deletion of the pichia pastoris ku70 homologue facilitates platform strain generation for gene expression and synthetic biology删除的毕赤酵母属pastoris ku70同系物促进平台应变一代基因表达和合成生物学.pdfVIP

Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology ¨ ¨ 1 2 1,2 1,2 1,2 Laura Naatsaari , Beate Mistlberger , Claudia Ruth , Tanja Hajek , Franz S. Hartner , Anton Glieder1,2* 1 Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria, 2 Austrian Centre of Industrial Biotechnology (ACIB GmbH), Graz, Austria Abstract Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new aux