We have isolated three types of cDNAs encoding novel ␤1,3-N-acetylglucosaminyltransferases (designated ␤3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the ␤1,3-galactosyltransferase family and a ␤1,3-N-acetylglucosaminyltransferase (designated ␤3Gn-T1), but show no structural similarity to another type of ␤1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed ␤1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not ␤1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with ␤3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these ␤3Gn-Ts were different among 35 human tissues. ␤3Gn-T2 was ubiquitously expressed, whereas expression of ␤3Gn-T3 and -T4 was relatively restricted. ␤3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. ␤3Gn-T4 was mainly expressed in brain. These results have revealed that several ␤1,3-Nacetylglucosaminyltransferases form a family with structural similarity to the ␤1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each ␤1,3-N-acetylglucosaminyltransferase may play different roles.A family of human ␤1,3-galactosyltransferases (␤3Gal-Ts) 1 consisting of five members (␤3Gal-T1, -T2, -T3, -T4, and -T5) was recently identified (1-4). The first ␤1,3-galactosyltransferase (␤3Gal-T1), which catalyzes the formation of type 1 oligosaccharides, was isolated by us using an expression cloning approach (1). Expression patterns of ␤3Gal-T1 and type 1 oligosaccharides strongly suggested the existence of ␤3Gal-T1 homologs. For instance, type 1-derived oligosaccharides such as sialyl-Le a were known to be expressed in colon and pancreatic cancer cell lines, whereas expression of ␤3Gal-T1 was detected in brain, but not in cancer cells. Our early approach using Southern hybridization failed to detect the existence of ␤3Gal-T1 homologous genes. However, recent accumulation of nucleotide sequence information on human cDNAs and genes such as expressed sequence tags (ESTs) enabled us to search homologous genes that do not have high similarity as detected by hybridization, but show significant similarity. A homology search based on the nucleotide or amino acid sequence of ␤3Gal-T1 led to the isolation of ␤3Gal-T2, -T3, and -T4, indicating that ␤3Gal-Ts form a family (1-3).␤3Gal-T2 catalyzed a similar reaction, but showed different substrate specificity compared with ␤3Gal-T1. The activity of ␤3Gal-T3 has not been detected, whereas the corresponding mouse enzyme exhibits weak ␤3Gal-T activity for both GlcNAc and GalNAc (5). On the other...

A large-scale production system of uridine 5'-diphospho-galactose (UDP-Gal) has been established by the combination of recombinant Escherichia coli and Corynebacterium ammoniagenes. Recombinant E. coli that overexpress the UDP-Gal biosynthetic genes galT, galK, and galU were generated. C. ammoniagenes contribute the production of uridine triphosphate (UTP), a substrate for UDP-Gal biosynthesis, from orotic acid, an inexpensive precursor of UTP. UDP-Gal accumulated to 72 mM (44 g/L) after a 21 h reaction starting with orotic acid and galactose. When E. coli cells that expressed the alpha1,4-galactosyltransferase gene of Neisseria gonorrhoeae were coupled with this UDP-Gal production system, 372 mM (188 g/L) globotriose (Galalpha1-4Galbeta1-4Glc), a trisaccharide portion of verotoxin receptor, was produced after a 36 h reaction starting with orotic acid, galactose, and lactose. No oligosaccharide by-products were observed in the reaction mixture. The production of globotriose was several times higher than that of UDP-Gal. The strategy of producing sugar nucleotides by combining metabolically engineered recombinant E. coli with a nucleoside 5'-triphosphate producing microorganism, and the concept of producing oligosaccharides by coupling sugar nucleotide production systems with glycosyltransferases, can be applied to the manufacture of other sugar nucleotides and oligosaccharides.

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A large-scale production system of cytidine 5'monophospho-N-acetylneuraminic acid (CMP-NeuAc) and sialyloligosaccharides was established by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammonia-genes. For the production of CMP-NeuAc, two recombinant E. coli strains were generated that overexpressed the genes of CMP-NeuAc synthetase and CTP synthetase, respectively. C. ammoniagenes contributed to the formation of UTP from orotic acid. CMP-NeuAc was accumulated at 27 mM (17 g/l) after a 27-h reaction starting with orotic acid and N-acetylneuraminic acid. When E. coli cells that overexpressed the alpha-(2-->3)-sialyltransferase gene of Neisseria gonorrhoeae were put into the CMP-NeuAc production system, 3'-sialyllactose was accumulated at 52 mM (33 g/l) after an 11-h reaction starting with orotic acid, N-acetylneuraminic acid, and lactose. Almost no oligosaccharide byproducts other than 3'-sialyllactose were observed after the reaction. The production of 3'-sialyllactose at a 5-l jar fermenter scale was almost the same as that at a beaker scale, which indicated the high potential of the 3'-sialyllactose production on an industrial scale.

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