Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT-1 cells cultured in serum-free medium

The inability to sialylate recombinant glycoproteins is a critical limitation of the baculovirus-insect cell expression system. This limitation is due, at least in part, to the absence of detectable sialyltransferase activities and CMP-sialic acids in the insect cell lines routinely used as hosts in this system. SfSWT-1 is a transgenic insect cell line encoding five mammalian glycosyltransferases, including sialyltransferases, which can contribute to sialylation of recombinant glycoproteins expressed by baculovirus vectors. However, sialylation of recombinant glycoproteins requires culturing SfSWT-1 cells in the presence of fetal bovine serum or another exogenous source of sialic acid. To eliminate this requirement and extend the utility of SfSWT-1 cells, we have isolated a new baculovirus vector, AcSWT-7B, designed to express two mammalian enzymes that can convert N-acetylmannosamine to CMP-sialic acid during the early phase of infection. AcSWT-7B was also designed to express a model recombinant glycoprotein during the very late phase of infection. Characterization of this new baculovirus vector showed that it induced high levels of intracellular CMP-sialic acid and sialylation of the recombinant N-glycoprotein upon infection of SfSWT-1 cells cultured in serum-free medium supplemented with N-acetylmannosamine. In addition, co-infection of SfSWT-1 cells with AcSWT-7B plus a conventional baculovirus vector encoding human tissue plasminogen activator resulted in sialylation of this recombinant N-glycoprotein under the same culture conditions. These results demonstrate that AcSWT-7B can be used in two different ways to support recombinant N-glycoprotein sialylation by SfSWT-1 cells in serum-free medium. Thus, AcSWT-7B can be used to extend the utility of this previously described transgenic insect cell line for recombinant sialoglycoprotein production.     

Evidence for a sialic acid salvaging pathway in lepidopteran insect cells

We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.     

Engineering the protein N-glycosylation pathway in insect cells for production of biantennary,complex N-glycans

Insect cells, like other eucaryotic cells, modify many of their proteins by N-glycosylation. However, the endogenous insect cell N-glycan processing machinery generally does not produce complex, terminally sialylated N-glycans such as those found in mammalian systems. This difference in the N-glycan processing pathways of insect cells and higher eucaryotes imposes a significant limitation on their use as hosts for baculovirus-mediated recombinant glycoprotein production. To address this problem, we previously isolated two transgenic insect cell lines that have mammalian beta1,4-galactosyltransferase or beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes. Unlike the parental insect cell line, both transgenic cell lines expressed the mammalian glycosyltransferases and were able to produce terminally galactosylated or sialylated N-glycans. The purpose of the present study was to investigate the structures of the N-glycans produced by these transgenic insect cell lines in further detail. Direct structural analyses revealed that the most extensively processed N-glycans produced by the transgenic insect cell lines were novel, monoantennary structures with elongation of only the alpha1,3 branch. This led to the hypothesis that the transgenic insect cell lines lacked adequate endogenous N-acetylglucosaminyltransferase II activity for biantennary N-glycan production. To test this hypothesis and further extend the N-glycan processing pathway in Sf9 cells, we produced a new transgenic line designed to constitutively express a more complete array of mammalian glycosyltransferases, including N-acetylglucosaminyltransferase II. This new transgenic insect cell line, designated SfSWT-1, has higher levels of five glycosyltransferase activities than the parental cells and supports baculovirus replication at normal levels. In addition, direct structural analyses showed that SfSWT-1 cells could produce biantennary, terminally sialylated N-glycans. Thus, this study provides new insight on the glycobiology of insect cells and describes a new transgenic insect cell line that will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors.     

Mammalian glycosyltransferase expression allows sialoglycoprotein production by baculovirus-infected insect cells.

The baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N-glycoprotein sialylation by the baculovirus-insect cell system. This was accomplished by using a novel baculovirus vector designed to express a mammalian alpha2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian beta1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the baculovirus-insect cell expression system can be metabolically engineered for N-glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.     

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.     

