In Vivo Plasmid Assembly in NHEJ-Deficient Pichia pastoris Strains.

Area of Science:

• Biotechnology
• Molecular Biology
• Yeast Genetics

Background:

• Linear plasmid integration into the Komagataella phaffii genome is common for genetic engineering but causes low transformation efficiency and expression variability.
• These issues stem from genomic locus effects and copy number variations, complicating mutant library screening and comparative studies.
• Autonomously replicating, extrachromosomal plasmids offer a solution to overcome these limitations.

Purpose of the Study:

• To develop a streamlined method for generating mutant libraries in Komagataella phaffii.
• To overcome the limitations of genomic integration, such as low transformation efficiency and clonal variation.
• To accelerate the plasmid cloning process for yeast genetic engineering.

Main Methods:

• Utilizing episomal plasmids for extrachromosomal maintenance in K. phaffii.
• Employing in vivo assembly of plasmids via homology-directed repair.
• Performing assembly in a non-homologous end-joining deficient Δku70 K. phaffii strain.
• Avoiding in vitro cloning and vector amplification steps.

Main Results:

• Direct in vivo assembly of episomal plasmids in K. phaffii was achieved.
• The method bypasses the need for in vitro cloning and E. coli-based vector amplification.
• High transformation efficiencies, yielding libraries of 10^5-10^6 CFU/μg DNA, were obtained.

Conclusions:

• In vivo episomal plasmid assembly in Δku70 K. phaffii is an efficient strategy for rapid library generation.
• This method overcomes limitations associated with genomic integration, enabling robust yeast genetic engineering.
• The protocol facilitates faster and more reliable mutant library screening and expression studies in K. phaffii.