A sucrase mutant of Zymomonas mobilis Zsuc1, which was unable to grow on sucrose medium, was isolated and characterized. This mutant did not produce extracellular sucrase (SacC) and levansucrase (SacB). A 1.7-kb DNA fragment from plasmid pLSS41 carrying sacB gene of Z. mobilis was subcloned into an Escherichia coli-Z. mobilis shuttle vector, pZA22. The resultant recombinant plasmid pLSD19 was transferred into the mutant Zsuc1. The transconjugant Zsuc1 (pLSD19) grew well on sucrose and produced a maximum of 5 g/l levan, whereas its parent strain Z, mobilis B14023 produced 6.8 g/l levan from sucrose fermentation. The recombinant strain produced an extracellular SacB activity of 48 U/ml, whereas its parent strain produced 119 U/ml. The parent strain was also transformed with the plasmid pLSD19; the resulting transconjugant B14023 (pLSD19) produced a high level of extracellular SacB activity (187 U/ml) and levan (10.7 g/l). In the absence of selection pressure, 75% of the transconjugants retained the recombinant plasmid pLSD19.