Acetate-mediated pH-stat fed-batch cultivation of transconjugant <Emphasis Type="Italic">Enterobacter</Emphasis> sp. BL-2S over-expressing <Emphasis Type="Italic">glmS</Emphasis> gene for excretive production of microbial polyglucosamine PGB-1

A unique cationic polyglucosamine biopolymer PGB-1 comprising more than 95% D-glucosamine was excretively produced from a new bacterial strain Enterobacter sp. BL-2 under acetate-mediated culture conditions. Since the biopolymer PGB-1 could be synthesized from the UDP-N-acetylglucosamine monomer derived from the hexosamine pathway, three glmS, glmM, and glmU genes in the hexosamine pathway were cloned from Enterobacter sp. BL-2, and their molecular structures were elucidated. The cloned glmS, glmM, and glmU genes were reintroduced into the parent strain Enterobacter sp. BL-2 through a conjugative transformation for the overproduction of the biopolymer PGB-1. The biopolymer production increased 1.5-fold in the transconjugant Enterobacter sp. BL-2S over-expressing the first-step glmS gene encoding glucosamine-6-phosphate synthase. The transconjugant Enterobacter sp. BL-2S was cultivated pH-stat fed-batch widely, while intermittently feeding an acetate solution to maintain a constant pH level of 8.0 for 72 h, resulting in 1.15 g/L of the extracellular polyglucosamine biopolymer PGB-1.