Engineering the E.coli UDP-Glucose Synthesis Pathway for Oligosaccharide Synthesis

摘要

A metabolic engineering strategy was successfully applied to engineer the UDP-glucose synthesis pathway in E.coli.Two key enzymes of the pathway,phosphoglucomutase and UDP-glucose pyrophosphorylase,were overexpressed to increase the carbon flux toward UDP-glucose synthesis.When additional enzymes (a UDP-galactose epimerase and a galactosyltransferease) were introduced to the engineered strain,the increased flux to UDP-glucose synthesis led to an enhanced UDP-galactose derived disaccharide synthesis.Specifically,close to 20 mM UDP-galactose derived disaccharides were synthesized in the engineered strain,whereas in the control strain only 2.5 mM products were obtained,indicating that the metabolic engineering strategy was successful in channeling carbon flux (8-fold more) into the UDP-glucose synthesis pathway.UDP-sugar synthesis and oligosaccharide synthesis were shown to increase according to the enzyme expression levels when inducer concentration was between 0 and 0.5 mM.However,this dependence on the enzyme expression stopped when expression level was further increased (IPTG concentration was increased from 0.5 to 1 mM),indicating that other factors emerged as bottlenecks of the synthesis.Several likely bottlenecks and possible engineering strategies to further improve the synthesis are discussed.