👤 Derek M Ayittey

Lysophosphatidic acid acyltransferase (LPAAT) is a pivotal enzyme in the de novo biosynthesis of phosphatidic acid (PA), playing a central role in glycerophospholipid assembly and triacylglycerol (TAG) accumulation. Myrmecia incisa is a green microalga notable for its high content of arachidonic acid (ArA), yet the molecular mechanism underlying ArA enrichment in TAG remains unclear. In this study, a putative LPAAT gene from M. incisa, designated MiLPAAT, was identified and cloned, followed by systematic structural and functional characterization. Sequence analysis revealed that MiLPAAT contains a conserved PlsC domain and the characteristic H(X)₄D and EGTR motifs. Bioinformatic predictions identified at least one transmembrane domain at the N-terminus, supporting its identity as an integral membrane protein. This was further confirmed by membrane fractionation and Western blot analysis, which demonstrated its association with the membrane fraction. Phylogenetic analysis further demonstrated its close evolutionary relationship to LPAAT homologs in other green algae. Heterologous expression in Escherichia coli, coupled with in vitro enzymatic assays, confirmed that the recombinant MiLPAAT protein possesses LPAAT activity, catalyzing the acylation of LPA with various acyl-CoAs. Among the substrates tested, MiLPAAT exhibited the highest catalytic efficiency toward ArA-CoA (104.8 ± 3.2 nmol/mg/min), followed by oleoyl-CoA (81.5 ± 2.7 nmol/mg/min) and palmitoyl-CoA (68.4 ± 2.1 nmol/mg/min), consistent with the ArA-rich TAG composition observed in M. incisa. Immunogold labeling and immunohistochemical localization experiments revealed that MiLPAAT is predominantly localized at the plasma membrane. Findings of the present study suggest that MiLPAAT plays a critical role in PA biosynthesis and assembly of ArA into TAG in M. incisa, providing a novel target for microalgal lipid metabolic engineering. Show less