Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Pichia pastoris is a methylotrophic yeast with high protein expression capabilities.
• Its ability to utilize methanol as a carbon source makes it a promising platform for C1 biotransformation.
• Limited synthetic biology tools hinder the manipulation of multigene pathways in P. pastoris.

Purpose of the Study:

• To establish a CRISPR/Cas9 system for efficient multiplex genome integration in P. pastoris.
• To develop a toolkit for constructing P. pastoris cell factories with multiple heterologous genes.
• To demonstrate the system's application in engineering P. pastoris for 2,3-butanediol (BDO) production.

Main Methods:

• Development of a CRISPR-based multiplex genome integration toolkit.
• Construction of donor plasmids for gene integration.
• Preparation of competent P. pastoris cells and transformation.
• Verification of transformants and pathway functionality.

Main Results:

• Successful establishment of the CRISPR/Cas9 system for efficient multi-gene integration in P. pastoris.
• Demonstration of the toolkit's utility by constructing engineered P. pastoris for methanol-to-BDO conversion.
• Detailed protocol provided for constructing P. pastoris cell factories.

Conclusions:

• The developed CRISPR toolkit significantly enhances the synthetic biology capabilities of P. pastoris.
• This facilitates the construction of robust P. pastoris cell factories for producing high-value compounds via multi-gene pathways.
• Lays the groundwork for advanced C1 biotransformation applications in P. pastoris.