Use of a new catabolite repression resistant promoter isolated from Bacillus subtilis KCC103 for hyper-production of recombinant enzymes.

Bacillus subtilis KCC103 hyper-produces alpha-amylase and the synthesis is resistant to carbon catabolite repression. The strain efficiently produced alpha-amylase in low cost agro-biomass based medium rich in simple sugars without catabolite repression. Here, the catabolite repression resistant promoter (amyR4) of alpha-amylase was isolated from KCC103 and used to synthesize recombinant enzymes in B. subtilis. When the bgaB gene encoding beta-galactosidase of Bacillus stearothermophilus was cloned and expressed under the amyR4 promoter, high level of beta-galactosidase activity was found in Escherichia coli (28 U/ml)) and B. subtilis (19 U/ml). Further, the genes encoding endoxylanase (xynA) and carboxymethyl cellulase (bglC) from B. subtilis were cloned with signal peptides and expressed with CCR-resistant amyR4 promoter. In E. coli, the expression was intracellular with activities of cellulase and xylanase at 76 and 105IU/ml respectively. The expression was extracellular in B. subtilis with activities at 17 and 17 IU/ml of cellulase and xylanase respectively in LB medium. When recombinant B. subtilis was cultured in LB-glucose medium, the synthesis of recombinant enzymes was not subject to catabolite repression and the expression was observed throughout the growth. This is important as glucose in the medium can prevent sporulation of the Bacillus and prevent activation of the other scavenger pathways that leads to degradation of recombinant proteins. The catabolite derepressed promoter of alpha-amylase from B. subtilis KCC103 can be efficiently used for overexpression of various industrial enzymes.