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Summary: 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. [ABSTRACT FROM AUTHOR]

Acyl-CoA:lysophosphatidylcholine acyltransferase from the unicellular diatom Phaeodactylum tricornutum (PtLPCAT1) is involved in triacylglycerol and galactoglycerolipid synthesis and enhances eicosapentaenoic acid accumulation in recombinant oleaginous yeast Keywords: CRISPR/Cas9; eicosapentaenoic acid; LPCAT; Phaeodactylum tricornutum; Yarrowia lipolytica EN CRISPR/Cas9 eicosapentaenoic acid LPCAT Phaeodactylum tricornutum Yarrowia lipolytica 238 240 3 01/31/23 20230201 NES 230201 Dietary supplementation of omega-3 very long-chain polyunsaturated fatty acids (VLC-PUFAs), particularly eicosapentaenoic acid (EPA, C20: 5 SP 5,8,11,14,17 sp ) and docosahexaenoic acid (DHA, C22: 6 SP 4,7,10,13,16,19 sp ), is well known to have diverse health benefits. Recently, the biochemical properties of one I P. tricornutum i LPCAT (PtLPCAT1) were reported and it can esterify a 18: 3-, 18: 4- or 20: 4-CoA (n-3) moiety to lysoPC I in vitro i , the later acyl being available for further desaturation to synthesize EPA, contrasting with plant LPCATs (Polo'nska I et al i ., [2]). [Extracted from the article]

Summary: 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. [ABSTRACT FROM AUTHOR]