A system for multilocus chromosomal integration and transformation-free selection marker rescue

Area of Science:

• Molecular Biology
• Synthetic Biology
• Yeast Genetics

Background:

• Yeast integrating plasmids (YIPs) are crucial for stable DNA integration in Saccharomyces cerevisiae.
• Existing YIP systems are often inefficient, requiring extensive time and labor for cloning and selection marker removal.

Purpose of the Study:

• To develop and validate a new YIP system that accelerates stable integration of multiple expression constructs into various yeast loci.
• To enable scarless integration and efficient selection marker rescue.

Main Methods:

• Design and construction of novel 'directed pop-out' YIPs.
• Utilizing a dedicated 'YIPout' fragment for marker rescue and target DNA preservation.
• Implementing a multifragment modular DNA assembly system for simplified cloning.
• Employing new counterselectable markers for serial integration and transformation-free marker rescue.

Main Results:

• Successfully constructed and tested directed pop-out YIPs for integrating fluorescent reporter genes into four distinct yeast loci.
• Validated the YIP design by integrating three reporter genes into three loci, achieving transformation-free selection marker rescue.
• Demonstrated scarless integration of expression constructs.

Conclusions:

• The new directed pop-out YIP system significantly streamlines the process of stable DNA integration in Saccharomyces cerevisiae.
• This technology facilitates the construction of yeast strains with complex heterologous metabolic pathways.
• The system offers a versatile and efficient tool for yeast genetic engineering.