Abstract

A series of mating- and sporulation-specific genes were identified in K. phaffii . • Deficiency of sporulation targets generated dominant and stable diploids. • Orthogonal induction enabled programmable control of the sexual life cycle. • In situ mating-sporulation shift platform explored for improved evolution in yeast. 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. Download: Download high-res image (283KB) Download: Download full-size image