Bacterial Coupling

The bacterial coupling technology, developed by Kyowa Hakko Kogyo Co. Ltd. in Japan (57,63,116-118), initiated a new era in large-scale enzymatic synthesis of oligosaccharides. The key to Kyowa Hakko's technology was a Corynebacterium ammoniagenes bacterial strain engineered to efficiently convert inexpensive orotic acid to UTP (Scheme 12). When combined with an E. coli strain engineered to overexpress UDP-Gal biosynthetic genes including galK, galT, galU, and ppa (pyrophosphatase), UDP-Gal was accumulated in the reaction solution (72 mM/ 21 h). When these two strains were combined with another recombinant E. coli strain, overexpression of the a-1,4-galactosyltransferase LgtC gene of Neisseria gonorrhoeae produced a high concentration of globotriose (188 g/liter) (118).

C. ammoniagenes DN510

Orotic acid

Glucose Fructose

Globotriose (188 g/L)

UDP VLgtC

E. coli NM522/pGT5

E. coli NM522/pNT25/pNT32

Lactose (100 g/L)

PPi 2Pi r

Glc-1-P UDP-Glc GalT

Galactose (100 g/L)

Scheme 12 Large-scale production of globotriose through coupling of engineered bacteria.

Kyowa Hakko also achieved large-scale production of other sugar nucleotides and related oligosaccharides with the utilization of this bacterial coupling concept. For fucosylated carbohydrate synthesis, the combination of genetically engineered E. coli overexpressing GDP-fucose biosynthetic genes and C. ammoniagenes produced 18.4 g/liter GDP-fucose after 22 h reaction. Total 21 g/liter of LewisX were synthesized using this system including a-1,3-fucosyltransferase

(121). Large-scale synthesis of sialylated carbohydrates was also reported (117). However, despite the striking feature of producing sugar nucleotides cost-effectively, the bacterial coupling method still suffers from the need of multiple fermentations of several bacterial strains and transport of substrates between different bacterial strains.

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