A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica

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.Results Nannochloropsis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0858-1) contains supplementary material, which is available to authorized users.

Plastidial and ER Triacylglycerol Biosynthesis in a Growth Phase-Dependent Manner in the Heterokont Nannochloropsis oceanica

“…In addition, DGAT, which catalyzes the final committed step of TAG biosynthesis, was remarkably increased during the late growth phase. Various previous studies have demonstrated the pivotal role of DGAT in elevating the TAG content of microalga, which is in accordance with our lipidomic data Klaitong et al, 2017;Wei et al, 2017).…”

Principles of plastid reductive evolution illuminated by nonphotosynthetic chrysophytes

“…4 and SI Appendix, Figs. S5, S13, and S16) and previous studies (5,(60)(61)(62)(63)(64). The threshold values were manipulated for each predictor to define optimal ones for the detection of experimental localizations, with the best trade-offs between sensitivity and specificity identified for the following: the default conditions for SignalP and ASAFind; modified mitochondria targeting thresholds of 0.7 and 0.35 for TargetP and MitoFates, respectively, as previously published (5,9); and a substantially lower chloroplast targeting threshold (0.0855, defined as the midpoint between the 90% sensitivity and 90% stringency; with the additional stipulation that the chloroplast targeting value be greater than the signal anchor targeting value) for HECTAR (SI Appendix, Fig.…”

Novel insights into salinity-induced lipogenesis and carotenogenesis in the oleaginous astaxanthin-producing alga Chromochloris zofingiensis: a multi-omics study

“…It has been reported in algae that membrane lipids undergo turnover to provide fatty acyls for TAG assembly under abiotic stress conditions such as nitrogen deficiency (ND) [15,[50][51][52][53][54]. Similarly, salt stress also caused a decrease in polar lipids, but predominantly in the chloroplastic membrane lipids (MGDG, DGDG, SQDG and PG) (Figs.…”

“…DGAT catalyzes the last committed step and has been demonstrated to play a critical role in TAG accumulation in various algal species [53,54,[60][61][62]. Recently, we showed by functional complementation in TAG-deficient yeast that seven out of the ten C. zofingiensis DGAT s were able to restore TAG synthesis, with DGAT1A being most functional followed by DGTT5 and DGAT1B [63].…”