| Summary: | Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the <i>Saccharomycotina</i> subphylum, including those already in use (<i>Pichia pastoris, Yarrowia lypolitica</i>, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the <i>Saccharomycotina</i> subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of <i>B. bruxellensis</i> can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the <i>Saccharomycotina</i> subphylum. To provide a proof of principle, we successfully transformed three species from the <i>Schwanniomyces</i> genus (<i>S. polymorphus var. polymorphus</i>, <i>S. polymorphus var. africanus</i> and <i>S. pseudopolymorphus</i>) with high efficiencies (up to 8 × 10<sup>3</sup> in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 10<sup>5</sup> in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from <i>Scheffersomyces stipitis</i> can be used as a replicative plasmid for these three <i>Schwanniomyces</i> species.
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