Construction of Plasmid Vectors and Transformation of the Marine Yeast Debaryomyces hansenii

Abstract

We have constructed two plasmid vectors (pMR95 and pMR96) with selectable markers for the marine yeast Debaryomyces hansenii. Plasmid pMR95 contains an autonomously replicating sequence previously isolated from Debaryomyces and a hygromycin B resistance gene from the plasmid pLG90 under the control of the isocytochrome C1 promoter and terminator sequences, while pMR96 has, in addition, the Saccharomyces URA3 gene. Transformation in Debaryomyces was accomplished by electroporation. Plasmid pMR95 was capable of transforming both Saccharomyces cerevisiae and D. hansenii to hygromycin resistance at low frequencies; pMR96 transformed both yeasts at low frequencies when selected for hygromycin B resistance and at very high efficiencies when selected for uracil prototrophy. The presence of the plasmids in the transformed yeast was confirmed by polymerase chain reaction. The plasmids could be recovered back in Escherichia coli when transformed with total DNA from the yeast transformants, indicating at least a partial autonomous existence of the plasmids in the marine yeast. To our knowledge this is the first successful attempt to transform D. hansenii.