Cocktail biosynthesis of triacylglycerol by rational modulation of diacylglycerol acyltransferases in industrial oleaginous Aurantiochytrium.
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ABSTRACT: Background
Triacylglycerol (TAG) is an important storage lipid in organisms, depending on the degree of unsaturation of fatty acid molecules attached to glycerol; it is usually used as the feedstock for nutrition or biodiesel. However, the mechanism of assembly of saturated fatty acids (SFAs) or polyunsaturated fatty acids (PUFAs) into TAGs remains unclear for industrial oleaginous microorganism.Results
Diacylglycerol acyltransferase (DGAT) is a key enzyme for TAG synthesis. Hence, ex vivo (in yeast), and in vivo functions of four DGAT2s (DGAT2A, DGAT2B, DGAT2C, and DGAT2D) in industrial oleaginous thraustochytrid Aurantiochytrium sp. SD116 were analyzed. Results revealed that DGAT2C was mainly responsible for connecting PUFA to the sn-3 position of TAG molecules. However, DGAT2A and DGAT2D target SFA and/or MUFA.Conclusions
There are two specific TAG assembly routes in Aurantiochytrium. The "saturated fatty acid (SFA) TAG lane" primarily produces SFA-TAGs mainly mediated by DGAT2D whose function is complemented by DGAT2A. And, the "polyunsaturated fatty acid (PUFA) TAG lane" primarily produces PUFA-TAGs via DGAT2C. In this study, we demonstrated the functional distribution pattern of four DGAT2s in oleaginous thraustochytrid Aurantiochytrium, and provided a promising target to rationally design TAG molecular with the desired characteristics.
SUBMITTER: Lan C
PROVIDER: S-EPMC8714446 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Diacylglycerol acyltransferases (DGATs) play a key role in plant triacylglycerol (TAG) biosynthesis. Two type 1 and 2 DGATs from soybean were characterized for their functions in TAG biosynthesis and physiological roles. GmDGAT1A is highly expressed in seeds while GmDGAT2D is mainly expressed in flower tissues. They showed different expression patterns in response to biotic and abiotic stresses. GmDGAT2D was up-regulated by cold and heat stress and ABA signaling, and repressed by insect biting and jasmonate, whereas GmDGAT1A show fewer responses. Both GmDGAT1A and GmDGAT2D were localized to the endoplasmic reticulum and complemented the TAG deficiency of a yeast mutant H1246. GmDGAT2D-transgenic hairy roots synthesized more 18:2- or 18:1-TAG, whereas GmDGAT1A prefers to use 18:3-acyl CoA for TAG synthesis. Overexpression of both GmDGATs in Arabidopsis seeds enhanced the TAG production; GmDGAT2D promoted 18:2-TAG in wild-type but enhanced 18:1-TAG production in rod1 mutant seeds, with a decreased 18:3-TAG. However, GmDGAT1A enhanced 18:3-TAG and reduced 20:1-TAG contents. The different substrate preferences of two DGATs may confer diverse fatty acid profiles in soybean oils. While GmDGAT1A may play a role in usual seed TAG production and GmDGAT2D is also involved in usual TAG biosynthesis in other tissues in responses to environmental and hormonal cues.
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Project description: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.
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Project description:BackgroundThraustochytrids have gained attention as a potential source for the production of docosahexaenoic acid (DHA), where DHA is predominantly stored in the form of triacylglycerol (TAG). The TAG biosynthesis pathways, including the acyl-CoA-dependent Kennedy pathway and the acyl-CoA-independent pathway, have been predicted in thraustochytrids, while the specific details regarding their roles are currently uncertain.ResultsPhospholipid:diacylglycerol acyltransferase (PDAT) plays a key role in the acyl-CoA-independent pathway by transferring acyl-group from phospholipids (PL) to diacylglycerol (DAG) to from TAG. In thraustochytrid Aurantiochytrium sp. SD116, an active AuPDAT was confirmed by heterologous expression in a TAG-deficient yeast strain H1246. Analysis of AuPDAT function in vivo revealed that deletion of AuPDAT led to slow growth and a significant decrease in cell number, but improved PL content in the single cell during the cell growth and lipid accumulation phases. Interestingly, deletion of AuPDAT did not affect total lipid and TAG content, but both were significantly increased within a single cell. Moreover, overexpression of AuPDAT also resulted in a decrease in cell number, while the total lipid and cell diameter of a single cell were markedly increased. Altogether, both up-regulation and down-regulation of AuPDAT expression affected the cell number, which further associated with the total lipid and TAG content in a single cell.ConclusionsOur study demonstrates that AuPDAT-mediated pathway play a minor role in TAG synthesis, and that the function of AuPDAT may be involved in regulating PL homeostasis by converting PL to TAG in a controlled manner. These findings expand our understanding of lipid biosynthesis in Aurantiochytrium sp. and open new avenues for developing "customized cell factory" for lipid production.
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Project description:BackgroundPhotosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail.ResultsBased on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds.ConclusionsTaken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.
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