Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris

Area of Science:

• Biotechnology and Synthetic Biology
• Microbial Engineering
• Molecular Biology

Background:

• Pichia pastoris is a methylotrophic yeast widely used for protein and fine chemical production.
• High cell densities and strong inducible promoters make it suitable for industrial applications.
• Current genome engineering protocols in P. pastoris are complex and time-consuming, with limited selection markers.

Purpose of the Study:

• To develop a rapid and efficient gene disruption method for Pichia pastoris.
• To enable repetitive genome engineering cycles through marker recycling.
• To simplify cloning procedures and accelerate knockout generation.

Main Methods:

• Development of a novel gene knockout strategy utilizing marker recycling.
• Creation of ready-to-use knockout vectors for P. pastoris.
• Application of the method for efficient gene disruption.

Main Results:

• Achieved fast and efficient gene disruption in P. pastoris.
• Demonstrated the utility of marker recycling for repetitive genome engineering.
• Streamlined cloning and knockout generation processes.

Conclusions:

• The described method significantly improves genome engineering efficiency in P. pastoris.
• Marker recycling facilitates extensive genetic modifications, advancing cell factory development.
• The ready-to-use vectors simplify and accelerate the generation of gene knockouts.