PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

The search for novel microbial lipases with robust and versatile biochemical properties remains a priority in industrial biotechnology. In this study, the gut microbiota of the Peruvian anchovy (Engraulis ringens) was explored as a potential source of lipase-producing bacteria. A total of 31 distinct bacterial strains were isolated, among which 10 exhibited extracellular lipase activity in qualitative plate assays. Molecular identification revealed representatives of the genera Staphylococcus, Serratia, and Aeromonas. Two promising strains, Staphylococcus ureilyticus LMB-06 and LMB-Ju02, were selected based on their superior lipase productivity and were further subjected to partial biochemical characterization. Their lipase-containing secretomes displayed activity across a broad temperature range, retained stability under mildly acidic conditions (pH 5.0–6.0), tolerated several organic solvents, and exhibited enhanced activity in the presence of Ca²⁺. Notably, the lipase activity of LMB-06 was positively influenced by Mg²⁺ and K⁺—a response not previously reported for Staphylococcus lipases—suggesting unique enzymatic properties. In addition, LMB-06 retained activity in the presence of H₂O₂, highlighting its suitability for biodiesel production from recycled oils. Furthermore, hydrolysis assays using various natural oils as substrates revealed a marked preference for plant-based oils, particularly olive oil. Altogether, these findings highlight the value of S. ureilyticus strains from anchovy gut microbiota as novel biocatalyst sources for sustainable oil bioprocessing and oleochemical applications.