Yeast flocculation offers advantages for fuel ethanol production such as high cell density fermentation through the self-immobilization of yeast cells within fermentors as well as cost-effective biomass recovery through sedimentation of yeast flocs. However, many ethanol-fermenting yeast strains are naturally non-flocculating. In this work, a new flocculating gene was isolated from the genomic library of the industrial flocculating yeast SPSC01. Analysis of the 8049 bp gene sequence indicated that the gene is highly similar to FLO1 gene from the model yeast strain Saccharomyces cerevisiae S288C, but with more repeated sequences. Disruption of FLO1(spsc) via homologous recombination led to a loss of the flocculating phenotype. Moreover, a non-flocculating industrial yeast strain 6525 transformed with an expression cassette containing the 5.2 kb PCR product of FLO1(spsc) under the transcriptional control of the PGK1 promoter exhibited strong flocculation property. Fermentations carried out with the transformants indicated that the same ethanol titer could be achieved with less biomass in the new flocculating yeast 6525 FLO1 using high concentration sugar (250 g/l). This is the first report that such a long FLO1-derivative gene of 8049 bp was identified, which provides basis for engineering yeast strains with the flocculating phenotype for more efficient fuel ethanol production. (c) 2012 Elsevier Ltd. All rights reserved.