Haematococcus pluvialis FACHB-712 was obtained from Freshwater Algae Culture Collection at the Institute of Hydrobiology, CAS, China. Algal cells were cultivated in 100 ml of BBM medium in a 250-mL Erlenmeyer flask under normal growth conditions (23±1°C, light intensity of 25 μmolm⁻² s⁻¹, and a diurnal cycle of 12 h light/12 h dark). The algal cultures at the later exponentially growing stage (biomass ~200 mg L⁻¹) were used for high light with blue (HL-B), high light with white (HL-W), nitrogen deficient (ND), high light with blue coupled nitrogen deficient (HL-B-ND), and high light with white coupled nitrogen deficient (HL-W-ND) treatments, respectively, according to our previous descriptions (Cui et al., 2021).

Total RNA extraction, quantification, and the first-strand cDNA synthesis were conducted using EasySpin RNA Extraction Kit (Aidlab Biotech, China), NanoDrop 2000c (Thermo Scientific, United States), and PrimeScript^® RT Enzyme Mix I Kit (TaKaRa DRR047A, China), respectively. The local BLAST program based on the H. pluvialis transcriptome database together with RACEs methods were employed to obtain the full-length cDNA sequence of HpDGAT1. The putative HpDGAT1 and other annotated DGATs were listed in Supplementary Table S1. All primers were listed in Supplementary Table S2. The physico-chemical properties of HpDGAT1 protein were predicted in ExPASy (Gasteiger et al., 2003), including molecular weight (Mw), isoelectronic point (pI), sub-cellular localization, signal peptides (SP), chloroplast transfer peptides (CTP), trans-membrane regions (TM), and phosphorylation sites (Phos). HpDGAT1 and other DGATs from plants and microalgae were aligned using ClustalX (Thompson et al., 2002). Maximum likelihood tree was constructed using PhyML (Guindon et al., 2010). The transcription expression of HpDGAT1 was examined using qRT-PCR running on a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, United States) with SYBR Green PCR Master Mix (Invitrogen) Kit as described in our previous study (Cui et al., 2021).

For yeast (Saccharomyces cerevisiae) mutant H1246, four genes (DGA1, LRO1, ARE1, and ARE2) contributing to TAG synthesis was knocked out, and thus, such mutant is TAG-deficient. This H1246 was used to investigate the function of HpDGAT1. The ORF of HpDGAT1 was cloned into the yeast expression vector pYES2.0 (Invitrogen) to construct the recombinant pYES2.0-HpDGAT1 vector. After sequence confirmation, the recombinant vector was transformed into both wild-type S. cerevisiae INVSc1 and mutant H1246 with the S.c. EasyComp Transformation Kit (Invitrogen, United States). PCR was used to verify the presence of pYES2.0-HpDGAT1 vector in the yeast cells. The expression of HpDGAT1 gene in the yeast strain was induced by 2% (w/v) galactose SD/−ura medium, followed by qRT-PCR analysis on the transcription level of HpDGAT1. The supplementation of each FA (C18:2n6, C18:3n3, C18:3n6, and C20:4n6) was added to the culture to a final concentration of 100 μm, respectively, at the beginning of galactose induction as described by Siloto et al. (2009). Briefly, FAs were dissolved in ethanol at 0.5 M. FAs solutions were diluted in a warm medium containing 0.01% (v/v) Tergitol NP-40 (Sigma-Aldrich, St. Louis, MO, United States) immediately before yeast inoculation. Control experiments without FAs contained the same volume of ethanol and 0.01% (v/v) of Tergitol NP-40. Yeast samples at an OD600 of 2.5 were harvested, and lipids were extracted for TAG separation by TLC and FA composition analysis by GC. Briefly, 50 mg of yeast cells was used to extract total lipids (Bligh and Dyer, 1959). Then, TAGs were separated by thin-layer chromatography (TLC) methods as descripted in previous study (Liu et al., 2019). Finally, TAGs were trans-esterified with 5% H₂SO₄ in methanol at 85°C for 1 h and the fatty acid methyl esters (FAMEs) were analyzed by an Agilent GC equipped with a flame ionization detector (FID) and a capillary column (HP-88100 m × 0.25 mm × 0.2 mm) with an appropriate add amount of C17:0 FAME (Sigma) as an internal standard (Liu et al., 2019; Cui et al., 2021).