Expression and functional characterization of a nucleotide sugar transporter from Drosophila melanogaster: relevance to protein glycosylation in insect cell expression systems

Insect cells are used routinely to express recombinant mammalian glycoproteins. However, insect protein glycosylation pathways are not well understood and appear to differ from those of mammalian cells. One way to more clearly evaluate the protein glycosylation potential of insect cells is to use the Drosophila melanogaster genome to identify genes that might encode relevant functions. These genes can then be expressed and the functions of the gene products directly evaluated by biochemical assays. In this study, we used this approach to determine the function of a putative Drosophila nucleotide sugar transporter gene. The results showed that this gene encodes a protein that can transport UDP-galactose, but not CMP-sialic acid. Thus, Drosophila encodes at least some of the infrastructure needed to produce glycoproteins with complex glycans, but this particular gene product does not directly support glycoprotein sialylation. These findings are relevant to insect cell biology and to an informed consideration of insect cell expression systems as tools for recombinant glycoprotein production.     

A CMP-sialic acid transporter cloned from Arabidopsis thaliana

Sialylation of glycans is ubiquitous in vertebrates, but was believed to be absent in plants, arthropods, and fungi. However, recently evidence has been provided for the presence of sialic acid in these evolutionary clades. In addition, homologs of mammalian genes involved in sialylation can be found in the genomes of these taxa and for some Drosophila enzymes, involvement in sialic acid metabolism has been documented. In plant genomes, homologs of sialyltransferase genes have been identified, but there activity could not be confirmed. Several mammalian cell lines exist with defects in the sialylation pathway. One of these is the Chinese hamster ovary cell line Lec2, deficient in CMP-sialic acid transport to the Golgi lumen. These mutants provide the possibility to clone genes by functional complementation. Using expression cloning, we have identified an Arabidopsis thaliana nucleotide sugar transporter that is able to complement the CMP-sialic acid transport deficiency of Lec2 cells. The isolated gene (At5g41760) is a member of the triose-phosphate/nucleotide sugar transporter gene family. Recombinant expression of the gene in yeast and testing in vitro confirmed its ability to transport CMP-sialic acid.     

Platelets Support Extracellular Sialylation by Supplying the Sugar Donor Substrate

Sizable pools of freely circulating glycosyltransferases are in blood, but understanding their physiologic contributions has been hampered because functional sources of sugar donor substrates needed to drive extracellular glycosylation have not been identified. The blood-borne ST6Gal-1 produced and secreted by the liver is the most noted among the circulatory glycosyltransferases, and decorates marrow hematopoietic progenitor cells with α2,6-linked sialic acids and restricts blood cell production. Platelets, upon activation, secrete a plethora of bioactive molecules including pro- and anti-inflammatory mediators. Cargos of sugar donor substrates for glycosyltransferase activity have also been reported in platelets. Here, we implemented a cell-based system to interrogate platelets for their ability to deliver effectively the sugar donor substrate for extracellular ST6Gal-1 to function. We report that thrombin-activated platelets, at physiologic concentration and pH, can efficiently and effectively substitute for CMP-sialic acid in extracellular ST6Gal-1-mediated sialylation of target cell surfaces. Activated platelets can also supply the sialic acid donor to sialylate the synthetic acceptor, Gal(β1,4)GlcNAcα-o-benzyl, with the product Sia(α2,6)Gal(β1,4)GlcNAcα-o-benzyl structurally confirmed by LC/MS. Platelet-secreted donor substrate was recovered in the 100,000 × g sediment, strongly suggesting the association of this otherwise soluble substrate, putatively CMP-sialic acid, within platelet microparticles. Sequestration within microparticles may facilitate delivery of glycosylation substrate at effective dosages to sites of extracellular glycosylation while minimizing excessive dilution.     

Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose.

Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.     

Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell

Plant cells have no beta1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured cells that produced human beta1,4-galactosyltransferase [N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692-4697]. In this study, we introduced two critical genes encoding human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.     

