This TSR1 gene associated with the secretion of Proteins of the yeast Yarrowia lipolytica was deleted. Transformation of a Y. lipolytica strain.with the disrupted tsr1 fragment resulted in the substitution of partially deleted TSR1 gene for the wild-type TSR1 gene on the chromosome. The result of southern hybridization show that the tsr1 mutants have been constructed. The isolation of these mutants would be useful for studing the function of TSR1 gene in yeast Y. lipolytica.