Phaeodactylum tricornutum is a well-studied oleaginous model diatom with high triacylglycerol (TAG) and omega3 eicosapentaenoic acid (EPA) contents, however, only a small portion of EPA is transferred from the polar lipids to TAG through phospholipid:diacylglycerol acyltransferase (PDAT). In this study, a Nannochloropsis oceanica PDAT (NoPDAT) was heterologously expressed in P. tricornutum to increase the EPA content in TAG. NoPDAT heterologous expression slightly disturbed the growth of the transgenic lines, but enhanced the TAG content by 16 - 18 %. Considering the localization of NoPDAT in P. tricornutum after heterologous expression together with our lipidomic results, NoPDAT is more likely to utilize phosphatidylcholine and phosphatidylethanolamine on the chloroplast endoplasmic reticulum membrane as the acyl donors. Especially, NoPDAT transferred the EPA acyl moiety from the sn -2 position of the two above-mentioned phospholipids to the sn -3 position of TAG, resulting in the increase of two major EPA-containing TAG molecules (16:1/16:0/20:5 and 16:1/16:1/20:5). Correspondingly, two major 16:0-containing TAG molecules (16,0/16:1/16:1 and 16:0/16:0/16:1) decreased in the transgenic strains. In conclusion, our attempt to express NoPDAT in P. tricornutum increases the EPA content in TAG by approximately one-fold, and demonstrates the versatility of diatom lipid metabolism and acyl editing.

Lysophosphatidic acid acyltransferases (LPAATs) catalyze the formation of phosphatidic acid (PA), a central metabolite in both prokaryotic and eukaryotic organisms for glycerolipid biosynthesis. Phaeodactylum tricornutum contains at least two plastid-localized LPAATs (ptATS2a and ptATS2b), but their roles in lipid synthesis remain unknown. Both ptATS2a and ptATS2b could complement the high temperature sensitivity of the bacterial plsC mutant deficient in LPAAT. In vitro enzyme assays showed that they prefer lysophosphatidic acid over other lysophospholipids. ptATS2a is localized in the plastid inner envelope membrane and CRISPR/Cas9-generated ptATS2a mutants showed compromised cell growth, significantly changed plastid and extra-plastidial membrane lipids at nitrogen-replete condition and reduced triacylglycerols (TAGs) under nitrogen-depleted condition. ptATS2b is localized in thylakoid membranes and its knockout led to reduced growth rate and TAG content but slightly altered molecular composition of membrane lipids. The changes in glycerolipid profiles are consistent with the role of both LPAATs in the sn-2 acylation of sn-1-acyl-glycerol-3-phosphate substrates harboring 20:5 at the sn-1 position. Our findings suggest that both LPAATs are important for membrane lipids and TAG biosynthesis in P. tricornutum and further highlight that 20:5-Lyso-PA is likely involved in the massive import of 20:5 back to the plastid to feed plastid glycerolipid syntheses.

Dietary supplementation of omega-3 very long-chain polyunsaturated fatty acids (VLC-PUFAs), particularly eicosapentaenoic acid (EPA, C20 : 5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, C22 : 6Δ4,7,10,13,16,19), is well known to have diverse health benefits. In addition to their major source of marine fish, metabolic engineering of oilseed crops could provide a sustainable alternative source for these pharmaceutically important FAs. The reconstruction of VLC-PUFAs biosynthetic pathway in plants involves the assembly of multiple desaturases and elongases from different organisms. However, when phospholipid-linked desaturases and acyl-CoA-dependent elongases were co-expressed, the so-called substrate dichotomy was predicted to generate a metabolic bottleneck (i.e. low efficiency in the channelling of lipid-linked desaturation intermediates into the acyl-CoA pool) for the accumulation of high levels of VLC-PUFAs in transgenic plants (Abbadi et al., 2004). Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, E.C. 2.3.1.23) has previously been thought to be the possible candidate enzyme regulating this bottleneck. The diatom Phaeodactylum tricornutum produces EPA up to ~30% of total fatty acids. Eicosapentaenoic acid biosynthesis proceeds predominantly via the so-called Δ6-pathway in the endoplasmic reticulum (ER), which prompts us to identify LPCAT from this microalga. Recently, the biochemical properties of one P. tricornutum LPCAT (PtLPCAT1) were reported and it can esterify a 18 : 3-, 18 : 4- or 20 : 4-CoA (n-3) moiety to lysoPC in vitro, the later acyl being available for further desaturation to synthesize EPA, contrasting with plant LPCATs (Połońska et al., 2021). However, its role in lipid synthesis and the potential to enhance VLC-PUFAs production remain unknown. Here, we found that PtLPCAT1 fused with green fluorescence protein was localized at the chloroplast ER membrane (Figure 1a). To disrupt PtLPCAT1, we constructed a multiplexed CRISPR/Cas9 plasmid (Figure 1b) and transformed into P. tricornutum. We obtained at least 3 independent mutant lines harbouring mutations at the intended target site based on PCR and DNA sequencing (Figure 1c,d). To analyse lipid-associated phenotypes, P. tricornutum wild-type (WT) and the PtLPCAT1 knockout mutants were grown in nutrient-replete and phosphorus-deprived conditions. During 10 days of growth, cell density and neutral lipid content in PtLPCAT1 were significantly decreased compared with WT (Figure 1e,f). The mutants also accumulated less triacylglycerols (TAGs) than WT (Figure 1g). Although there was no difference in cell densities between WT and PtLPCAT1 grown in phosphorus-deprived condition for 7 days, the mutants showed a significant decrease in Nile red fluorescence and TAG accumulation (Figure 1h–j), which suggesting that disruption of PtLPCAT1 led to compromised cell growth and impaired TAG synthesis. Additionally, EPA was significantly decreased in PtLPCAT1 at Day 9 (P < 0.05), and the levels of C16 : 0 and C16 : 1 were reduced in PtLPCAT1 at both Day 6 and Day 9 (Figure 1k). Glycerolipidomic analysis revealed that inactivation of PtLPCAT1 caused a significant decrease in the levels of TAGs (30%, P < 0.001) and monogalactosyldiacylglycerol (MGDG, 20%, P < 0.05) at 9 day, and an increase in the amounts of phosphatidylcholine (PC) (50%, P < 0.01) and phosphatidylethanolamine (PE) (50%, P < 0.01) compared with WT (Figure 1l). Quantitative analysis of molecular species in each lipid class revealed that molecular composition of the major chloroplast galactoglycerolipids was influenced in PtLPCAT1. The most abundant 20 : 5/16 : 3 species in MGDG was decreased from 30% in WT to 15% in the mutants at 6 d (P < 0.0001). In addition, almost all other molecular species harbouring a 20 : 5 at the sn-1 position also showed a significant decrease in the mutants correlated with a dramatic increase in the 16 : 0/16 : 0 and 16 : 1/16 : 0 species (P < 0.0001; Figure 1m). Similarly, a significant decrease in C16-FA was observed in 20 : 5-containing molecular species of digalactosyldiacylglycerol (DGDG) in the PtLPCAT1 mutants (Figure 1n). For extra-plastidic glycerolipids, disruption of PtLPCAT1 caused a decrease in the levels of 18 : 2/18 : 1 and 18 : 2/18 : 2 in PC and 20 : 5/20 : 5 in diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) (Table S1). Both 18 : 3 and 18 : 4 accumulated in PC and DGTA, suggesting that PtLPCAT1 was not involved in their transfer and was more specific for 20 : 4 in vivo. Phosphatidylcholine and DGTA are the putative extra-plastidic platforms for FA desaturation, and their turnover may provide substrates for chloroplast galactolipid synthesis. In PE, the PtLPCAT1 mutants also showed a significant decrease in the level of 20 : 5/20 : 5 in the exponential phase when compared to WT. The changes in molecular composition of PC, DGTA and chloroplast galactolipids are consistent with a role of PtLPCAT1 in acyl editing of PC and possibly DGTA. The most significant alterations of TAGs were observed in the reduction of C20 : 5-containing species, balanced by an increase in C16-containing species, at both Day 6 and Day 9 (Figure S1, Table S1). These changes demonstrate that disruption of PtLPCAT1 caused a decrease in the C20 : 5-containing molecular species in galactoglycerolipids and TAGs. To evaluate the potential of PtLPCAT1 to enhance VLC-PUFA production, we reconstructed EPA biosynthetic pathway in the nonconventional oleaginous yeast Yarrowia lipolytica, which is an attractive host for the production of oils and FA-derived compounds. We