Programmable and controllable sexual life cycle for improved evolution in Komegataella phaffii

Area of Science:

• Microbiology
• Synthetic Biology
• Industrial Biotechnology

Background:

• The sexual life cycle is crucial for genetic diversity in yeast but challenging to control in industrial strains.
• Developing controllable sexual reproduction methods is key for rapid, directed evolution of industrial yeast.

Purpose of the Study:

• To develop quantitative mating and sporulation methods for industrial yeast Komagataella phaffii.
• To engineer stable diploid yeast strains with enhanced traits like stress tolerance and recombinant protein production.
• To create a programmable life-cycle system for accelerated microbial evolution.

Main Methods:

• Quantitative mating and sporulation assays were established for Komagataella phaffii.
• Key genes regulating the sexual life cycle were identified and manipulated.
• A programmable life-cycle system with orthogonal inducible controls for mating and sporulation was constructed.
• An in situ mating-sporulation shift (iMASS) platform was developed for liquid cultures.

Main Results:

• Functional genes for the yeast sexual life cycle were identified.
• Disrupting key sporulation genes yielded stable diploids with improved stress tolerance and protein production.
• The iMASS platform accelerated cell evolution under various selection pressures.
• Engineered strains exhibited enhanced robustness and protein production.

Conclusions:

• A controllable sexual reproduction system for industrial yeast was successfully developed.
• This system enables directed evolution for improved industrial yeast strains.
• The integration of rational design and controlled sexual reproduction offers a new paradigm for microbial evolution.