remains ambiguous.ResultsNannochloropsis oceanica IMET1 possesses two DGAT1 genes: NoDGAT1A and NoDGAT1B. Functional complementation demonstrated the capability of NoDGAT1A rather than NoDGAT1B to restore TAG synthesis in a TAG-deficient yeast strain. In vitro DGAT assays revealed that NoDGAT1A preferred saturated/monounsaturated acyl-CoAs and eukaryotic diacylglycerols (DAGs) for TAG synthesis, while NoDGAT1B had no detectable enzymatic activity. Assisted with green fluorescence protein (GFP) fusion, fluorescence microscopy analysis indicated the localization of NoDGAT1A in the chloroplast endoplasmic reticulum (cER) of N. oceanica. NoDGAT1A knockdown caused ~25% decline in TAG content upon nitrogen depletion, accompanied by the reduced C16:0, C18:0, and C18:1 in TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. NoDGAT1A overexpression, on the other hand, led to ~39% increase in TAG content upon nitrogen depletion, accompanied by the enhanced C16:0 and C18:1 in the TAG sn-1/sn-3 positions and C18:1 in the TAG sn-2 position. Interestingly, NoDGAT1A overexpression also promoted TAG accumulation (by ~2.4-fold) under nitrogen-replete conditions without compromising cell growth, and TAG yield of the overexpression line reached 0.49 g L−1 at the end of a 10-day batch culture, 47% greater than that of the control line.ConclusionsTaken together, our work demonstrates the functional role of NoDGAT1A and sheds light on the underlying mechanism for the biosynthesis of various TAG species in N. oceanica. NoDGAT1A resides likely in cER and prefers to transfer C16 and C18 saturated/monounsaturated fatty acids to eukaryotic DAGs for TAG assembly. This work also provides insights into the rational genetic engineering of microalgae by manipulating rate-limiting enzymes such as DGAT to modulate TAG biosynthesis and fatty acid composition for biofuel production.
Highlights
Photosynthetic oleaginous microalgae are considered promising feedstocks for biofuels
The two genes were differentially regulated by nitrogen depletion: NoDGAT1A was upregulated moderately, while NoDGAT1B had a much lower level of transcripts than NoDGAT1A and remained relatively stable in response to nitrogen depletion (Fig. 1e), suggesting that NoDGAT1A is more involved in the nitrogen-depletion-induced TAG biosynthesis
The induced TAG accumulation was accompanied by a decrease in membrane polar lipids including monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), diacylglycerolN,N,N-trimethylhomoserine (DGTS), sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) (Fig. 1f ), consistent with previous results in Nannochloropsis strains [30, 33] and indicative of the turnover of membrane lipids for TAG assembly under nitrogen-depleted conditions
Summary
Photosynthetic oleaginous microalgae are considered promising feedstocks for biofuels. TAG biosynthesis has been documented and is believed to be mediated mainly via two pathways, acyl-CoA independent pathway and acyl-CoAdependent Kennedy pathway [12]. The latter pathway starts from glycerol-3-phosphate with three sequential acylation steps, with the last step being mediated by a diacylglycerol acyltransferase (DGAT), which employs an acyl-CoA as the acyl donor and transfers the acyl moiety to the sn-3 position of DAG for TAG assembly [13]. Previous studies in higher plants have revealed distinct roles of different types of DGAT in TAG synthesis in different organisms [16, 17]. Chlamydomonas is generally not considered as an oleaginous organism for lipid production and may differ from oleaginous algae in lipid metabolism, driving the research interest to industrially important algae such as Chlorella and Nannochloropsis [21, 22, 27, 28]
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