A rational approach for multicopy delta integration in Saccharomyces cerevisiae targeting a conserved Ty1 LTR sequence

Genomic integration is a foundation in yeast-based cell factories, ensuring phenotypic stability and adequate gene dosage to sustain desired metabolic fluxes. Retrotransposon-associated sequences are attractive genomic integration targets for metabolic engineering, as they are present in multiple copies throughout the genome and their disruption should not jeopardize the cell. In S. cerevisiae, multiple strategies have been proposed to target Ty elements, but little attention has been given to the homology sequence itself. Through analysis of all long terminal repeats in S. cerevisiae, we propose a novel consensus {delta} sequence for efficient multicopy gene integration. Reducing homology length to favor a more conserved sequence greatly increases the number of potential genomic integration sites - reaching 85 targets in this work. The designed integration sequence was validated by introducing a non-native xylose fermentation pathway into this yeast, achieving a single-step simultaneous integration of 15 xylose isomerase copies, enabling efficient ethanol production in the engineered strain.