Conversion of L-arabinose to L-ribose by genetically engineered Candida tropicalis.

L-Ribose, a starting material for the synthesis of L-nucleoside, has attracted lots of attention since L-nucleoside is responsible for the antiviral activities of the racemic mixtures of nucleoside enantiomers. In this study, the L-ribulose-producing Candida tropicalis strain was engineered for the conversion of L-arabinose to L-ribose. For the construction of a uracil auxotroph, the URA3 gene was excised by homologous recombination. The expression cassette of codon-optimized L-ribose isomerase gene from Acinetobacter calcoaceticus DL-28 under the control of the GAPDH promoter was integrated to the uracil auxotroph. The resulting strain, K1 CoSTP2 LsaAraA AcLRI, was cultivated with the glucose/L-arabinose mixture. At 45.5 h of fermentation, 6.0 g/L of L-ribose and 3.2 g/L of L-ribulose were produced from 30 g/L of L-arabinose. The proportion between L-ribose and L-ribulose was approximately 2:1 and the conversion yield of L-arabinose to L-ribose was about 20% (w/w). The L-ribose-producing yeast strain was successfully constructed for the first time and could convert L-arabinose to L-ribose in one-pot fermentation using the mixture of glucose and L-arabinose.

Antimicrobial Activity of Amino-Derivatized Cationic Polysaccharides.

To improve the antimicrobial property of chitosan, water-soluble chitosan modified in their quaternary ammonium groups were synthesized. The antimicrobial properties were evaluated against Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Candida tropicalis. The activities increased with increasing cationic charges and the length of the alkyl chain as follows amino-chitosan, dimethylaminoethyl-chitosan, dimethylpropyl amino-chitosan, dimethylamino-1-propyl-chitosan, diethylaminoethyl (DEAE)-chitosan, and quaternized DEAE-chitosan. The modified cationic chitosans showed high antimicrobial property against B. subtilis-Gram-positive bacteria, but were less active towards yeast (C. tropicalis and S. cerevisiae) and E. coli-Gram-negative bacteria. The simple structure of the Gram-positive bacteria may explain why the cationic chitosan derivatives are more active towards B. subtilis than yeast and E. coli. The target sites of the chitosan derivatives are assumed to be the cytoplasmic membranes of microorganisms. The antimicrobial activities were strongly dependent on the cationic charge and the molecular weight. It can be suggested that these cationic chitosan derivatives have potential as antimicrobial agents.

Designation of fingerprint glycopeptides for targeted glycoproteomic analysis of serum haptoglobin: insights into gastric cancer biomarker discovery.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography-mass spectrometry and nano liquid chromatography-tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery. Graphical abstract ᅟ.

Globoside promotes activation of ERK by interaction with the epidermal growth factor receptor.

BACKGROUND: Globoside (Gb4), a globo-series glycosphingolipid (GSL), has been characterized as a stage-specific embryonic antigen (SSEA), and is highly expressed during embryogenesis as well as in cancer tissues. However, the functional role and molecular mechanism of Gb4 are so far unknown. METHODS: GSLs were preferentially inhibited by treatment with D-threo-1-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4), a nanomolar inhibitor of GSL synthesis, in two carcinoma cell lines, HCT116 and MCF7. The effect of EtDO-P4 was examined by MTT assay, FACS, wound assay, western blotting, and RTK array analysis. The functional role of Gb4 was determined by the exogenous addition of various GSLs, and an assay utilizing GSL-coated latex beads. RESULTS: Both cell lines contained higher levels of neutral GSLs than of sialic acid-containing GSLs. Gb4 was one of the major neutral GSLs. The depletion of total GSLs caused significant reduction of cell proliferation, but had less effect on cell apoptosis or motility. EtDO-P4 treatment also suppressed activation of the epidermal growth factor receptor (EGFR)-induced ERK pathway and various receptor tyrosine kinases (RTKs). The reduced activation of ERK was restored by the exogenous addition of Gb4, but not by the addition of gangliosides (GM1, GM2, GM3, and GD1a). The GSL-coated bead assay indicated that Gb4 forms a complex with EGFR, but not with other RTKs. Taken together, Gb4 promotes activation of EGFR-induced ERK signaling through direct interaction with EGFR. GENERAL SIGNIFICANCE: A globo-series GSL, Gb4, promotes EGFR-induced MAPK signaling, resulting in cancer cell proliferation. These findings suggest a possible application of Gb4 in cancer diagnostics and drug targeting.

