Functional characterization of the plastidial enzyme GPAT in microalgae and its potential role in lipid biosynthesis.

Microalgae are a rich source of high-value natural products. The green microalga Chlamydomonas reinhardtii has long been used as a model organism for studying lipid metabolism in photosynthetic organisms. Here, we comprehensively characterized the enzymatic activity and substrate preferences of the plastidial glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT1) from C. reinhardtii. Our results revealed that, in addition to GPAT activity, recombinant GPAT1 is associated with lysophosphatidic acid:acyl-CoA acyltransferase (LPAAT) activity. Notably, the membrane-bound form of GPAT1 displayed distinct acyl donor preferences, favoring both C18:1 and C16:0 substrates in its LPAAT function. Knockdown of GPAT1 resulted in a reduced triacylglycerol content, particularly C16 species, under mixotrophic growth and nitrogen deprivation. Interestingly, GPAT1 knockdown triggered a compensatory upregulation of the endoplasmic reticulum-localized GPAT2, resulting in a significant increase in the content and yield of 1,3-olein-2-palmitin (OPO), an essential functional lipid used in infant formula. These findings provide insights into the function and physiological role of microalgal plastidial GPAT1 and highlight its potential as a biotechnological target for enhancing OPO production in microalgae.