Previous studies have reported that insect cell lines lack the capacity to generate endogenously the
nucleotide sugar,
CMP-Neu5Ac, required for sialylation of
glycoconjugates. In this study, the biosynthesis of this activated form of
sialic acid completely from endogenous metabolites is demonstrated for the first time in insect cells by expressing the mammalian genes required for the multistep conversion of endogenous
UDP-GlcNAc to
CMP-Neu5Ac. The genes for
UDP-
GlcNAc-2-
epimerase/ManNAc
kinase (EK),
sialic acid 9-phosphate synthase (SAS), and
CMP-sialic acid synthetase (CSAS) were coexpressed in insect cells using baculovirus expression vectors, but the
CMP-Neu5Ac and precursor Neu5Ac levels synthesized were found to be lower than those achieved with ManNAc supplementation due to feedback inhibition of the EK
enzyme by
CMP-Neu5Ac. When
sialuria-like mutant EK genes, in which the site for feedback regulation has been mutated, were used,
CMP-Neu5Ac was synthesized at levels more than 4 times higher than that achieved with the wild-type EK and 2.5 times higher than that achieved with ManNAc feeding. Addition of
N-acetylglucosamine (GlcNAc), a precursor for
UDP-GlcNAc, to the media increased the levels of
CMP-Neu5Ac even more to a level 7.5 times higher than that achieved with ManNAc supplementation, creating a bottleneck in the conversion of Neu5Ac to
CMP-Neu5Ac at higher levels of
UDP-GlcNAc. The present study provides a useful biochemical strategy to synthesize and enhance the levels of the sialylation donor molecule,
CMP-Neu5Ac, a critical limiting substrate for the generation of complex
glycoproteins in insect cells and other cell culture systems.