HIGHLY-EFFICIENT ELECTROTRANSFORMATION OF THE YEAST HANSENULA-PALYMORPHA

A highly-efficient method for transformation of the methylotrophic yea st Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7 x 10(6)/mu g plasmid DNA were obtained by app lying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformati on was dependent on: (1) pretreatment of the cells with the reducing a gent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to th e mid-logarithmic phase. Important parameters for optimizing the trans formation frequencies were field strength, pulse duration, and cell co ncentration during the electric pulse. In contrast to electrotransform ation protocols described for Saccharomyces cerevisiae and Candida mal tosa, transformation frequencies (transformants per mu g DNA) for H. p olymorpha remained high when large amounts (up to 10 mu g) of plasmid DNA were added. This feature renders this procedure pre-eminently adva ntageous for gene cloning experiments when high numbers of transforman ts are needed.