Reconstructing an Inducible Homologous Recombination System in Ogataea Polymorpha

ABSTRACT 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.