CLONING OF THE ACETOBACTER-XYLINUM CELLULASE GENE AND ITS EXPRESSION IN ESCHERICHIA-COLI AND ZYMOMONAS-MOBILIS

A DNA fragment corresponding to carboxy-methylcellulase activity of Acetobacter, xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201-3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.