Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.

Area of Science:

• Microbial biotechnology
• Molecular biology
• Yeast genetics

Background:

• Plasmids are essential tools for recombinant DNA delivery in microbial biotechnology for producing valuable compounds.
• Instability of circular episomal plasmids in Komagataella phaffii necessitates genomic integration, limiting transformation rates and expression uniformity.
• Existing episomal plasmid systems in P. pastoris (formerly K. phaffii) lack the stability and high transformation rates seen in Saccharomyces cerevisiae.

Purpose of the Study:

• To develop and validate a stable episomal plasmid system for Komagataella phaffii.
• To overcome limitations of genomic integration methods for gene expression and strain engineering.
• To enable high-throughput applications and reduce reliance on selective markers.

Main Methods:

• Utilized ARS1 autonomously replicating sequences (ARS) for plasmid replication and partitioning in K. phaffii.
• Implemented various selection markers including antibiotic resistance, auxotrophy, and carbon source utilization.
• Applied ARS1 plasmids for antibiotic-free selection, knockout strain complementation, and transient CRISPR/Cas gene expression.

Main Results:

• Demonstrated successful and stable episomal maintenance of ARS1-containing plasmids in K. phaffii.
• Achieved high transformation rates and low clonal variability comparable to S. cerevisiae episomal systems.
• Validated the utility of ARS1 plasmids for diverse applications, including antibiotic-free selection and CRISPR/Cas systems.

Conclusions:

• ARS1-containing plasmids offer a stable and efficient alternative to genomic integration in K. phaffii.
• This system enhances K. phaffii's utility in research and industrial applications by improving transformation efficiency and expression consistency.
• The developed plasmid system broadens the toolkit for genetic manipulation and protein production in K. phaffii.