Citation: Matanovi´ c, A.; Arambaši´ c, K.; Žunar, B.; Štafa, A.; Svetec Mikleni´ c, M.; Šantek, B.; Svetec, I.-K. Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus. J. Fungi 2022, 8, 531. https:// doi.org/10.3390/jof8050531 Academic Editor: Long Liu Received: 24 April 2022 Accepted: 17 May 2022 Published: 20 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Fungi Journal of Article Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus Angela Matanovi´ c † , Kristian Arambaši´ c † , Bojan Žunar , Anamarija Štafa , Marina Svetec Mikleni´ c, Božidar Šantek and Ivan-Krešimir Svetec * Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, Croatia; amatanovic@pbf.hr (A.M.); kristian.arambasic@outlook.com (K.A.); bzunar@pbf.hr (B.Ž.); anamarija.stafa@gmail.com (A.Š.); mmiklenic@pbf.hr (M.S.M.); bsantek@pbf.hr (B.Š.) * Correspondence: iksvetec@pbf.hr; Tel.: +385-14836016 † These authors contribute equally to this work. Abstract: 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 Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, 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 Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis 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 Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 10 3 in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 10 5 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 Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species. Keywords: non-conventional yeasts; Saccharomycotina subphylum; Schwanniomyces species; genetic transformation; electroporation; yeast plasmids 1. Introduction In recent years non-conventional yeasts, i.e., all those yeast species beyond well- established model organisms Saccharomyces cerevisiae and Schizosaccharomyces pombe, are rapidly gaining the interest of researchers and industry [1]. It is becoming clear that the diversity of yeasts offers vast resources in terms of new compounds and biochemical pathways, as well as other biotechnologically interesting features such as thermotolerance, halotolerance, etc. For example, Yarrowia lipolytica has been used in single-cell protein production from hydrocarbons and in production of citric acid and lipases also produced by Candida cylndracea, Eremothecium gossypii is a producer of riboflavin, Kluyveromyces marx- ianus produces lactase, Komatogella (Pichia) pastoris and Ogatea (Hansenula) polymorpha are important systems for heterologous protein expression, while Pachysolen tannophilus, Schef- fersomyces (Pichia) stipitis, Candida shehatae, Spathaspora passalidarum and Candida jeffriesii can ferment D-xylose to ethanol and are being researched as possible producers of bioethanol J. Fungi 2022, 8, 531. https://doi.org/10.3390/jof8050531 https://www.mdpi.com/journal/jof