Molecular cloning and bioinformatics analysis of HpDGAT2 genes
Based on the H. lacustris transcriptome database , five putative DGAT2 genes were predicted by the BLAST method using other DGAT2s from different algal species (Additional file 1: Table S1) as queries. The full-length mRNA sequences of the five genes were obtained by the rapid amplification of cDNA ends (RACEs) method, and the initiation codon, termination codon, 5′-untranslated region (5′-UTR), 3′-untranslated region (3′-UTR), and poly (A) characteristic tail were determined. Five putative DGAT2 genes were designed, HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E, by multiple sequence alignment with CrDGAT2s, four of which, HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E, contained a full-length open reading frame (ORF), while HpDGAT2C was a partial sequence (Additional file 2: Table S2 and Additional file 3: Table S3). Then, the full-length ORFs were cloned and sequenced by PCR with primers (Additional file 4: Table S4), which were renamed and deposited in NCBI GenBank (HpDGAT2A: MT875161; HpDGAT2B: MT875162; HpDGAT2C: MT875163; HpDGAT2D: MT875164; HpDGAT2E: MT875165). To date, this is the highest dose of DGAT2s reported in the green alga H. lacustris. Based on a comparison with gene models of HpDGAT2s reported by Nguyen et al. , our results confirmed that there were five HpDGAT2s members in H. lacustris. Generally, only one or two alleles of DGAT1s are identified in a number of microalgae, whereas multiple alleles of DGAT2s are typically present .
To gain insights into the biochemical characteristics of HpDGAT2s, the molecular weight (MW), isoelectric point (pI), subcellular location, transmembrane domain (TM), signal peptide (SP), chloroplast transfer peptide (CTP), and phosphorylation site (Phos) were analyzed. No SP or CTP was present in HpDGAT2s protein sequences except for CTP in HpDGAT2C (Additional file 2: Table S2). There were two TMs in all pDGAT2s protein sequences except for three TMs in HpDGAT2B (Additional file 2: Table S2 and Additional file 5: Figure S1), which is consistent with the membrane bound forms of DGAT1 and DGAT2 . In addition, 14-30 phosphorylation sites were predicted in HpDGAT2 protein sequences (Additional file 2: Table S2 and Additional file 6: Figure S2), indicating that phosphorylation plays important roles in DGAT2 enzyme activity because DGAT1 enzyme activity is affected by serine phosphorylation sites in mouse DGAT1 , TmDGAT1 , and BnDGAT1 . It remains to be determined whether these phosphorylation sites are important for the functional regulation of HpDGAT2 in vivo.
To further analyze the conserved domains (CDs) and evolutionary relationship between HpDGAT2s and other algal DGAT2s, multiple sequence alignment and a phylogenetic tree were reconstructed. CDs analysis showed that HaeDGAT2s contained 7 CDs [26, 47, 48], including YF/YFP block (CD1), which is essential for DGAT2 activity; HPHG/EPHS block (CD2), which is proposed to partially consist of the active site; PxxR (x=random amino acid) block (CD3); xGGxAE block (CD4); RxGFx(K/R)xAxxxGxx(L/V)VPxxxFG block (CD5), which is the longest conserved sequence in plants and animals; PxxxVVGxPIxVP block (CD6); and RHK block (CD7) (Additional file 7: Figure S3). As shown in Additional file 7: Figure S3, there were two completely conserved amino acid residues (proline, P and phenylalanine, F) among all DGAT2s, which is consistent with previous reports indicating that these two highly conserved residues may be located at the active sites of the enzymes and make significant contributions to their enzymatic activities . The phylogenetic analysis of the HpDGAT2s and other DGAT orthologues from eukaryotic algae and plants is illustrated in Additional file 8: Figure S4, which is consistent with most previous results [20-26]. Briefly, all HpDGAT2s clustered with the algal DGAT2s orthologues, which are distinct from other DGAT subfamilies, including DGAT1, DGAT3, and DGAT/WSD. Of the five HpDGAT2s, HpDGAT2A formed a monophyletic subgroup (BS: 100%) with CrDGAT2A, CzDGAT2A, CzDGAT2B, LiDGAT2A, and LiDGAT2B. HpDGAT2B and HpDGAT2E were highly close (BS: 98%) to CrDGAT2B, CzDGAT2E and CrDGAT2C. HpDGAT2C was evolutionarily close (BS: 100%) to CzDGAT2C and LiDGAT2C. HpDGAT2D built a monophyletic subgroup (BS: 73%) with CrDGAT2D and CzDGAT2D.