Yeast microsome was prepared as described by Siloto et al. (2009) and Liu et al. (2016). Briefly, induced yeast cultures were collected at 3,000 g for 5 min and washed twice with 1 ml of buffer containing 20 mm Tris–HCl pH 7.5, 10 mm MgCl₂, 1 mm EDTA, 5% (v/v) glycerol, 300 mm ammonium sulfate, and 2 mm dithiothreitol and lysed by a bead beater (Biospec, Bartlesville, United States) using 0.5 mm glass beads. Then, cell debris was removed from the suspension by centrifugation at 10,000 g for 10 min at 4°C, and the supernatant was recovered by centrifuging further at 100,000 g for 1 h at 4°C. The resulting microsomal membrane pellets were resuspended in microsomal storage buffer containing 50 mm Tris–HCl, pH 7.5, 10% (v/v) glycerol with a protein concentration of 10 μg μL⁻¹ for immediate use or storage at −80°C. The in vitro acyl-CoA substrate specificity of HpDGAT1 assay was performed in a 200 μl volume of mixture containing 40 μg microsomal membrane protein, 25 mm sucrose, 0.1 mm EDTA, 15 mm Tris–HCl pH 7.5, 125 μgml⁻¹ BSA (FA-free), 250 μm acyl CoA, and 250 μm DAG (eukaryotic type C16:0/C18:1n9 DAG and prokaryotic type C18:1n9/C16:0 DAG were used as the acyl acceptor) as described method by Liu et al. (2016). The DAGs were purchased from Larodan Fine Chemicals.¹ The other lipid standards were purchased from Avanti Polar Lipids.² The reactions were incubated at 30°C for 1 h, and the lipids were extracted and detected as described above. The microsome fraction alone and the microsome fraction with different DAGs were used as control. The assays were performed in three technical replicates.

For genetic engineering in microalgae, the ORF of HpDGAT1 was optimized for codon preference according to microalga C. reinhardtii CC849 and then cloned into the algal nuclear transformation expression vector pDB124 with the Pml I and Bmt I sites in the form of HpDGAT1-His fusion gene as described in our previous study (Cui et al., 2021). Briefly, after sequence confirmation, the resulting pDB124-HpDGAT1-His vector was linearized by Xba I digestion, and subsequently transformed into the C. reinhardtii CC849 strain via the glass beads method. Algal transformants were selected on Tris-acetate-phosphate (TAP) plates with 10 μgml⁻¹ bleomycin (Sigma-Aldrich). C. reinhardtii CC849 cells at the later exponentially growth stage (biomass ~420 mg L⁻¹) were treated for ND stress using methods described above. The integration of HpDGAT1 gene into the Chlamydomonas genome was verified by genomic PCR. Afterward, the transcription and protein expression levels of HpDGAT1 were verified by qRT-PCR and Western blotting using his-tagged antibodies, respectively, according to the method reported previously (Cui et al., 2021).

For genetic engineering in higher plants, the codon optimized ORF of HpDGAT1 was cloned into Nco I and Spe I sites of pCAMBIA1303 to yield pCAMBIA1303-HpDGAT1 vector under the expression cassette of CaMV 35S promoter and NOS terminator. This recombinant plant expression vector was transferred into Agrobacterium tumefaciens strain GV3101 by the freeze–thaw method (Holsters et al., 1978). After verification, the positive clones were cultured in liquid medium and then used to infect Arabidopsis thaliana by vacuum infiltration (Clough and Bent, 1998). T1 generation seeds were selected on hygromycin (50 mg L⁻¹), and then, the selected transformed plants were transferred to soil in pot for growing to maturation. T2 transgenic A. thaliana lines (At-HpDGAT1-4, At-HpDGAT1-7, and At-HpDGAT1-9) were used for seed and oil analyses. The stable integration of pCAMBIA1303-HpDGAT1 in A. thaliana genome and the transcription expression of HpDGAT1 were checked by genomic PCR and qRT-PCR, respectively.

AutoDock tool is a powerful method for identifying potential binding sites between 3D structure and ligand (Forli et al., 2016). The symmetrical half of the AST molecule (C20) was selected in docking process due to the main reasons that (1) the oversized C40 structure (compare to C16-C22 fatty acids); (2) AST esterification occurred on the hydroxyl group of the six-membered rings at both ends. In addition, FAs (C16:1 and C18:1n9) were used for AutoDock analysis as positive control because DGAT1 enzyme should have these FA-binding sites (Wang et al., 2020).

The HPLC method was applied to quantify the contents of different AST forms in H. pluvialis using the standard curve of AST (purchased from Sigma-Aldrich) at known concentrations (Cui et al., 2013). Total lipids extraction, TAG separation, and FA analysis from microalgae and higher plants were performed according to our previously described procedures (Liu et al., 2019; Cui et al., 2021). The Nile red staining was used to visualize the intracellular lipid bodies as an indicator of TAG formation according to previously described procedures (Siloto et al., 2009; Cui et al., 2021).

All experiments were biologically repeated three times to ensure reproducibility. The data were obtained as the mean value ± SD. Statistical analyses were performed using the SPSS statistical package (SPSS Inc., Chicago, IL, United States). Significant differences between treatments were statistically analyzed by paired-samples t-test. Statistical significance was achieved when p < 0.01.

Based on the predicted partial coding sequence of DGAT1 homolog (Supplementary Figure S1) from H. pluvialis transcriptome, a full-length cDNA fragment of HpDGAT1 (GenBank no. MT612720) was cloned. The 1,811bp HpDGAT1 in length contained a 1,560bp open reading frame (ORF) encoding 519 amino acids with MW of 58.7 kDa and pI of 9.8 (Supplementary Figure S2A). This protein was detected to have several conserved domains, such as chloroplast transduction peptide (CTP, 56 aa), TM helices (9 TMs), and phosphorylation sites (18) (Supplementary Figures S2B,E). All these analyses suggested that HpDGAT1 might be a membrane-bound DGAT1 enzyme localized in the chloroplast.

Sequence alignment of HpDGAT1 with other functional-known DGAT1s revealed 10 conserved motifs (CMs) in these proteins (Supplementary Figure S3). Four CMs were detected in the N-terminal of amino acids, including CM1 (the acyl-CoA binding), CM2 (putative thiolase acyl-enzyme intermediate signature), CM3, and CM4. Furthermore, a leucine zipper repeat motif with signature residues was also present in N-terminal of amino acids. However, HpDGAT1 lacked the N-terminal basic motif RRR present in higher plants or motif KRS substituted in other algae tested (Guihéneuf et al., 2011). The N-linked glycosylation sites (N-X-S/T) existed in DGAT1s from Tropaeolum majus, Vernicia fordii, and Nicotiana tabacum were absent in HpDGAT1, AtDGAT1, and BnDGAT1 (Caldo et al., 2017). The other six CMs (CM5-10) were discovered in the C-terminal of these DGAT1s, including CM6 involved in FA binding with a conserved tyrosine phosphorylation site, CM7 responsible for DAG-binding, and CM10, a putative C-terminal ER retrieval motif. In addition, CM5, CM8, and CM9 were found to locate near the active sites. All the above results indicate that HpDGAT1 is a member of DGAT1 family. The phylogenetic tree was constructed, which contained membrane-bound DGAT1s and DGAT2s, cytosolic DGAT3s, and dual-function WS/DGATs from algae and higher plants. HpDGAT1 was clustered into the DGAT1 family at bootstrap value of 100%, evidencing that the cloned cDNA encodes a typical DGAT1 protein (Supplementary Figure S4).

To further understand the possible role of HpDGAT1 in AST and TAG accumulation, several physiological-biochemical assays were employed to examine the transcriptional expression of HpDGAT1, and the accumulation patterns of total AST (T-AST), total TAG (T-TAG), and total FA (T-FA) in H. pluvialis under HL-B, HL-W, ND, HL-B-ND, and HL-W-ND stresses (Figure 1).

