The aim of this study was to develop anew fungal strain that simultaneously amplifies the carbon source spectrum and increases arachidonic acid (AA) productivity using genome shuffling between Diasporangium sp and inactive Aspergillus niger. Through three rounds of genome shuffling. one of the stable daughter strains (F-1) acquired the ability to produce arachidonic acid and utilize various carbon sources. Compared to the parental Diasporangium sp.. which could only use four out of eight carbon Sources tested, F, could utilize all eight carbon sources During fermentation with CMC-Na as the carbon Source, F, was able to obtain 30 16% of lipid effectively whereas the parental Diasporangium; sp. was not able to grow at all. When glucose was used as the carbon Source, the CMCase activity of F, was 879 36 U, 298 23% higher than that of the parental Diasporangium sp. Under optimized fermentation conditions in a 5-L fermentation container, the AA yield of F, reached 0 81 g/l, 94.78% higher than hat of the parental generation. These results indicate that inter-kingdom genome shuffling can be used Successfully in eukaryotic microorganisms and that it can effectively improve the production of desired metabolites within a short period of time The findings of this Study may be useful for extending the application of genome shuffling in eukaryotic microbial breeding. (C) 2009 Elsevier Inc. All rights reserved