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.

No abstract

crobiol. 62:1903-1907, 1996). The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35°C, respectively. The K m values were 0.56 and 0.11 mM for L-proline and 2-oxoglutarate, respectively. The k cat value of hydroxylation was 3.2 s ؊1 . Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database. A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence. With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR. A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe. The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158. No sequence homolog was found in EMBL, GenBank, and DDBJ databases. ORF 3 was expressed in Escherichia coli DH1. Recombinants showed hydroxylating activity five times higher than that of the original bacterium, Streptomyces sp. strain TH1. It was concluded that the ORF 3 encodes functional proline 3-hydroxylase.Among eight stereoisomers of hydroxyprolines, only trans-4-hydroxy-L-proline is abundant in nature as a component of collagens produced by higher organisms (7). It is known that procollagen-proline dioxygenase (prolyl 4-hydroxylase; EC 1.14.11.2) hydroxylates L-proline residues of procollagen posttranslationally to trans-4-hydroxy-L-proline residues during collagen biosynthesis (7,14,21). Procollagen-proline 3-dioxygenase (prolyl 3-hydroxylase; EC 1.14.11.7), which hydroxylates peptidic L-proline residues to trans-3-hydroxy-L-proline, is also involved in collagen biosynthesis (11,17). However, free Lproline is not accepted as a substrate for these prolyl hydroxylases. These enzymes belong to a family of 2-oxoglutaratedependent dioxygenases which requires 2-oxoglutarate and O 2 as cosubstrates and ferrous ion as a cofactor for the reaction.In contrast, hydroxylation of free L-proline to free trans-4-hydroxy-L-proline was reported for microorganisms producing the peptide antibiotic etamycin, which contains 4-hydroxyproline as its component (5, 15). Recently, Lawrence et al. have purified 4-hydroxylase of free L-proline from Streptomyces griseoviridus P8648, which produces etamycin (9). They reported that microbial proline 4-hydroxylase belongs to a family of 2-oxoglutarate-dependent dioxygenases as well as prolyl hydroxylases involved in collagen biosynthesis (9).Independently, we have screened microbial proline 4-hydroxylases from more than 3,000 strains and have found 8 actinomycete strains to produce proline 4-hydroxylase (unpublished results). In this screening, we found a novel activity which hydroxylates free L-proline to cis-3-hydroxy-L-proline in Streptomyces sp. strain TH1, Streptomyces canus AT...

No abstract

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.