哺乳动物N-糖基化途径中关键酶唾液酸合酶和CMP-唾液酸合成酶基因在转基因家蚕中的表达

对昆虫的N-糖基化途径进行修饰改变是扩展昆虫蛋白表达系统应用范围的重要途径。本研究利用基于piggyBac转座子的家蚕Bombyx mori转基因技术表达昆虫所缺乏的哺乳类糖基化途径中的关键基因, 构建了可以同时表达小鼠Mus musculus唾液酸合酶和小鼠CMP-唾液酸合成酶两个基因的piggyBac表达载体, 选用家蚕肌动蛋白A3启动子控制基因的表达, 并导入3×P3启动子控制下的增强绿色荧光蛋白EGFP作为分子标记。在得到的G1代转基因家蚕中对转入的基因进行了分子水平的鉴定和分析, 为在家蚕这种模式昆虫中模拟哺乳类糖基化途径奠定了基础。     

Improvement of glycosylation in insect cells with mammalian glycosyltransferases

The N-glycans of recombinant glycoproteins expressed in insect cells mainly contain high mannose or tri-mannose structures, which are truncated forms of the sialylated N-glycans found in mammalian cells. Because asialylated glycoproteins have a shorter half-life in blood circulation, we investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells. We co-expressed in two insect cell lines, Sf9 and Ea4, the human alpha1-antitrypsin (halpha1AT) protein with a series of key glycosyltransferases, including GlcNAc transferase II (GnT2), beta1,4-galactosyltransferase (beta14GT), and alpha2,6-sialyltransferase (alpha26ST) by a single recombinant baculovirus. We demonstrated that the enhancement of N-glycosylation is cell type-dependent and is more efficient in Ea4 than Sf9 cells. Glycan analysis indicated that sialylated halpha1AT proteins were produced in Ea4 insect cells expressing the above-mentioned exogenous glycosyltransferases. Therefore, our expression strategy may simplify the production of humanized therapeutic glycoproteins by improving the N-glycosylation pathway in specific insect cells, with an ensemble of exogenous glycosyltransferases in a single recombinant baculovirus.     

Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.     

Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids

Four common sialic acids (Sia), NeuAc, N-glycolyl-neuraminic acid (NeuGc), 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc), and 9-O-Ac-NeuAc were examined for activation to their corresponding CMP-sialic acid conjugates and subsequently for their transfer to glycoprotein oligosaccharides by purified mammalian sialyltransferases. CMP-sialic acid synthetases from calf brain and from bovine and equine submaxillary glands were found to convert NeuAc, NeuGc, and 9-O-Ac-NeuAc to their corresponding CMP-sailic acids. In contrast, no conversion of 4-O-Ac-NeuAc to CMP-4-O-Ac-NeuAc was observed for any of the three synthetases examined. A new procedure for the preparation of CMP-9-O-Ac-NeuAc, CMP-NeuGc, and CMP-NeuAc in high yield and purity was developed, using the calf brain CMP-sialic acid synthetase. Each of these derivatives was tested as donor substrates for six mammalian sialyltransferases purified from porcine, rat, and bovine tissues, including a bovine GalNAc alpha 2,6 sialyltransferase whose purification is described in this report. The sialyltransferases examined represent those which form the Sia alpha 2,6Gal beta 1,4-GlcNAc-, Sia alpha 2,3Gal beta 1,3(4)GlcNAc-, Sia alpha 2,3Gal beta 1,3-GalNAc- and Sia alpha 2,6GalNAc- sequences found on N-linked and O-linked oligosaccharides of glycoproteins. CMP-NeuAc and CMP-NeuGc were equally good donor substrates for all six sialyltransferases. However, transfer of 9-O-Ac-NeuAc from CMP-9-O-Ac-NeuAc varied from only 10% to nearly 70% that of the transfer of NeuAc from CMP-NeuAc. Results are viewed to define the relative roles of direct transfer of these sialic acids and modification of glycosidically bound NeuAc in glycoproteins.     

Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes

Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.     

Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.

Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance.     

Structure of O-glycosidically linked oligosaccharides synthesized by the insect cell line Sf9

The O-glycosidically linked oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Gal beta 1-3GalNAc, either unsubstituted or substituted with one or two sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Gal beta 1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal:N-acetylgalactosamine, beta 1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.     

Engineering intracellular CMP-sialic acid metabolism into insect cells and methods to enhance its generation

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.