Abstract
Interspecific genetic exchanges caused by natural hybridization or horizontal gene transfer can lead to enhanced phenotypes, which are often of interest for industrial applications and evolutionary research. However, transferring genetic materials between distantly related species, such as intergeneric yeasts, presents technical challenges. In this study, we establish a method to transfer individual chromosomes from Saccharomyces cerevisiae (Sc) into Kluyveromyces marxianus (Km), an emerging model for bioproduction. The Sc chromosome of interest is circularized, genetically modified to carry Km centromeres and replication origins, and transformed into Km via protoplast transformation. Using this method, we generate two synthetic strains, each containing a full set of Km chromosomes and either Sc chromosome I or III. The Sc chromosomes exhibit normal replication, segregation, and active transcription after the transfer. The synthetic strains display enhanced phenotypes in flocculation and salt tolerance, which is found to be caused by transgressive expression of FLO9 and SPS22 on the transferred Sc chromosomes, respectively. Transcriptomic analysis reveal that transgressive expression is prevalent among the transferred Sc genes, suggesting evolution of lineage-specific cis- and trans-regulatory interactions across a long evolutionary timescale. Our strategy has potential applications in optimizing cell factories, constructing synthetic genomes, and advancing evolutionary research.