Enhancement of xylitol production in glycerol kinase disrupted Candida tropicalis by co-expression of three genes involved in glycerol metabolic pathway.

Glycerol can be used as a primary carbon source by yeasts, little is known regarding glycerol metabolism in Candida tropicalis. In this study, glycerol kinase gene (gk) was disrupted from xylitol dehydrogenase gene (XYL2) knockout C. tropicalis strain BSXDH-3. The resultant gk knockout C. tropicalis strain was incapable to grow on glycerol. The cells growth on glycerol was resumed by co-expressing Scheffersomyces stipitis gcy1, 2 and 3 genes, which respectively encode NADP(+)-dependent glycerol dehydrogenase 1, 2 and 3, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "GK", than in BSXDH-3. In fermentation experiments using glycerol as co-substrate with xylose, strain GK produced xylitol 0.85 and 1.28 g l(-1) h(-1) at the time periods of 16 and 24 h, respectively, which is 30 and 18 % higher at same time intervals in BSXDH-3. This is the first report of gk gene disruption and co-expression of gcy1, 2 and 3 genes for NADPH regeneration and enhanced xylitol production in C. tropicalis.

Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate.

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37-73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.

Variations in pain management outcomes among palliative care centers and the impact of organizational factors.

BACKGROUND: The assessment of pain management outcomes is important for the quality assurance of palliative care. The objective of this study was to determine whether there are significant variations in pain management outcomes among palliative care centers and whether they are affected by organizational factors. METHODS: Data used in this investigation were from the 2009 Korean Terminal Cancer Patient Information System and administrative records of the 34 inpatient palliative care centers designated by the Korean Ministry of Health and Welfare in 2009. Self-reported pain scores (range, from 0 to 10) at admission and 1 week after admission were prospectively collected. Multilevel mixed-effect regression models were used to analyze the variations and the impact of organizational-level factors on 2 pain management outcomes (ie, reduction in average pain score and achievement of adequate pain control at 1 week after admission). RESULTS: In total, 1711 patients with terminal cancer were included in the analyses. The mean reduction in the pain score was 0.69 to 1.91 after 1 week, and most patients (82.8%) achieved adequate pain control. There were significant variations in pain management outcomes among palliative care centers. Higher composite scores for human resources adequacy were associated significantly with a greater reduction in pain score (ß, 0.11; 95% confidence interval, 0.01-0.21), and achievement of adequate pain control (adjusted odds ratio, 1.26; 95% confidence interval, 1.10-1.45). CONCLUSIONS: There were significant variations in pain management outcomes among inpatient palliative care centers, and they were affected by organizational factors, such as human resources adequacy.

Variations in process and outcome in inpatient palliative care services in Korea.

PURPOSES: Hospice programs in Korea have been largely based on volunteer activity, religious services, or social services. Recent government policy of designating medically based inpatient palliative care services and per diem payment system made it necessary to monitor the quality of these services. We examined the variation in the process and outcomes of palliative care services, using 2009 data obtained from the Korean Terminal Cancer Patient Information System. METHODS: Data were collected from 3,867 patients with terminal cancer who were registered in 34 inpatient palliative care centers designated by the Ministry of Health and Welfare. We used the mean length of stay and the subsequent place of care as process indicators, and change in average pain score as an outcome indicator. The data were analyzed using descriptive statistics, and analysis of covariance for the case-mix adjustment. RESULTS: There were considerable variations among services with regards to the mean length of stay (i.e., 10.5 to 32.6 days for each admission) and subsequent place of care (i.e., 39.8% to 92.6% ended in death at the first admission), even after stratification by service level. The mean change in average pain score varied from -1.48 to 2.16, and remained significant after case-mix adjustment. CONCLUSION: We found considerable variations among palliative care services with regard to the mean length of stay, subsequent place of care, and change in average pain score. Continued assessment of the variations in process and outcomes will assist in developing the national benchmarking system and the evaluation of the government policy.

Factors associated with medical cost among patients with terminal cancer in hospice units.

