In Vivo Plasmid Assembly in NHEJ-Deficient Pichia pastoris Strains

The integration of linear plasmids into the genome is the most common method for genetic engineering of Komagataella phaffii (reclassified from Pichia pastoris). This, however, results in the drawbacks of lower transformation efficiencies and diverse clonal variations of expression levels compared to typical experiments with baker's yeast. This is due to the affected genomic loci or copy number effects, making mutant library screenings or comparative expression studies challenging. The use of autonomously replicating plasmids, which are maintained extrachromosomally, can overcome these limitations. To speed up the library generation process and reduce the bottleneck of plasmid cloning, the episomal plasmids can be directly assembled in vivo in K. phaffii via homology-directed repair in a non-homologous end-joining deficient Δku70 strain. Time-consuming cloning in vitro followed by vector amplification can be avoided. At the same time, in principle, no specific sequences for selection and replication in E. coli are needed, enabling a vector design solely employing yeast DNA. Typically, libraries in the range of 10⁵-10⁶ CFU/μg DNA can be obtained by following the protocol described in this chapter.