As shown in Figure 1A, the HL-B, HL-W, ND, HL-B-ND, and HL-W-ND stresses intensively inhibited the algal growth in different degrees. Free AST (F-AST) content increased on 1 d under HL-B-ND, HL-W-ND, HL-B, and HL-W conditions and then decreased along with the induction time (2–4 d; Figure 1C). M-EAST content continuously increased along with the induction time (1–4 d), with the peak levels of 33.8, 27.6.22.3, 23.8, and 22.3 mg g⁻¹ under stresses of HL-B-ND, HL-W-ND, ND, HL-B, and HL-W, respectively, on the final day (Figure 1D). This dynamic trend was also observed for D-EAST content despite the highest value was less than 3.0 mg g⁻¹ (Figure 1E). T-AST content had the similar trend as M-EAST because M-EAST was the main form of ASTs (Figure 1F). Total TAG (T-TAG) content slowly increased from day 1 to 4 and then reached its maximum value of 29.5, 28.7, 25.2, 26.8, and 24.8%, respectively, for HL-B-ND, HL-W-ND, HL-B, HL-W, and ND treatments on 4 d, which were 159.5, 155.1, 136.2, 144.9, and 134.1% higher than that in the control (Figure 1H). The trend of change in T-FA content was highly similar with Total TAG accumulation (Figure 1G). The simultaneous accumulation of TAG and AST under HL-B, HL-W, ND, HL-B-ND, and HL-W-ND stresses encourages us to further explore the correlation between them and to identify the enzyme responsible for both EAST and TAG biosynthesis in H. pluvialis. Upon exposure of H. pluvialis cells to HL-B-ND, HL-W-ND, ND, HL-B, and HL-W stresses, the HpDGAT1 mRNAs rapidly increased up to their maximum levels at 3 d exposure, with 3.1-, 2.5-, 1.7-, 2.1-, and 1.6-fold higher than that in the control, respectively (Figure 1B). The upregulation of HpDGAT1 expression was positively concomitant with the increase of T-AST, T-TAG, and T-FA contents under the tested stress conditions, indicating that HpDGAT1 might play an important role in both TAG and AST accumulation in H. pluvialis, particularly under stress conditions.

To identify the HpDGAT1 enzymatic activity, the HpDGAT1 coding sequence was heterologously overexpressed in the TAG-deficient yeast quadruple mutant strain H1246. The wild-type yeast strain INVSc1 and the mutant H1246 strain carrying the empty vector (H1246-EV) were used as positive and negative controls, respectively. A qRT-PCR analysis displayed that HpDGAT1 gene was effectively expressed in the transgenic yeast cells. TLC separation of total lipids (Figure 2A) and Nile Red staining (Figure 2B) of oil bodies both revealed that TAG were largely accumulated in the yeast cells overexpressing HpDGAT1. TAG content and FA composition were similar in both wild-type INVSc1 and INVSc1-EV cells (Figure 2C). Notably, compared to wild-type INVSc1, higher levels of TAG (26.6%) were obtained in the INVSc1 cells overexpressing HpDGAT1 due to the contribution of both endogenous yeast DGAT and exogenous algal HpDGAT1. However, TAG content (18.8%) in the HpDGAT1-transformed H1246 was lower than that in INVSc1 and INVSc1-EV (21.9%) although HpDGAT1 restored TAG synthesis in the transgenic H1246 cells. The possible reason may be that acyl-CoA substrates in yeast cells are not suitable for HpDGAT1. Fatty acid profiles in TAGs detected by GC showed that levels of monounsaturated fatty acyl-CoAs (MUFAs, C16:1 and C18:1) were much higher in both the transgenic H1246 and INVSc1 cells overexpressing HpDGAT1 than that in the wild-type INVSc1 and INVSc1- EV cells, indicating that HpDGAT1 may have higher substrate preference for MUFAs (C16:1 and C18:1) than saturated fatty acids (SFAs, C16:0 and C18:0) in yeast cells.

Normally, the host yeast cells just synthesized four major fatty acids including C16:0, C18:0, C16:1, and C18:1, but not the PUFAs, such as C18:2n6, C18:3n3, C18:3n6, and C20:4n6 enriched in H. pluvialis (Chen et al., 2015; Ma et al., 2018). In order to further identify substrate preference to those PUFAs synthesized in H. pluvialis, the HpDGAT1-transgenic yeast H1246 cells were cultured in the medium supplemented with each of those PUFAs, respectively. Interestingly, compared to yeast cells without feeding of any exogenous fatty acids, the HpDGAT1-transgenic yeast H1246 cells with PUFAs supplement accumulated high levels of TAGs with different degrees of enhancement for various PUFAs feeding. The increased level of TAGs was ordered as C18:3n3>C18:2n6>C20:4n6>C18:3n6 (Figure 2D). The above results indicated that HpDGAT1 could import these free PUFAs for sequestration into TAG. Additionally, PUFAs supplement also adjusted the FA profiles in TAGs. Much higher levels of PUFAs (C18:3n3 and C18:2n6) were incorporated into TAGs in the HpDGAT1-transgenic H1246 cells when fed with C18:3n3 or C18:2n6, respectively (Figure 2D), which were at the cost of lower levels of MUFAs (C16:1 and C18:1) and SFAs (C16:0 and C18:0). Conversely, the FA profiles in the HpDGAT1-transgenic H1246 cells were similar between exogenous C20:4n6 or C18:3n6 fed, respectively, and non-fed treatments (Figure 2D). Overall, these FA profiles demonstrated that HpDGAT1 had a stronger substrate preference for C18:3n3 and C18:2n6 than C20:4n6 and C18:3n6.