AST and TAG accumulation and HpDGAT2s gene transcription upon exposure to high light and nitrogen deficient stresses
High light (HL) and nitrogen deficient (nitrogen-free, ND) stresses can effectively promote the accumulation of AST and TAG in H. lacustris [32-34, 50-53]. However, under such circumstances, the growth of algae was completely restricted [51-53]. Recently, our team completed research investigating the effects of nitrogen deficiency (nitrogen content compared to growth in control BBM medium, e.g., 0, 1/4 N, 1/2 N, and 3/4 N) on algal growth and AST and TAG accumulation. The results indicated that the highest AST productivity was achieved under 1/4 N stress due to a certain level of algal growth. Therefore, in the current manuscript, the 1/4N condition was selected as the nitrogen deficient stress for further experiments. To understand the relationship between HpDGAT2s transcription and TAG and AST biosynthesis, time-course patterns of algal biomass, expression, total AST (T-AST), and total TAG (T-TAG) contents in photoautotrophic cultures of H. lacustris under HL, 1/4N, and double HL-1/4N stresses were studied (Fig. 1).
As shown in Fig. 1a, compared to the control, HL, 1/4N, and double HL-1/4N stresses inhibited algal growth. The T-AST production and composition are summarized in Fig. 1b-1e. From these results, we could draw the conclusions that (1) M-AST is the main form; (2) compared to 1/4N stress, HL is more effective at inducing AST accumulation, especially under high blue light (HLB) conditions; and (3) coupled HL and 1/4N dual stimulation might be a better choice for improving AST accumulation. Moreover, T-TAG contents slowly increased from day 1 to day 4 and reached maximum values of 29.5%, 28.7%, 26.8%, 25.2%, and 24.8% under HLB-1/4N, HLW-1/4N, HLB, 1/4N, and HLW conditions, respectively, which were 159.5%, 155.1%, 144.9%, 136.2%, and 134.1% higher than the values of the control (Fig. 1f). The effects of HL, 1/4N and double HL-1/4N stresses on TAG and AST accumulation were largely consistent with previous studies showing that AST and lipid biosynthesis were enhanced and that the former was coordinated with later biosynthesis under HL and ND conditions [34, 41]. Previous studies have indicated that DGAT enzymes are probably responsible for both AST esterification and TAG biosynthesis in H. lacustris [33, 34]. As revealed by qRT-PCR results (Fig. 2), the HpDGAT2 gene transcription expression levels exhibited distinct patterns under HL, 1/4N and double HL-1/4N stresses. Of the five HpDGAT2s, the HpDGAT2B and HpDGAT2C expression levels decreased and remained constant (Fig. 2b and 2c). The HpDGAT2A and HpDGAT2E expression levels increased and reached their maximum at 4 d of exposure, and they were HL and 1/4N stress-dependent (Fig. 2a and 2e), respectively, while the HpDGAT2D expression level increased and was stress dependent (Fig. 2d). These results suggested that these HpDGAT2A, HpDGAT2D, and HpDGAT2E genes were together involved in AST and TAG biosynthesis under stress.
Functional complementation of HpDGAT2s in yeast
To verify the function of the putative HpDGAT2s enzymes, the ORF-encoding sequences were cloned (Additional file 4: Table S4) into the pYES2.0 plasmid and heterologously expressed in the quadruple mutant yeast strain S. cerevisiae H1246 (∆dga1∆lro1∆are1∆are2), which lacks TAG synthesis activity. Mutant type (H1246) yeast can form TAG when at least one of these four genes is expressed. Furthermore, wild-type (INVSc1) and H1246-EV (H1246 harbouring empty vector pYES2.0) yeast strains were used as positive and negative controls, respectively.
