Koizumi S, Endo T, Tabata K, Ozaki A
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Machida, Japan.
Nat Biotechnol. 1998 Sep;16(9):847-50. doi: 10.1038/nbt0998-847.
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.
通过重组大肠杆菌和产氨棒杆菌的组合，建立了一个大规模生产尿苷5'-二磷酸半乳糖（UDP-半乳糖）的系统。构建了过表达UDP-半乳糖生物合成基因galT、galK和galU的重组大肠杆菌。产氨棒杆菌利用乳清酸（一种廉价的UTP前体）生产尿苷三磷酸（UTP），UTP是UDP-半乳糖生物合成的底物。以乳清酸和半乳糖为起始原料，经过21小时反应后，UDP-半乳糖积累至72 mM（44 g/L）。当将表达淋病奈瑟菌α1,4-半乳糖基转移酶基因的大肠杆菌细胞与该UDP-半乳糖生产系统偶联时，以乳清酸、半乳糖和乳糖为起始原料，经过36小时反应后，产生了372 mM（188 g/L）的异麦芽糖三糖（Galα1-4Galβ1-4Glc），它是维罗毒素受体的三糖部分。反应混合物中未观察到寡糖副产物。异麦芽糖三糖的产量比UDP-半乳糖高几倍。将代谢工程改造的重组大肠杆菌与核苷5'-三磷酸生产微生物相结合来生产糖核苷酸的策略，以及将糖核苷酸生产系统与糖基转移酶偶联来生产寡糖的概念，可应用于其他糖核苷酸和寡糖的制造。