Alternatively, the import capacities for various PUFAs in yeast might be responsible for the different FA profiles. To test this possibility, two strategies were applied. Firstly, the yeast microsome was prepared and in vitro acyl-CoA substrate specificity for HpDGAT1 was analyzed. The prokaryotic DGA (C18:1n9/C16:0) and eukaryotic DAG (C16:0/C18:1n9) were used as an acyl acceptor for the in vitro assay. The PUFAs including C18:2n6, C18:3n3, C18:3n6, and C20:4n6 were examined. As shown in Figure 2E, C18:2n6 and C18:3n3 but not C18:3n6 and C20:4n6 were the preferred substrates for HpDGAT1 indicated by the obvious TAG spots. HpDGAT1 had the greatest activity toward C18:3n3, followed by C18:2n6, C18:3n6, and C20:4n6 when eukaryotic DAG (C16:0/C18:1n9) was used. The similar trend was also presented when prokaryotic DGA (C18:1n9/C16:0) was used. Again, these above results implied that HpDGAT1 had a stronger substrate preference for C18:3n3 and C18:2n6 than C20:4n6 and C18:3n6 in vitro. Secondly, the wild-type INVSc1 yeast cells were cultured in the medium supplemented with each of those PUFAs, respectively (Figure 2F). Higher levels of TAG were obtained in the INVSc1 cells when fed with C18:2n6 and C18:3n3 than no fed treatment. Meanwhile, the FA profiles in the INVSc1 cells were changed. Briefly, the contents of C18:3n3 and C18:2n6 were increased at the expensive of MUFAs (C16:1 and C18:1) while the SFAs (C16:0 and C18:0) contents remained constant. However, the lower levels of TAG were observed in the INVSc1 cells when fed with C18:3n6 and C20:4n6 than no fed treatment. The FA profiles in these feeding INVSc1 cells remained unchanged. It is important to note as well that the TAG accumulation was obviously restricted when the HpDGAT1-transgenic yeast H1246 cells were cultured in the medium supplemented with a mixture of four PUFAs. Overall, these FA profiles demonstrated that HpDGAT enzyme activity and import capacities for various PUFAs together determined the acyl-CoA substrate specificity for HpDGAT1 in yeast cells.

To explore engineering potential of HpDGAT1 to modulate TAG biosynthesis in microalgae, the HpDGAT1 gene with codon preference optimized (Supplementary Figure S5) was transformed into model algal C. reinhardtii CC849 using the nuclear transformation expression vector pDB124 under the control of the endogenous promoter and terminator (Figure 3A; Supplementary Figure S6). A number of transgenic C. reinhardtii lines were successfully obtained. After screening over 20 putative algal transformants and identification by genomic PCR, three HpDGAT1-transgenic algal lines (Cr-HpDGAT1-7, Cr-HpDGAT1-13, and Cr-HpDGAT1-18) were selected for further analysis (Figure 3B). The qRT-PCR analysis showed that heterologous HpDGAT1 gene was effectively expressed in these three transgenic algal lines (Figures 3C,D). Under ND conditions, the expression level of HpDGAT1 was approximately 5.5-fold higher in these three algal lines than that of the control, without detectably affecting algal growth (Figures 3C,D). In addition, the expression of HpDGAT1 protein was examined using His-tagged antibodies via Western blot analysis. As shown in Figure 3E, the target bands were present in the membrane protein fractions prepared from the three transgenic algal lines, but absent in the soluble protein samples, evidencing that HpDGAT1 is a trans-membrane enzyme (Figure 3E; Supplementary Figure S7).