The expression of HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E restored TAG biosynthesis at different levels in H1246 cells, as indicated by the remarkable TAG spot on a TLC plate (Fig. 3a). In contrast, HpDGAT2B expression in H1246 cells produced inconspicuous TAG levels, indicating a nonfunctional encoded protein considering the low transcription expression levels in H1246 cells (Fig. 3b) and H. lacustris cells (Fig. 2b). Nevertheless, the limited FA composition in Saccharomyces cerevisiae might lead to low TAG content for HpDGAT2B. The ability of HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E to restore TAG biosynthesis in yeast led us to examine FA substrate specificity. As indicated in Fig. 3b and 3c, the HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E genes were heterologously expressed in H1246 and INVSc1 cells. The changes in TAG content and FA composition of TAGs extracted from the transformed H1246 and INVSc1 cells were similar. As shown in Fig. 3d, the TAG contents of expressed HpDGAT2A and HpDGAT2B in H1246 cells were 78.3% and 56.5% lower, respectively, than those of the control (INVSc1 and INVSc1+EV). The TAG contents of expressed HpDGAT2D and HpDGAT2E were 108.7% and 122.7% higher, respectively, than the control. To further test FA substrate specificity, FAs from transformed H1246 and INVSc1 cells were analyzed by GC. As shown in Fig. 3d, compared to the control, the MUFAs palmitoleic acid (C16:1) and oleic acid (C18:1) abundances increased in HpDGAT2A-, HpDGAT2D-, and HpDGAT2E-expressing H1246 cells at the expense of saturated fatty acids (SFAs), including palmitic acid (C16:0) and stearic acid (C18:0). Such a tendency, however, at different levels was observed for almost all transformed lines of H1246 for various DGAT enzymes [20, 23-28].
Considering the limited FA composition in yeast strains (C16:0, C18:0, C16:1, and C18:1), some PUFAs enriched in H. lacustris, including linoleic acid (C18:2n6), α-linolenic acid (C18:3n3), γ-linolenic acid (C18:3n6), and parinaric acid (C18:4n3), were tested for substrate specificity for the HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E enzymes by employing a feeding strategy. HpDGAT2A, HpDGAT2D, and HpDGAT2E had similar tendencies to incorporate these PUFAs into TAG at the expense of C16:1 and C18:1 with the following patterns: C18:2n6 > C18:3n3 > C18:3n6 > C18:4n3 (Fig. 3e). Considering that C18:2n6 and C18:3n3 were rich in H. lacustris, it is reasonable to speculate that these HpDGAT2s may have potential in C18:2n6- and C18:3n3-enriched TAG production [32-34]. The HpDGAT2A, HpDGAT2D, and HpDGAT2E enzymes showed a stronger preference for PUFAs than MUFAs due to the higher feeding content of PUFAs than endogenous MUFAs content. This phenomenon was also confirmed by Zienkiewicz et al. (2018), who incorporated some PUFAs into TAG at the expense of 16:1 and 18:1 in LiDGAT1-, LiDGAT2.1-, LiDGAT2.2-, and LiDGAT2.3-expressing yeast  and in CzDGAT2C-expressing mutant H1246 yeast cells  by feeding tests. However, FA profiles of the TAG fraction from yeast cells expressing HpDGAT2B showed no obvious changes, implying a nonfunctional protein (Fig. 3e).
HpDGAT2D heterologous expression promotes TAG biosynthesis and its relative MUFAs and PUFAs abundance in C. reinhardtii
To investigate the possible biological role of HpDGAT2s and their engineering potential to modulate TAG biosynthesis in algae, we generated HpDGAT2D heterologous expression lines in the evolutionarily close green alga C. reinhardtii CC849. HpDGAT2D was selected for further experiments due to the relatively strong TAG biosynthetic activity in yeast cells (Fig. 3) and high transcription expression level in H. lacustris under stress conditions (Fig. 2d).
