To direct the secretory expression of Zymomonas mobilis levansucrase (LevU) in the methylotrophic yeast Hansenula polymorpha, the Z mobilis levU gene was fused to the inulinase signal sequence under the control of the methanol oxidase promoter (P-MOX). The vector containing the P-MOX-levU expression cassette was introduced into H. polymorpha and multiply integrated into the host chromosomes. Recombinant LevU was expressed as an active and soluble form in H. polymorpha transformants, but the recombinant protein accumulated mainly inside the cells, representing about 5% of total cell proteins in shake-flask cultivations. Interestingly, a significant fraction of the recombinant LevU was secreted into the culture supernatant with a maximum yield of 12,200 U/l using fed-batch fermentations with methanol feeding. H. polymorpha-derived recombinant LevU displayed catalytic activities comparable to those of the Escherichia coli-derived recombinant LevU, producing levans with high molecular weights. (C) 2003 Elsevier Inc. All rights reserved.