The Importance of the Lipid Biosynthetic Pathway for Glycolipids Production in Engineered E. coli Cells

The production of glycolipids as biosurfactants is attracting much interest due to their potential advantages over their synthetic counterparts but large-scale application is limited by the high production cost and low yields¹. Of special interest for industrial use are microbial glycolipids and among them, we focus on glycoglycerolipids (GGL) for their drug delivery applications. Our aim is to turn E. coli into a platform host for GGL industrial production. E. coli does not produce GGL but when the glycolipid synthase MG517 from Mycoplasma genitalium is expressed in E. coli, this enzyme is functional^2,3. It is able to produce glycoglycerolipids from UDPGlc and diacylglycerol, precursors which are E. coli common metabolites. The metabolic engineering design was based on the introduction of the glycolipid synthase MG517 and the overproduction of the sugar nucleotide and the phosphatidic acid, which is precursor of diacylglycerol⁴. For the modulation of the glycolytic flux, the overexpression of the GalU enzyme that forms UDP-Glc from glucose-1-phosphate was chosen. For diacylglycerol overproduction, the biosynthetic pathway that converts glycerol-3-phosphate into phosphatidic acid was evaluated. Among the different enzymes involved, the PlsC acyltransferase that incorporates the second acyl chain to form phosphatidic acid was selected to avoid an increase of the anionic phospholipid biosynthesis and guarantee the availability of phosphatidic acid for DAG formation.

Seven engineered strains were obtained combining mg517, galU and plsC genes. While the UDPGlc pool was enough and not limiting, the increased flux to phosphatidic acid was the keypoint to enhance DAG availability and GGL synthesis. The strain overexpressing the mg517 and plsC genes produced the highest GGL yield, with a 39% increase of GGL compared to the strain only expressing MG517⁴. Surprisingly, the MG517 glycosyltransferase activity is significantly decreased compared to other strains and the strain coexpressing the three genes is not the highest producer. The presence of GGL contributes to membrane properties and perturbs phospholipid biosynthesis decreasing the phosphatidylethanolamine amounts and giving new membrane properties to these E. colicells. Therefore this engineered strain has proven to be effective on GGL production and a suitable microorganism platform to generate a variety of glycolipids with different regio- and stereoselectivity using their corresponding glycolipid synthases.

 

1 Banat, I.M., Franzetti, A., Gandolfi, I., Bestetti, G., Martinotti, M.G., Frachhia, L., Smyth, T.J., Marchant, R., Appl. Microbiol. Biotechnol. 2010, 87, 427-444.

2 Andrés, E., Martínez, N., Planas, A., J. Biol. Chem. 2011, 13, 286 (41), 35367-79.

3 Andrés, E., Biarnés, X., Faijes, M., Planas, A., Biocat. Biotransform. 2011, 30, 274-287.

4 Mora-Buyé, N., Faijes, M., Planas, A., Metab. Eng. 2012, 14, 551-559.