FASTOP─Fast Editing Toolkit for Top Expression Sites in Yeast

Budding yeast Saccharomyces cerevisiae is a widely used chassis for producing food- and agriculture-related compounds, but construction of multienzyme pathways often requires repeated genomic integration. We developed FASTOP, a genome-editing toolkit for rapid engineering of eight transcriptionally active genomic hot-spot loci. FASTOP combines selectable markers, extended homology arms, and Cas9/gRNA-assisted integration, and enables PCR-free release of donor cassettes by restriction digestion to reduce sequence errors introduced during amplification. After characterizing the sites, we integrated a five-gene naringenin biosynthetic pathway into the hot spots as a proof of concept. Our results demonstrated that, upon expressing the five essential genes in simple shake-flask culture, naringenin production reached 505.7 mg/L, 6-fold higher than an episomal control expressing the same pathway genes and 4.5-fold higher than the previously reported strain using additional precursor engineering and controlled bioreactor cultivation. FASTOP therefore provides a practical platform for the rapid assembly of multienzyme pathways in yeast for stable heterologous gene expression.