ABSTRACT

After the first demonstration of “recombinant” oligosaccharide production, this technology was carried forward by scientists from

Tokyo Research Laboratories of Kyowa Hakko Kogyo Co. Ltd. in Japan. A series of research reports have been published starting with an article in Nature Biotechnology in 1998 (Koizumi et al., 1998). There are two distinct characteristics of their technology as compared with the original one developed by Samain and co-workers. Firstly, three strains of two different microorganisms were used in the production process: two recombinant E. coli strains and one Corynebacterium ammoniagenes strain. Four genes encoding enzymes involved in the UDP-galactose (UDP-Gal) biosynthesis pathway (galK for galactokinase, galT for galactose-1-phosphate uridyltransferase, galU for glucose-1-phoshpate uridyltransferase, and ppa for pyrophosphatase) were overexpressed in E. coli NM522 strain, an auxotrophic strain that lacks lactose metabolism pathway. UTP, an indispensable substrate for UDP-Gal synthesis, was produced from an inexpensive raw material, orotic acid, by C. ammoniagenes DN510, taking advantage of its strong ATP regeneration activity. The other recombinant E. coli NM522 strain harboring an α1,4-galactosyltransferase from Neisseria gonorrhoeae was used as a biocatalyst for the glycosylation reaction.Secondly, cell fermentation and oligosaccharide synthesis

reaction were allowed to proceed successively. The cells of different bacterial strains were collected after separate fermentation and then mixed together as biocatalysts with the supply of raw materials and reagents to permeabilize the cells (Fig. 14.3). The permeabilization facilitates the substrate intake and product expulsion of the cells. Although the permeabilized cells were not healthy, they could

Figure 14.2 The production of chitopentaose by recombinant E. coli strain harboring NodB and NodC. GlcNAc: N-acetylglucosamine; NodC: chitooligosaccharide synthase (an N-acetylglucosaminyl transferase); NodB: chitooligosaccharide N-deacetylase.