The nuclear transformation expression vector pDB124 (Additional file 9: Figure S5), characterized in C. reinhardtii CC849 and gifted by professor Zhangli Hu from Shenzhen University, was used in this study after modification because it contained overexpression cassettes of the HpDGAT2D-His fusion and bleomycin resistance Ble genes under the control of the verified endogenous promoter and terminator of the PsaD and RBCS2 genes, respectively (Fig. 4a). The codon preference (HpDGAT2D) was optimized according to the alga C. reinhardtii (Additional file 10: Figure S6) before constructing the expression vector. Transformants (screening over 20 putative transformants) were selected on TAP plates supplemented with bleomycin and confirmed by genomic PCR. The exogenous HpDGAT2D-His fusion gene was integrated into the alga chromosome due to the clear band using the HpDGAT2D-Cr gene as primers in transformation lines, whereas no signal was detected in WT cells (Fig. 4b and Additional file 11: Figure S7a). Three heterologous expression lines, HpDGAT2D-4, HpDGAT2D-7, and HpDGAT2D-9, exhibited a maximum increase in transcription levels (by ~ 5.5-fold higher than the control) under ND conditions in a 4-day batch culture, with no significant difference in cell growth between the transgenic lines and the control (Fig. 4c and 4d). Furthermore, in vivo heterologous expression of the HpDGAT2D protein was validated by using His-tagged antibodies via western blot analysis. Bands were present in the membrane proteins of three heterologous expression lines (HpDGAT2D-4, HpDGAT2D-7, and HpDGAT2D-9), but were absent from the soluble proteins, which was consistent with HpDGAT2D being a transmembrane enzyme (Fig. 4e and Additional file 11: Figure S7b). HpDGAT2D heterologous expression led to considerable increases (by ~ 1.4-fold) in TAG content under ND conditions (Fig. 4f). HpDGAT2D heterologous expression also affected the FA profiles in TAGs (Fig. 4f). A significant increase was observed in the relative abundance of MUFAs (C16:1 and C18:1) and PUFAs (C18:2n6 and C18:3n3), accompanied by a significant decrease in SFAs (C16:0 and C18:0) and some PUFAs (C16:2, C16:3, C18:3n6, and C18:4n3). These results indicated that (1) HpDGAT2D showed a stronger preference for MUFAs and PUFAs than SFAs; (2) of all PUFAs, HpDGAT2D chose C18:2n6 and C18:3n3 as the first option rather than C16:2, C16:3, C18:3n6, and C18:4n3; and (3) these preferred substrates were enriched in C. reinhardtii. This trend was consistent with results from yeast cells obtained by feeding test (Fig. 3d and 3e) and previous studies of NoDGAT1A expression in C. reinhardtii UVM4 and CzDGAT1A expression in oleaginous alga N. oceanica by Wei et al. (2017) and Mao et al. (2019), respectively [20, 22].
HpDGAT2D heterologous expression enhances seed oil content and its relative MUFAs and PUFAs abundance in A. thaliana
To explore HpDGAT2s as a tool to manipulate acyl-CoA pools and to engineer TAG biosynthesis in higher plants, HpDGAT2D was heterologously expressed in Arabidopsis thaliana. Three A. thaliana independent expression T2 generation lines (At-HpDGAT2D-3, At-HpDGAT2D-6, and At-HpDGAT2D-8) were selected for further detailed analysis. There were no visible morphological difference (e.g., 1000-seed weight) between the transgenic lines and untransformed control A. thaliana (Fig. 5a). The qRT-PCR results showed that the HpDGAT2D transcript was expressed in transgenic lines in different tissue organs, including roots, tubers, leaves, siliques, and seeds, to different extents (Fig. 5b). The transformation of wild-type A. thaliana with HpDGAT2D resulted in higher (120.0-126.4%) seed TAG content than the control (Fig. 5c). Again, further GC analysis of FA profiles from TAGs revealed that PUFAs and MUFAs significantly increased, accompanied by a significant decrease in SFAs (Fig. 5c). However, the exact alteration process was much more complicated than those in yeast and C. reinhardtii cells. Specifically, of the SFAs, C16:0 and C22:0 decreased while C18:0 and C20:0 remained stable. Of MUFAs and PUFAs, HpDGAT2D preferred C18:1, C18:2n6, and C18:3n3 rather than C20:1, C20:2 and C22:1 in TAG biosynthesis. These results were largely in agreement with those from yeast cells (Figs. 3d and 3e) and C. reinhardtii cells (Fig. 4c). Guo et al. (2017) indicated that the CeDGAT1 gene can stimulate FA biosynthesis and enhance seed weight and oil content when expressed in A. thaliana and B. napus .