Reconstructing an Inducible Homologous Recombination System in Ogataea Polymorpha

Ogataea polymorpha is a promising host for industrial production due to its broad substrate spectrum, thermotolerance, and high-density fermentation capability. While recombination machinery engineering has improved genome editing via homologous recombination (HR), constitutive expression of HR-related proteins often causes growth defects. In this study, we divided the complex and multistep HR pathway into three stages and systematically evaluated genes under control of a rhamnose-inducible promoter (P_LRA3). We reconstructed a dynamically regulated HR repair system through co-expression of genes ScRAD51, ScRAD52, and ScSLX4, achieving HR rates up to 58%—a 2-fold increase over the starting strain. Notably, coordinated expression of these genes enhanced simultaneous deletion of two genes with a positive rate of 29%. Our tools enable precise genome editing while maintaining normal cell growth in O. polymorpha, providing a solid foundation for studying recombination machinery in non-conventional yeasts.