A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial...

Area of Science:

• Synthetic biology
• Microbial engineering
• Biotechnology

Background:

• Industrial yeast strain development is vital for efficient biomanufacturing.
• Current methods face limitations due to long timelines and low mutation rates.
• Optimizing yeast hosts requires overcoming challenges in generating diverse mutants.

Purpose of the Study:

• To develop a novel system for rapid generation of abundant yeast mutants.
• To engineer a hypermutation yeast host for industrial applications.
• To enhance the robustness and productivity of industrial yeast strains.

Main Methods:

• Introduction of the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system.
• Utilizing nonhomologous-end-joining (NHEJ) repair for accelerated mutagenesis.
• Modification of rad52 in *Saccharomyces cerevisiae* to create a hypermutation host.
• Iterative evolution under multiple environmental stresses.
• High-throughput screening for desired yeast phenotypes.

Main Results:

• Achieved a 400-fold enhancement in mutation ability in the engineered yeast host.
• Rapidly generated millions of mutants within a few rounds of iterative evolution.
• Isolated a robust industrial *Saccharomyces cerevisiae* strain (G4-72) with higher productivity.
• Demonstrated stable growth and efficient ethanol production under high temperature and osmotic stress.
• Pilot-scale fermentation showed a 6.9% increase in ethanol production under industrial conditions.

Conclusions:

• The GEbCD system is a powerful tool for rapid mutant generation and host optimization.
• The engineered hypermutation yeast host significantly improves industrial biomanufacturing efficiency.
• This strategy offers a novel approach for tailoring microbial chassis to industrial demands.
• Developed a robust yeast strain (G4-72) suitable for high-stress industrial fermentation environments.