title: Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis.
abstract: Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants μg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8 × 10(3) transformants μg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non-homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available.
authors.affiliation: Department for Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia.
authors.forename: Marina
authors.initials: M
authors.lastname: Miklenić
authors.affiliation: Department for Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia.
authors.forename: Bojan
authors.initials: B
authors.lastname: Žunar
authors.affiliation: Department for Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia.
authors.forename: Anamarija
authors.initials: A
authors.lastname: Štafa
authors.affiliation: Department for Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia iksvetec@pbf.hr.
authors.forename: Ivan-Krešimir
authors.initials: IK
authors.lastname: Svetec
mesh_terms: D055168:Brettanomyces / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D014170:Transformation, Genetic
keywords: Dekkera/Brettanomyces bruxellensis; LiAc/DTT electroporation; genetic transformation; non-Saccharomyces yeast transformation