An expanded library of 76 integration sites for gene expression in <i>Saccharomyces cerevisiae</i>

Abstract Constructing efficient yeast cell factories involves introducing heterologous biosynthetic pathways and overexpressing key genes. Chromosomal integration of recombinant genes is preferred over episomal plasmids for greater stability during large‐scale industrial cultivation. The expression of complex pathways in engineered microbes necessitates the activation of an increasing number of genes, a process limited by the availability of suitable integration sites. To address this challenge, we investigated 125 potential chromosomal sites in Saccharomyces cerevisiae by inserting mCherry using the CRISPR/Cas9 technique to evaluate their capacity to integrate and express heterologous genes. Subsequently, 76 sites were identified to support effective integration with genomic stability. Furthermore, to demonstrate the potential for multiplexed engineering, we successfully performed a one‐step, four‐locus integration of the β‐carotene pathway using the characterized sites. The expanded integration sites are expected to be valuable for constructing yeast cell factories for applications in synthetic biology and metabolic engineering.