Pullulan is a water-soluble extracelluler polysaccharide produced aerobically by fungus Aureobasidium pullulans and some other species. Current work focused on developing an A. pullulans strain with enhanced pullulan productivity and raw material utilization ratio using a new method for genome shuffling of A. pullulans N3.387. After the third cycle of the protoplast fusion, a mutant, designated as F3-2, producing 179.7% (20.7 g/L) more pullulan than the wild-type strain was obtained. Furthermore, the raw material utilization ratio of F3-2 was determined as 97.9%, which was 29.0% higher than the wild-type strain. Genetic stability analysis of F3-2 showed that ranges of the pullulan production and the raw material utilization ratio of the generation were 0.13 g/L and 1.93%, respectively, suggesting that F3-2 was genetically stable. Elucidation of this new genome shuffling process is also interesting for enhancing production of extracellular polysaccharide in both A. pullulans and other fungus. (C) 2010 Elsevier Ltd. All rights reserved.