LEU2 directed expression of beta-galactosidase activity and phleomycin resistance in Yarrowia lipolytica.

The nucleotide sequence of a 968 bp DNA fragment spanning the promoter and the 5' upstream sequence of the LEU2 coding sequence of the yeast Yarrowia lipolytica has been determined. A LEU2::lacZ fusion has been constructed and expressed in transformed yeast cells, showing that as few as 232 bp of the LEU2 promotor were sufficient to direct gene expression. In order to develop new markers for transformation of this yeast, the LEU2 initiation codon was destroyed by in vitro mutagenesis and replaced by a cloning site. A gene conferring phleomycin resistance in E. coli was attached to the LEU2 promoter and shown to be efficiently expressed in yeast: direct selection of phleomycin resistant transformants was possible.