Sialic acid plays important roles in stabilization and modulation of the interaction of molecules and membranes in organisms. Due to its high electronegativity, sialic acid can promote binding effects of molecules and support the transportation of drugs and ions in cells. This also strengthens cells against degradation from glycosidases and pro teases. Hence sialic acid helps glycoproteins extend their half-lives and bioactivity. On the other hand, Chinese hamster ovary (CHO) cells have been widely used as a workhorse in biopharmaceutical fields in part due to the similarity between their glycan properties and those in humans. Thus, a high sialylation produced by CHO host cell line is strongly desired. In this study, we simultaneously overexpressed two key sialylated-based enzymes human beta-galactoside alpha(2,6) sialyltransferase I and UDP-G1cNAc 2-epimerase/ManNAc kinase to achieve greater sialylation pattern produced host cells. The single-cell line thus-generated produced an approximately 41.6% higher level of total free sialic acid, and the glycan profiles showed a significant increase of more than 7-fold in the relative amount of total sialylated N-glycan as compared to the wild-type. These results demonstrated that co-expression of these two sialylated-based key enzymes yielded a cell line that effectively produced glycoproteins with superior sialylation and achievable human-like glycoforms. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.