Artificial Biosynthesis Of Ganglioside Oligosaccharides In Escherichia Coli

Gangliosides are glycosphingolipids composed of sialic acid-containing oligosaccharides and ceramides,which are widely present in all vertebrate cells,especially in nerve cells.They are involved in a range of physiological processes,such as cell recognition,cellular immunity,and apoptosis.Some of them such as GM1 still have important pharmaceutical activities.Consequtly,a highly efficiency approach for producing gangliosides is urgently needed and it will contribute to the studying of physiological functions,mechanism and new drugs.The gangliosides are complex in structure and difficult to synthesize on a large scale with traditional chemical catalysis methods.At present,the manufacture of gangliosides mainly relies on extracted from natural biological tissues such as pig brain and bovine brain,but it has several notable limitations,including poor yielding and serious pollution during the extraction process.In the previous work of our laboratory,a new ganglioside multi-module assembly synthesis system has been proposed based on the concept of synthetic biology.The efficient synthesis of ganglioside oligosaccharide modules is a critical step.We have focused on the artificial biosynthesis of gangliosides GM3,GM2 and GM1 oligosaccharides,including the construction of artificial synthesis pathways,establishing quantitative detection methods and optimization of engineered strains.First,we recruited CMP-Neu Ac synthase(neu A)derived from Neisseria meningitidis,α-2,3 sialyltransferase(cst),β-1,4 Gal NAc transferase(cgt A),β-1,3galactosyltransferase(cgt B)and UDP-Glc NAc C4 epimerase(gne)derived from Campylobacter jejuni to construct artificial biosynthetic pathways of GM3,GM2 and GM1 oligosaccharides.The engineered strains Ja,Jb and Jc of were successfully constructed using Escherichia coli JM107 as the chassis cells,which using exogenous sialic acid and lactose as substrates.Further,additional N-acetylglucosamine C2epimerase(neu C)and sialic acid synthase(neu B)derived from N.meningitidis were introduced into the cells to construct strains Ja-1 Jb-1 and Jc-1,which can supply endogenous sialic acid and use lactose as substrate.To improve the efficiency of the artificial biosynthetic pathway,several key parameters including fermentation temperature,substrate concentration,medium composition and sample crushing method were preliminarily optimized.Engineered strains were modificated with peroxidase overexpression and promoter replacement based on the means of metabolic engineering.Compared with the previous strains,optimized strains Ja-2,Jb-2,Jc-2 increased by 18.9 times,11.4 times,22.8 times and the yield of GM3,GM2,GM1 oligosaccharides reached 89.2 mg/L,20.5 mg/L,18.3mg/L.Finally,through Mass Spectrometry analysis based on UPLC-QQQ/MS,a series of rate-limiting steps and limiting factors in the pathway were discovered such as low activity of catalytic elements neu A and gne,insufficient supply of critical precursors UDP-Gal NAc and CTP,which providing important guiding significance for further optimization.On this basis,in order to couple with the gangliosidase synthesis system established in the early stage in our lab,we replaced the lactose substrate with lactose fluoride and first got three kinds of oligosaccharide derivative,which laid a foundation for the complete biosynthesis of gangliosides.