Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus

Abstract

Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl 2-mediated transformation of protoplasts. Electroporation yielded 100–200 transformants per μg of DNA per 10 8 cells after 3 days on selective medium. For its part, ATMT yielded 60–160 transformants per 10 6 input cfu after 5–10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl 2-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.