Cloning of Phanerochaete chrysosporium leu2 by complementation of bacterial auxotrophs and transformation of fungal auxotrophs

Abstract

A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in both Escherichia coli and Saccharomyces cerevisiae. This expression vector, λYES, contains the lacZ promoter for expression in E. coli and the GALl promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from P. chrysosporium was characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.