Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus
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&...
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| Format: | Article |
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MDPI AG
2022-05-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/8/5/531 |
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| author | Angela Matanović Kristian Arambašić Bojan Žunar Anamarija Štafa Marina Svetec Miklenić Božidar Šantek Ivan-Krešimir Svetec |
| author_facet | Angela Matanović Kristian Arambašić Bojan Žunar Anamarija Štafa Marina Svetec Miklenić Božidar Šantek Ivan-Krešimir Svetec |
| author_sort | Angela Matanović |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T03:36:18Z |
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| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T03:36:18Z |
| publishDate | 2022-05-01 |
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| series | Journal of Fungi |
| spelling | doaj.art-083f09f75ed34711b91fa27eed35b0682023-11-23T11:42:27ZengMDPI AGJournal of Fungi2309-608X2022-05-018553110.3390/jof8050531Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> GenusAngela Matanović0Kristian Arambašić1Bojan Žunar2Anamarija Štafa3Marina Svetec Miklenić4Božidar Šantek5Ivan-Krešimir Svetec6Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaDepartment of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, CroatiaNon-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.https://www.mdpi.com/2309-608X/8/5/531non-conventional yeasts<i>Saccharomycotina</i> subphylum<i>Schwanniomyces</i> speciesgenetic transformationelectroporationyeast plasmids |
| spellingShingle | Angela Matanović Kristian Arambašić Bojan Žunar Anamarija Štafa Marina Svetec Miklenić Božidar Šantek Ivan-Krešimir Svetec Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus Journal of Fungi non-conventional yeasts <i>Saccharomycotina</i> subphylum <i>Schwanniomyces</i> species genetic transformation electroporation yeast plasmids |
| title | Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus |
| title_full | Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus |
| title_fullStr | Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus |
| title_full_unstemmed | Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus |
| title_short | Toolbox for Genetic Transformation of Non-Conventional <i>Saccharomycotina</i> Yeasts: High Efficiency Transformation of Yeasts Belonging to the <i>Schwanniomyces</i> Genus |
| title_sort | toolbox for genetic transformation of non conventional i saccharomycotina i yeasts high efficiency transformation of yeasts belonging to the i schwanniomyces i genus |
| topic | non-conventional yeasts <i>Saccharomycotina</i> subphylum <i>Schwanniomyces</i> species genetic transformation electroporation yeast plasmids |
| url | https://www.mdpi.com/2309-608X/8/5/531 |
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