UDP-galactose limitation impairs efficient Monogalactosyldiacylglycerol production in Escherichia coli expression...

Monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in photosynthetic membranes, playing critical roles in its architecture and protein function. Studying these roles is challenging in native systems, making heterologous expression in Escherichia coli an attractive alternative. This research aimed to establish a method for MGDG synthesis in common E. coli expression strains. We investigated the use of plant MGDG synthesis pathway utilising MGDG synthase from Cucumis sativus which was shown before to function in the XL1-Blue strain, but failed in common expression strains like BL21(DE3). We identified the absence of UDP-galactose 4'-epimerase (galE), an enzyme producing UDP-galactose, as the limiting factor. Co-expression of MGD1 and galE successfully enabled MGDG synthesis in BL21(DE3) and other expression strains, with optimized conditions yielding higher production than shown in literature before. Using transmission electron microscopy with a quantitative image analysis pipeline, we measured effect of MGDG accumulation on E. coli morphology, showing a significant increase in cell cross-sectional area, indicating cell swelling. This work establishes a method for producing MGDG in widely used E. coli expression strains, providing a platform for future in vivo studies of galactolipid-dependent processes.