Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling

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.

P1 Trisaccharide (Galα1,4Galβ1,4GlcNAc) Synthesis by Enzyme Glycosylation Reactions Using Recombinant Escherichia coli

“…In such experiments, several recombinant bacterial strains expressing enzymes involved in the catalytic pathway of particular carbohydrates were coupled. The development of a metabolically engineered strain of Corynebacterium ammoniagenes with highlevel UTP production and UDP-galactose (UDP-Gal) regeneration was a key contribution to the present work (11)(12)(13)20). Subsequently, an alternate pathway for UDP-Gal formation from sucrose and UDP has been developed.…”

Enzymatic Synthesis of Cytidine 5′-Monophospho-N-acetylneuraminic Acid

“…To solve these problems, Ichikawa et al 33) developed an in situ regeneration system from CMP and NeuAc, and Endo et al 19) also established a production system from orotic acid and NeuAc using bacterial coupling. Although both production systems appeared to be profitable, they still require NeuAc as a primary material.…”

“…Recently Endo et al also reported a CMP-NeuAc production system from orotic acid and NeuAc by bacterial coupling. 19) y To whom correspondence should be addressed. Fax: +81-479-22-9865; E-mail: noguchit@yamasa.com Abbreviations: CMP, cytidine 5 0 -monophosphate; CMP-NeuAc, cytidine 5 0 -monophospho-N-acetylneuraminic acid; CTP, cytidine 5 0 -triphosphate; NeuAc, N-acetyl-D-neuraminic acid; PEP, phosphoenolpyruvate…”

Biosynthesis of hexaand pentameric chitooligosaccharides using n-acetylglucoseaminyl transferase from rhizobial bacteria

“…В процессе биосинтеза могут быть решены такие про-блемы синтеза олигосахаридов как региоспецифичность (формирование олигомеров, состоящих только из одного типа аномеров) и стереоспецифичность (образование только β-1,4-гликозидной связи между мономерами), что позволяет получать соединения со строго опреде-ленной структурой. Для биосинтеза преимущественно используют ферменты гликозилтрансферазы, контро-лирующие образование гликозидной связи в результате трансгликозилирования, при этом донорами гликозиль-ных остатков могут быть нуклеотиды и фосфаты сахаров (Endo, 2000;Perugino et al, 2004;Murata, Usui, 2006). Основные трудности при проведении ферментативного синтеза связаны с выделением ферментов из природных источников.…”