It is not surprising that the TAG contents in the transgenic algal lines overexpressing HpDGAT1 were significantly increased by ~1.5-fold under ND conditions (Figure 3F), which may be partially due to the increased enzyme protein resulted from high transcription expression of HpDGAT1 (Figure 3D). In addition, the present data also indicated that the heterologous overexpression of HpDGAT1 also affected the FA profiles in TAGs (Figure 3F). The accumulation of MUFAs (C16:1 and C18:1) and PUFAs (C18:2n6 and C18:3n3) was significantly increased in the HpDGAT1-transgenic algal lines, accompanied by reduction of SFAs (C16:0 and C18:0) and PUFAs (C16:2, C18:3n6, and C18:4n3). There was no significant change in C16:4 and C16:3 contents between the transgenic algal lines and the controls. These results again indicated that HpDGAT1 had a stronger preference for MUFAs (C16:1 and C18:1) and PUFAs (C18:2n6 and C18:3n3) over SFAs (C16:0 and C18:0) and PUFAs (C16:2, C18:3n6, and C18:4n3), showing the application of HpDGAT1 in algal lipid engineering.

To explore engineering potential of HpDGAT1 to improve TAG yield and FA composition in higher plants, HpDGAT1 gene was heterologously expressed in A. thaliana. Three T2 transgenic lines (At-HpDGAT1-4, At-HpDGAT1-7, and At-HpDGAT1-9) were selected for further analysis. The 1,000-seed weight was largely enhanced compared to the controls (Figures 4A,B). This increase may be resulted from high activities of HpDGAT1 which pulled large amount of carbon flux into TAG biosynthesis in seed. The qRT-PCR results indicated that the HpDGAT1 transcripts were highly accumulated in the tested organs of the transgenic lines despite of different expression patterns (Figure 4C). Accordingly, the total TAG contents in seeds were increased from 25.23% in the control plants up to 30.13–32.64% in the HpDGAT1-overexpressed Arabidopsis lines (Figure 4D). Moreover, the heterologous overexpression of HpDGAT1 gene also resulted in alternation of FA profiles in TAGs in seeds of the transgenic Arabidopsis (Figure 4D), just like the cases in the yeast assay with feeding of exogenous FAs and HpDGAT1-overexpressed C. reinhardtii lines, respectively, described above (Figures 2D, 3F). Briefly, the abundances of C18:1, C18:2n6, and C18:3n3 were dramatically increased in the transgenic Arabidopsis seeds while C20:1, C20:2, and C22:1 maintained stable in both the transgenic lines and the control plants. For SFAs, C16:0 and C22:0 content decreased in the transgenic lines while C18:0 and C20:0 levels were not changed between the transgenic and control plants. These results revealed that the HpDGAT1-overexpression can increase both seeds weight and TAG enrichment in Arabidopsis, once again indicating that HpDGAT1 has a high substrate specificity for C18:1, C18:2n6, and C18:3n3. In summary, our data evidence that HpDGAT1 gene can be served as the target in genetic engineering to effectively improve oil yield and fatty acid composition in oilseeds or other higher plants.

Molecular docking (AutoDock analysis) was performed to explore the binding sites between AST structure and 3D structure of HpDGAT1. SWISS-MODEL server was employed to successfully generate the 3D structure for HpDGAT1 using crystal structure of human DGAT1 in complex with an oleoyl-CoA substrate as the template (Figure 5A; Wang et al., 2020). The symmetrical half of the AST molecule (C20, Figure 5B) got docked into the predicted 3D model of HpDGAT1 (Figure 5C) by van der waals force, conventional hydrogen bond, alkyl, Pi-alkyl, and Pi-sigma. The predicted binding amino acids sites were listed in Figure 5C, which were located in CM6-8 (Supplementary Figure S3). Interestingly, there were some organism-specific amino acid sequences and amino acid substitutions in the above CMs, which might play important role in AST esterification for HpDGAT1. In order to test the reliability of the AutoDock analysis, some other binding sites between fatty acyl-CoA (C16:1 and C18:1) and 3D structure of HpDGAT1 were also predicted. These binding sites should be existed in the DGAT1 enzyme (Wang et al., 2020). As expected, some fatty acyl-CoA-binding sites were predicted, which were listed in Figures 5D,E. These predicted sites were highly consistent with the reported CoA-binding signature in human DGAT1 (Wang et al., 2020). All these results provide another key clue for potentially functional role of HpDGAT1 in AST esterification despite further biological experiments, e.g., the expression of HpDGAT1 in AST-producing yeast, algal, and bacteria strains, are necessary in future to support this hypothesis.