We constructed dihomo-gamma-linolenic acid (DGLA)-producing strains with disruption of the Delta 5-desaturase (Delta 5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Delta lig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the A lig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Delta lig4 strain was constructed, and then transformed for disruption of Delta 5ds. The isolation of nine Delta 5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Delta 5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.