Abstract
High-copy integration of key genes offers a promising strategy for efficient biosynthesis of valuable natural products in Saccharomyces cerevisiae. However, traditional multi-copy gene integration methods meet challenges including low efficiency and labor-intensive screening processes. In this study, we developed the IMIGE (Iterative Multi-copy Integration by Gene Editing) system, a CRISPR/Cas9-based approach that exploits both δ and rDNA repetitive sequences for simultaneous multi-copy integrations in S. cerevisiae. This system combines the mixture of Cas9-sgRNA expression vectors with a split-marker strategy for efficient donor DNA assembly in vivo and enables rapid, iterative screening through growth-related phenotypes. When applied to the biosynthesis of ergothioneine and cordycepin, the IMIGE system achieved significant yield improvements, with titers of 105.31 ± 1.53 mg/L and 62.01 ± 2.4 mg/L, respectively, within just two screening cycles (5.5-6 days in total). These yields represent increases of 407.39 % and 222.13 %, respectively, compared to the strains with episomal expression. By streamlining the integration process, utilizing growth-based selection, and minimizing screening demands in both equipment and labor, the IMIGE system could provide an efficient and scalable platform for high-throughput strain engineering, facilitating enhanced microbial production of a wide range of bioproducts.