Overexpression of an endogenous type 2 diacylglycerol acyltransferase in the marine diatom Phaeodactylum tricornutum enhances lipid production and omega-3 long-chain polyunsaturated fatty acid content

Abstract

Background: Oleaginous microalgae represent a valuable resource for the production of high-value molecules. Considering the importance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) for human health and nutrition the yields of high-value eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) require significant improvement to meet demand; however, the current cost of production remains high. A promising approach is to metabolically engineer strains with enhanced levels of triacylglycerols (TAGs) enriched in EPA and DHA.

Results: Recently, we have engineered the marine diatom Phaeodactylum tricornutum to accumulate enhanced levels of DHA in TAG. To further improve the incorporation of omega-3 LC-PUFAs in TAG, we focused our effort on the identification of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT) capable of improving lipid production and the incorporation of DHA in TAG. DGAT is a key enzyme in lipid synthesis. Following a diatom based in vivo screen of candidate DGATs, a native P. tricornutum DGAT2B was taken forward for detailed characterisation. Overexpression of the endogenous P. tricornutum DGAT2B was confirmed by qRT-PCR and the transgenic strain grew successfully in comparison to wildtype. PtDGAT2B has broad substrate specificity with preferences for C16 and LC-PUFAs acyl groups. Moreover, the overexpression of an endogenous DGAT2B resulted in higher lipid yields and enhanced levels of DHA in TAG. Furthermore, a combined overexpression of the endogenous DGAT2B and ectopic expression of a Δ5-elongase showed how iterative metabolic engineering can be used to increase DHA and TAG content, irrespective of nitrogen treatment.

Conclusion: This study provides further insight into lipid metabolism in P. tricornutum and suggests a metabolic engineering approach for the efficient production of EPA and DHA in microalgae.

Keywords: Acyl-CoA:diacylglycerol acyltransferase (DGAT); Docosahexaenoic acid; Eicosapentaenoic acid; Omega-3 fatty acids; Phaeodactylum tricornutum; Triacylgycerol.

Fig. 1

Overexpression of DGAT2 isoforms in P. tricornutum. a Fatty acid composition (Mol%) of independent transgenic lines overexpressing DGAT2 genes during S phase. Each measurement is the average of three technical replicas (± standard error). b Neutral lipid content of transgenic cells overexpressing different DGAT2 isoforms assessed by Nile Red fluorescence. Each data point represents an average of three biological replicas. c Quantitative RT-PCR analysis of the transcript levels of DGAT2 isoforms. Transgenic algal cultures overexpressing TpDGAT2, PtDGAT2A and PtDGAT2B were grown under continuous light for 3 days in standard F/2 medium. Relative expression levels were normalised to that of the housekeeping gene RPS

Fig. 2

Functional characterisation of transgenic P. tricornutum lines coexpressing DGAT2B and OtElo5. a Fatty acid composition of WT and DGATElo5 cells in the S phase of growth in F/2 media. Each measurement is the average of three biological replicas (± standard error). b Neutral lipid content of DGATElo transgenic clones assessed by Nile Red fluorescence (refer to “Methods” ***section for experimental description). Relative level of fluorescence in the transformants compared to WT cells. Values are the average of three replicated experiments. Error bars indicate standard error. Fatty acids which are more abundant in transgenic cells compared to WT are indicated by an asterisk (*)

Fig. 3

Cell growth and relative expression of transgenes in WT and transgenic P. tricornutum strains. a, b Cellular growth of WT and transgenic Pt_OtElo5, DGAT2B and DGATElo strains. Cells grown under N+ and N− conditions were harvested for lipid analysis where indicated. Values are the average of four experiments (± standard error). c, d The relative expression of OtElo5 and DGAT2B transcript levels analysed by qRT-PCR in transformants growing under N + and N− conditions. Algal cultures were grown under continuous light for 72 h in standard F/2 medium containing nitrogen (N +) or in N-deprived (N−) conditions. Relative transcript abundance was normalised to that of housekeeping gene RPS and calculated using the 2^−∆∆CT method. Values are the average of three replicated experiments. Error bars indicate standard error

Fig. 4

Fatty acid composition (Mol  %) of WT and transgenic (Pt_OtElo5, DGAT2B and DGATElo) P. tricornutum. Cells cultivated in N-replete (N + , dark bars) and N-deplete (N−, pale bars) medium at 24 (a), 48 (b) and 72 (c) hours. Each measurement is the average of minimum four technical replicates. Error bars indicate standard error

Fig. 5

PCA plots of targeted lipidomic analysis (ESI-MS/MS) of P. tricornutum strains. Variate biplots of the first two principal axes derived from a principal component analysis characterising the response of cellular lipids over time of P. tricornutum (WT, Pt_OtElo5, DGAT2B & DGATElo) lines grown in N-replete and N-deplete conditions of the log transformed data are shown. In both panels, points represent the treatment variates and are coloured in either by (a) treatment combinations (strain, time in hours and nitrogen treatment) or (b) lipid group (DGDG, MGDG, SQDG, PC, DGTA, LPC, PE, PI, PG, and TAG). The arrows represent the loadings associated with each principal vector

Fig. 6

Quantitative analysis of TAG accumulation in P. tricornutum cells over a 72 h time course. WT and transgenic (Pt_OtElo5, DGAT2B and DGATElo) P. tricornutum lines were grown in (a) N-replete (N+) or (b) N-deplete (N−) conditions. Total TAG content was determined by ESI-MS/MS analysis. Each measurement is the average of minimum four technical replicates. Error bars indicate standard error

Fig. 7

Compositional analysis of fatty acids and molecular species in TAG for P. tricornutum cells. Lipids were analysed after 72 h of cultivation in N-replete (+N) and N-deplete (−N) medium. a, b TAG fatty acid composition; c, d TAG molecular species composition. Each measurement represents the average of at least four technical replicates. Error bars indicate standard error. Asterisks indicate dominant TAG species, which are significantly different between strains and those which are significantly altered by N stress. Black asterisks indicate TAG species containing 16:0 and 16:1 fatty acids; green asterisks indicate TAG species containing C16–C18 fatty acids; and blue asterisks indicate TAG species containing 20:5, 22:5 and 22:6

Fig. 8

Targeted ESI-MS/MS analysis of Lyso-PC in WT and transgenic P. tricornutum. Lipids were analysed in WT, Pt_OtElo5, DGAT2B and DGATElo cells grown in N-replete (+N) and N-deplete (−N) medium at 72 h. Each measurement represents the average of at least four technical replicates. Error bars indicate standard error. FA species that are significantly different to WT, new species or increased under N stress are denoted by an asterisk (*)