Cloning, sequence, and phylogenetic analysis of HaeDGAT2E
A full-length cDNA sequences of HaeDGAT2E (GenBank MN073495) was obtained by the strategy of combining homologous cloning and rapid amplification of cDNA ends (RACEs) method. The HaeDGAT2E cDNA sequence was 1,193 base pairs (bp) in length, which contained a 1,017 bp open reading frame, a 133-bp 5′-untranslated region (UTR), and a 43-bp 3′-UTR with the characteristic of the poly (A) tail (Fig. 1a). The deduced protein had a calculated molecular mass of 38.83 kDa with an estimated isoelectric point of 9.7. The putative HaeDGAT2E protein shared 54.1% and 57.6% identities with DGAT2E from green algae Chromochloris zofingiensis (QB0559.1) and Chlamydomonas reinhardtii (XP_001694904.1), respectively. However, it shared low identity with DGAT2 from higher plants, such as Brassica napus (37.2%, XP_013737142.1), Gossypium mustelinum (32.2%, TYI54295.1), and Arabidopsis thaliana (31.9%, NP_566952.1). HaeDGAT2E possessed 3 strongly hydrophobic trans-membrane regions (Fig. 1b) and also had the LPLAT superfamily and DAGAT functional domain, which was consistent with DGAT2s from other species (Fig. 1c).
Conserved domain analysis showed that HaeDGAT2E contained 7 conserved motifs, including YFP block, PR block, PHG block, GGE block, RGFA block, VPFG block, and G block (Fig. 2). In the first block, only CzDGAT2D and CrDGAT2D had the complete Tyr-Phe-Pro (YFP) block like those of higher plants (AtDGAT2 and GmDGAT2), which suggested that these two proteins were plant-type DGAT2, while HaeDGAT2E from H. pluvialis was not. The first two amino acids (YF) were highly conserved among all DGAT2s examined, while the third residue was variable in microalgae. Interestingly, in the PHG block, the first two continuous residues Pro-His (PH) were obviously conserved among all DGAT2s examined, whereas the third residue was Gly (G) or Ser (S). It was worth mentioning that the Tyr-Ile-Phe (YIF) motif was conserved in this region and was replaced by Leu-Val-Met (LVM) in HaeDGAT2E. In the following PR block, HaeDGAT2E had a conserved PxxR motif as well as other DGAT2s examined. Similar with PR block, the core GGxxE motif in the GGE block was highly conserved. Basing on this block, CzDGAT2D and CrDGAT2D proteins belonged to plant-type DGAT2. The RGFA block and VPFG block were also conserved among all DGAT2s. In the last block, the alignment result showed that NoDGAT2B was special because it had no G block.
In order to further investigate the evolutionary relationship of HaeDGAT2E, phylogenetic analysis was performed using proteins of DGATs (DGAT1, DGAT2, DGAT3, and WS/DGAT) from different higher plants and microalgae (Fig. 3). Four groups including DGAT1, DGAT2, DGAT3, and WS/DGAT were clustered in this tree. As expected, HaDGAT2E was separated into DGAT2 subgroup with other DGAT2 from algae, and it was clearly separated with those from fungal and higher plants. In addition, HaeDGAT2E also had a close evolutionary relationship with CrDGAT2B, CrDGAT2C, and CzDGAT2E, which implied that they have the same origin and function.
Recovery the TAG synthesis in quadruple mutant yeast strain H1246 with HaeDGAT2E
To verify the function of the putative HaeDGAT2E enzyme, the ORF encoding sequences was heterologously expressed in the quadruple mutant yeast strain S. cerevisiae H1246 (∆dga1∆lro1∆are1∆are2) that lacks the activity of TAG synthesis. This yeast mutant strain contains knockout mutations in four TAG biosynthesis-related genes (dga1, lro1, are1, and are2) and is unable to synthesize TAG. The mutant type (MT) yeast can formed TAG when at least one of these four genes was expressed. Furthermore, WT (INVSc1) and H-EV (H1246 harboring empty vector pYES2.0) yeast strains were used as positive and negative controls, respectively.
As shown in Fig. 4, HaeDGAT2E was able to restore yeast TAG biosynthesis. There was a prominent TAG spot on the TLC plate from WT and the H-HaeDGAT2E strains respectively, but no TAG spot was detected in both H-EV and MT strains (Fig. 4a). Nile red can specifically stain the lipid molecule in cells, resulting in an orange fluorescence that can be used to quantify the lipid accumulation. As shown in Fig. 4b, the fluorescence in the cells of H-EV and MT strains was difficult to observe and exhibited a shaded orange. However, the lipid droplets were easier to observe, and they were large, clear, and bright in the MT and H-HaeDGAT2E strains. These results suggested that expression of HaeDAGT2 in the quadruple mutant strain H1246 can recover its ability to form neutral lipids through interaction with yeast lipids biosynthesis pathway and confirm that HaeDGAT2E indeed encoded a functional protein with DGAT activity.
Analysis of total lipids and fatty acid composition in H-HaeDGAT2E yeast strain
The changes of total lipids content and fatty acid composition were studied in different yeast strains. As shown in Fig. 5a, the total lipids content of MT strain still remained as low as that in H-EV strain, whereas the total lipids content in the yeast transformed with HaeDGAT2E significantly increased and was 404.5% higher than that of the H-EV or MT strain. However, the total lipids content of H-HaeDGAT2E strain is still lower (77.6%) than that of the WT yeast INVSc1.
To further test the change of fatty acid composition in different yeast strains, the TAG extracted from different cells was analyzed by GC-MS. Since MT and H-EV had almost the same fatty acid composition, we only choosed H-EV as the negative control for the subsequent analysis, and selected WT as the positive control (Fig. 5b). In addition, the C18:2 and C18:3 were fed to test the substrate specificity of HaeDGAT2E to polyunsaturated fatty acids. As shown in Fig. 5b, the C16:1 and C18:1 fatty acid was principle component which was 58%, 48%, and 45% in WT, MT, and H-EV strains respectively. However, in H-HaeDGAT2E strain, the principle component was C16:0 and C18:1 fatty acid and reached 55%. Moreover, compared to WT, MT, and H-EV strains, the polyunsaturated fatty acid (C18:2 and C18:3) content decreased.
Transient expression of HaeDGAT2E in Nicotiana benthamiana
To explore HaeDGAT2E as a tool to manipulate acyl-CoA pools and to engineer TAG in higher plants, HaeDGAT2E was over-expressed in the leaves of Nicotiana benthamiana by injecting the Agrobacterium (GV3101) strain harboring binary vector (pCAMBIA1303) with the targeted gene. RT-PCR results showed that the HaeDGAT2E transcript was expressed (6.7 fold) in transgenic lines (Fig. 6a). The total lipids content significantly increased and was 138.9% higher than that in WT (Fig. 6b). Transgenic N. benthamiana lines did not show any visible difference on the total starch and protein contents from wild-type plants (Fig. 6c and 6d). In addition, the overe-xpression of HaeDGAT2E resulted in increased C16:0 and C18:1 fatty acid content, which was consistent with results from yeast strain (Fig. 6e).