This study identified factors associated with higher medical costs for patients with terminal cancer in hospice units in order to develop a daily payment system for hospice services within Korea's National Health Insurance (NHI) program. Through chart reviews conducted by staff nurses, medical information and costs were obtained for 274 patients with terminal cancer in 20 hospice units in October 2007. The daily medical cost per patient was calculated based on the fee-for-service scheme. The characteristics of the hospice units were examined by means of a semistructured questionnaire administered to hospice unit coordinators. Higher daily costs were associated with general hospital-based hospice units (as compared with free-standing units: p<0.01), low Palliative Performance Scale scores (PPS<50, p<0.05), and the presence of fever (p<0.01). In multivariate analysis, hospice unit type was found to be the factor most strongly associated with medical cost. A hospice payment system based on patient characteristics should be thoroughly considered.

Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis.

Xylose reductase (XR) is the first enzyme in D: -xylose metabolism, catalyzing the reduction of D: -xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)(-1)), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L(-1) h(-1) and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L(-1) h(-1); yield 59%).

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway.

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.

Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes.

Therapeutic glycoproteins with exposed galactose (Gal) residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase the content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases the half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (uridine diphosphate-N-acetyl glucosamine 2-epimerase)/MNK (N-acetyl mannosamine kinase), which plays a key role in the initial two steps of sialic acid biosynthesis, is regulated by cytidine monophosphate (CMP)-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared with wild type and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter and human α2,3-sialyltransferase (α2,3-ST) were transfected simultaneously into recombinant human (rh) EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. The sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased ∼32%, but ratios of asialo- and mono-sialylated glycans were decreased ∼50%, compared with control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases the intracellular CMP-sialic acid level. The newly constructed host CHO cell lines produced more highly sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT and α2,3-ST.

L-arabinose pathway engineering for arabitol-free xylitol production in Candida tropicalis.

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce L-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from L-arabinose to arabitol, the L-arabinose-preferring, endogenous XR was replaced by a D-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of L-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g L-arabinose l(-1) into 9.5 and 8.3 g arabitol l(-1), respectively, whereas the recombinant strain JY consumed 10.5 g L-arabinose l(-1) for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.

Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in Candida tropicalis.

To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62 g l(-1) h(-1), respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.

N-glycosylation status of beta-haptoglobin in sera of patients with colon cancer, chronic inflammatory diseases and normal subjects.

N-glycosylation status of purified beta-haptoglobin from sera of 17 patients, and from sera of 14 healthy volunteer subjects, was compared by blotting with various lectins and antibodies. Patients in this study were diagnosed as having colon cancer through histological examination of each tumor tissue by biopsy. Blotting index of serum beta-haptoglobin with Aleuria aurantia lectin (AAL) was clearly higher for cancer patients than for healthy subjects. No such distinction was observed for blotting with three other lectins and two monoclonal antibodies. To determine tumor-associated reactivity of AAL binding as compared to inflammatory processes in colonic tissues, beta-haptoglobin separated from sera of 5 patients with Crohn's disease (CD), and 4 patients with ulcerative colitis (UC), was studied. All these cases, except one case of UC, showed AAL index lower than that in cancer cases, similarly to healthy subjects. The higher AAL binding of beta-haptoglobin in colon cancer patients than in healthy subjects appeared to be due to alpha-L-fucosyl residue, since it was eliminated by bovine kidney alpha-fucosidase treatment. N-linked glycans of serum haptoglobin from colon cancer patients vs. healthy subjects were released by N-glycanase, fluorescence-labeled, and subjected to normal-phase high performance liquid chromatography (NP-HPLC). Glycan structures were determined based on glucose unit (GU) values and their changes upon sequential treatment with various exoglycosidases. Glycosyl sequences and their branching status of glycans from 14 cases of serum beta-haptoglobin were characterized. The identified glycans were sialylated or nonsialylated, bi-antennary or tri-antennary structures, with or without terminal fucosylation.

Array-based analysis of secreted glycoproteins for rapid selection of a single cell producing a glycoprotein with desired glycosylation.

The therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile. For the development of glycoproteins with therapeutic applications, selection of cell lines producing a glycoprotein with adequate glycoform is crucial. Here, we demonstrate an array-based analysis of secreted glycoproteins for rapid and efficient selection of a single cell producing a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of glycoproteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. On the basis of statistical analysis of the interrogation, single cells which are predicted to produce a desired glycoprotein are selected, retrieved, and expanded. We applied the approach to human recombinant erythropoietin (rhEPO)-producing CHO cells and verified the selection of a single CHO cell that produces rhEPO with a high sialylation degree. Human erythropoietin (hEPO) bearing highly sialylated oligosaccharide was shown to display a much longer plasma half-life, resulting in high therapeutic efficacy. This method may find widespread use in the clonal selection for the production of other glycoproteins with specific glycosylation as well as analysis of the heterogeneity in cell populations in a high-throughput manner.

Antitumor activity of methylan polysaccharide derivatives.

Methylan polysaccharide derivatives were prepared by dialkylaminoalkylation and reductive amination followed by quaternization. Their antitumor activity was investigated and a relationship between structure and activity is suggested. For quaternized DEAE-methylan at only 75 mug ml(-1), tumor cell proliferation was suppressed by 58-84% in three cell lines tested in the order Colo < Hela < HepG2.

Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production.

BACKGROUND: Deletion of large blocks of nonessential genes that are not needed for metabolic pathways of interest can reduce the production of unwanted by-products, increase genome stability, and streamline metabolism without physiological compromise. Researchers have recently constructed a reduced-genome Escherichia coli strain MDS42 that lacks 14.3% of its chromosome. RESULTS: Here we describe the reengineering of the MDS42 genome to increase the production of the essential amino acid L-threonine. To this end, we over-expressed a feedback-resistant threonine operon (thrA*BC), deleted the genes that encode threonine dehydrogenase (tdh) and threonine transporters (tdcC and sstT), and introduced a mutant threonine exporter (rhtA23) in MDS42. The resulting strain, MDS-205, shows an ~83% increase in L-threonine production when cells are grown by flask fermentation, compared to a wild-type E. coli strain MG1655 engineered with the same threonine-specific modifications described above. And transcriptional analysis revealed the effect of the deletion of non-essential genes on the central metabolism and threonine pathways in MDS-205. CONCLUSION: This result demonstrates that the elimination of genes unnecessary for cell growth can increase the productivity of an industrial strain, most likely by reducing the metabolic burden and improving the metabolic efficiency of cells.

Enhanced sialylation of recombinant erythropoietin in genetically engineered Chinese-hamster ovary cells.

Sialic acid, the terminal sugar in N-linked complex glycans, is usually found in glycoproteins and plays a major role in determining the circulatory lifespan of glycoproteins. In the present study we attempted to enhance the sialylation of recombinant EPO (erythropoietin) in CHO (Chinese-hamster ovary) cells. To enhance EPO sialylation, we introduced human alpha2,3-ST (alpha2,3-sialyltransferase) and CMP-SAS (CMP-sialic acid synthase) into recombinant human EPO-producing CHO cells. The sialylation of EPO was increased by the expression of alpha2,3-ST alone. Although the co-expression of alpha2,3-ST and CMP-SAS did not further increase sialylation, an increase in the intracellular pool of CMP-sialic acid was noted. On the basis of these observations, it was postulated that the transport capacity of CMP-sialic acid into the Golgi lumen was limited, thereby causing the reduced availability of CMP-sialic acid substrate for sialylation. Therefore, we co-expressed human alpha2,3-ST and CMP-SAS, as well as overexpress Chinese hamster CMP-sialic acid transporter (CMP-SAT) in CHO cells, which produced recombinant human EPO. When alpha2,3-ST, CMP-SAS, and CMP-SAT were overexpressed in CHO cells, there was a corresponding increase in sialylation compared with the co-expression of alpha2,3-ST and CMP-SAS. The present study provides a useful strategy for enhancing the sialylation of therapeutic glycoproteins produced in CHO cells.

Enhanced sialylation of recombinant erythropoietin in CHO cells by human glycosyltransferase expression.

Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human alpha2,3- sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of alpha2,3-ST and beta1,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When alpha2,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1%. When alpha2,3-ST and beta1,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of alpha2,3- ST and beta1,4-GT is more effective than the expression of alpha2,3-ST alone. Coexpression of alpha2,3-ST and beta1,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of alpha2,3-ST and beta1,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.