Matanović Angela, Arambašić Kristian, Žunar Bojan, Štafa Anamarija, Svetec Miklenić Marina, Šantek Božidar, Svetec Ivan-Krešimir
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10000 Zagreb, Croatia.
J Fungi (Basel). 2022 May 20;8(5):531. doi: 10.3390/jof8050531.
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 subphylum, including those already in use (, 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 subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of 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 subphylum. To provide a proof of principle, we successfully transformed three species from the genus (, and ) with high efficiencies (up to 8 × 10 in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 10 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 can be used as a replicative plasmid for these three species.
非传统酵母越来越多地被研究并用作生物技术过程中的生产者，与传统的成熟系统相比，它们往往具有优势。大多数具有生物技术意义的非传统酵母属于亚门，包括那些已经在使用的酵母（如 等），以及那些有前景但尚未得到充分表征的酵母。此外，对于许多这类酵母来说，构建菌株所需的基因工程基本工具，包括高效基因转化、异源蛋白表达和精确基因修饰的程序，都很缺乏。本研究的首要目标是构建一套整合型和复制型质粒，可用于亚门中的各种酵母。此外，我们在此证明，我们之前开发的用于 转化的电穿孔程序可应用于各种酵母，这与构建的质粒一起，为对亚门酵母进行转化提供了坚实的起点。为了提供原理证明，我们成功地高效转化了 属的三个物种（ 、 和 ）（非同源线性 DNA 非法整合时效率高达 8×10 ，复制型质粒时效率高达 4.7×10 ）。对于后两个物种，这是首次报道的基因转化。此外，我们发现携带 复制起点的质粒可作为这三个 物种的复制型质粒。
