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

The sexual life cycle represents a natural engine for promoting genetic diversity in yeast. However, harnessing it for controllable, rapid evolution in industrial yeast strains remains a big challenge. Here, we developed quantitative mating and sporulation methods for an industrial yeast Komagataella phaffii. Functional genes for its sexual life cycle were identified, and deficiency of key sporulation targets generated stable diploids with enhanced stress tolerance and recombinant protein production. A fully programmable life-cycle system was constructed, for which mating and sporulation are orthogonally controlled by different inducible signals. Leveraging this system, we developed an in situ mating-sporulation shift (iMASS) platform in liquid culture. Applications of iMASS under diverse selection pressures accelerated cell evolution, generating strains with improved robustness and protein productions. Our work proposes a paradigm that integrates rational design with controlled sexual reproduction for directed microbial evolution.