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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 auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known  P. pastoris AOX1 ,  ARG4  and  HIS4  genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the  ku70  deletion strain will further support protein production strain generation and synthetic biology using  P. pastoris  strains as platform hosts.

Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology