Expression of 2,6-sialyltransferase in Escherichia coli BL21(DE3) and endotoxin-free ClearColi® under fed-batch fermentation

Sialosides are intermediate compounds in the synthesis of sialic acid recognizing immunoglobulin lectins, a family of carbohydrate-binding proteins associated with immune function.  The synthesis of sialosides often requires sialyltransferases (SAT), a group of enzymes specifically link sialic acid to a sugar molecule. Previously, we successfully expressed the gene encoding 2,6-SAT from Photobacterium damsela in Escherichia coli BL21(DE3) under low cell density batch fermentation conditions.  Because of the therapeutic applications of sialosides, the expression of 2,6-SAT in an endotoxin-free system is desirable.  ClearColi^® BL21(DE3) is an E. coli expression system with a genetically-modified lipopolysaccharide pathway, resulting in the production of little to no endotoxin.  We expressed 2,6-SAT in ClearColi under batch and fed-batch fermentation conditions, and compared 2,6-SAT production and activity with batch and fed-batch production in E. coli BL21(DE3).  Although the growth conditions of ClearColi necessitate some fermentation process modifications, such as total incubation time and medium formulation, 2,6-SAT was produced in ClearColi through fed-batch fermentation at levels at least comparable to the commonly-used BL21(DE3) expression system run under similar fed-batch conditions.  On a per biomass basis, ClearColi produced up to 2.23 U/mg biomass under fed-batch conditions, while E. coli BL21(DE3) produced up to 1.64 U/mg biomass.  Volumetric activity up to 153.4 U/L whole broth in ClearColi and up to 152.6 U/L whole broth in E. coli BL21(DE3) was achieved.  Expression of 2,6-SAT in ClearColi under fed-batch conditions is a viable, endotoxin-free alternative to E. coli BL21(DE3).