DNA Double-Strand Break-Induced Gene Amplification in Yeast

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Precise control of gene copy number in Saccharomyces cerevisiae is crucial for understanding enzyme functions and optimizing cell factories.
• Existing methods offer high gene expression but lack accurate gene dosage control and genetic stability.
• Instability leads to gene copy loss during extended yeast cultivation.

Purpose of the Study:

• To develop a novel method for constructing yeast strains with defined and stable gene copy numbers.
• To enable precise gene dosage control for applications in enzyme function elucidation and metabolic engineering.

Main Methods:

• Introduction of the CASCADE system for gene amplification in Saccharomyces cerevisiae.
• Amplification of genes of interest up to nine copies using the CASCADE system.
• Assessment of strain stability during prolonged cultivation in selection-free media.

Main Results:

• The CASCADE system allows for the construction of yeast strains with defined gene copy numbers.
• Genes can be amplified up to nine copies, with potential for further expansion.
• Generated yeast strains exhibit stable propagation without the need for selective pressure.

Conclusions:

• CASCADE provides a robust solution for achieving precise and stable gene copy number control in yeast.
• This method facilitates accurate gene dosage essential for both fundamental research and biotechnological applications.
• Stable, defined-copy strains are valuable for advancing synthetic biology and metabolic engineering efforts.