Enzymatic preparation of GD3-type disialyl oligosaccharides Neu5Gc/KDNα2–8Neu5Gc/KDNα2–3LacβProN3 containing the combination of Neu5Gc and KDN. Reagents and conditions: (i) Pyruvate, CTP, Mg2+, Tris-HCl buffer (pH 8.5), E. coli K12 sialic acid aldolase, NmCSS, and PmST1; (ii) Pyruvate, CTP, Mg2+, Tris-HCl buffer (pH 8.5), E. coli K12 sialic acid aldolase, NmCSS, and CstIIΔ32I53S.