Fatty acyl-CoA synthetases are critical enzymes involved in lipid metabolism. The oleaginous yeast Yarrowia lipolytica is currently generating interest in biofuel research due to its ability to convert raw materials into value-added end products. In this study, the putative acyl-CoA synthetase gene YAL1 in Y. lipolytica was interrupted with the copper resistance (CRF1) gene to allow selection without antibiotics to facilitate industrial applications. Deletion of YAL1 led to reduced acyl-CoA synthetase activity. Furthermore, the fatty acid profile and lipid content of the mutant were different from the wild-type strain. The ratio of saturated to unsaturated fatty acids increased 6-fold, and the total lipid production of the mutant strain increased to 1.47-fold of the wild-type strain. The results indicate that YAL1 in Y. lipolytica is involved in fatty acid elongation and desaturation, whereas the homologous, highly conserved FAA1 gene from Saccharomyces cerevisiae was shown to be responsible for fatty acid activation. The increased ratio of saturated to unsaturated fatty acids would result in a higher combustion value and better oxidative stability for biofuel products obtained from the fatty acids from the engineered Y. lipolytica mutant. (C) 2011 Elsevier Ltd. All rights reserved.