first constructed an EPA-producing starting strain (YALI_P0) through integration of multiple gene expression cassettes into the PO1f strain. The FA profile of Y. lipolytica PO1f strain revealed that it can synthesize linoleic acid (C18 : 2 n-6) and oleic acid (C18 : 1 n-9) as the major FAs. To produce EPA from linoleic acid through an alternative pathway relying on the initiating Δ9-elongation step in the acyl-CoA pool followed by phospholipid-linked desaturations, we introduced a Δ9-elongase, Δ8-desaturase, Δ5-desaturase from Euglena gracilis and a Δ17-desaturase from Pythium aphanidermatum, all of which were codon-optimized for expression in Y. lipolytica. Insertion of these DNA fragments into the Y. lipolytica genome occurred by homologous recombination, and this led to the accumulation of EPA up to 7% of total FAs in the YALI_P0 strain. We then used pCfB-PtLPCAT1 and pCfB-2 × PtLPCAT1, which harbour one copy and two copies of a PtLPCAT1 gene, respectively, to transform strain YALI_P0. The resulting strains YALI_P1 and YALI_P2 produced EPA at 12.06% and 18.23% of total FAs (Figure 1o), respectively, which indicated that overexpression of PtLPCAT1 could significantly enhance EPA production in recombinant Y. lipolytica and the final percentage of EPA in total FAs was associated with the copy number of PtLPCAT1 gene. Moreover, compared with YALI_P0 strain, we observed a marked reduction in the amount of C20-FA intermediates such as Δ9-elongation products (C20 : 2Δ11,14 and C20 : 3Δ11,14,17) in PtLPCAT1-overexpressed strains YALI_P1 and YALI_P2, indicating that introduction of additional PtLPCAT1 activity may accelerate, at least in part, the exchange of Δ9-elongation product (C20 : 3Δ11,14,17) between phospholipids and the acyl-CoA pool and thereby push the intermediates towards the synthesis of the end product, EPA. In summary, our work reveals the important role of PtLPCAT1 in lipid synthesis in P. tricornutum and demonstrates the enhanced EPA accumulation in the yeast Y. lipolytica through overexpression of PtLPCAT1, which may also have the potential to enhance the accumulation of VLC-PUFAs in transgenic oilseed plants. This work was supported by the grant from the National Natural Science Foundation of China (31961133008 to YG), the National Science Center (NCN, Poland; No. UMO-2018/30/Q/NZ3/00497 to AB) and the Agence Nationale de la Recherche (ANR-10-LABEX-04 GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology; ANR-11-BTBR-0008 Océanomics; IDEX UGA CDP [email protected]; Institut Carnot 3BCAR). A related patent had been submitted to the State Intellectual Property Office of China. L.Y., X.H. and D.Z. performed the experiments. J.J., E.M., A.A. and A.B. analysed the data. Y.G. designed the study and wrote the manuscript. Figure S1 Profiling of molecular species of TAGs. Table S1 Lipidomic dataset. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown. In P. tricornutum, a family of five genes (ptACSL1-ptACSL5) encodes LACS activities. We generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned "Phatr3_J20460" gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).

Low biomass and lipid productivities of microalgae are main reasons for the difficulty in commercial production of microalgal oils. Newly established gene editing techniques, TALEN and CRISPR/Cas9, have great potential in analysis of key genes in lipid biosynthesis pathway and further genetic modification ofoil-producing algal strains. This review not only introduced TALEN and CRISPR/Cas9 techniques and their application in microalgal research, but also elaborated their importance in gene function study of oleaginous microalgae and construction of industrial strains.

In photosynthetic oleaginous microalgae, acyl-CoA molecules are used as substrates for the biosynthesis of membrane glycerolipids, triacylglycerol (TAG) and other acylated molecules. Acyl-CoA can also be directed to beta-oxidative catabolism. They can be utilized by a number of lipid metabolic enzymes including endogenous thioesterases, which catalyze their hydrolysis to release free fatty acids. Acyl-CoA availability thus plays fundamental roles in determining the quantity and composition of membrane lipids and storage lipids.