In order to understand the biological importance of naturally occurring sialic acid variations on disialyl structures in nature, we developed an efficient two-step multienzyme approach for the synthesis of a series of GD3 ganglioside oligosaccharides and other disialyl glycans containing a terminal Sia alpha 2-8Sia component with different natural and non-natural sialic acids. In the first step, alpha 2-3- or alpha 2-6-linked monosialylated oligosaccharides were obtained using a one-pot three-enzyme approach. These compounds were then used as acceptors for the alpha 2-8-sialyltransferase activity of a recombinant truncated multifunctional Campylobacter jejuni sialyltransferase CstII mutant, CstII Delta 32(153S), to produce disialyl oligosaccharides. The alpha 2-8-sialyltransferase activity of CstII Delta(153S) has promiscuous donor substrate specificity and can tolerate various substitutions at C-5 or C-9 of the sialic acid in CMP-sialic acid, while its acceptor substrate specificity is relatively restricted. The terminal sialic acid residues in the acceptable monosialylated oligosaccharide acceptors are restricted to Neu5Ac, Neu5Gc, KDN, and some of their C-9-modified forms but not their C-5 derivatives. The disialyl oligosaccharides obtained are valuable probes for their biological studies.