Transferring functional chromosomes between intergeneric yeast generates monochromosomal hybrids with improved phenotypes and transcriptional responses

Interspecific hybrids can exhibit phenotypes that surpass those of their parents, known as heterosis, which is often of interest for industrial applications and evolutionary genetics research. However, constructing hybrids between distantly related species, such as intergeneric yeasts, presents technical challenges. In this study, we established a method to transfer individual chromosomes from Saccharomyces cerevisiae (Sc) into Kluyveromyces marxianus (Km), an emerging model for bioproduction. The Sc chromosome of interest was circularized, genetically modified to carry Km centromeres and replication origins, and transformed into Km via protoplast transformation. With this method, we generated monochromosomal hybrids with eight Km chromosomes and Sc chromosome I or III. The Sc chromosomes exhibited normal replication, segregation, and active transcription in the hybrids. The hybrids displayed heterosis in flocculation and salt tolerance due to the overexpression of FLO9 and SPS22 , respectively. Transcriptomic analysis revealed that both cis- and trans -regulatory changes contributed to the divergence of gene expression between the two species, with the cis and trans effects often acting in a compensatory manner. Our strategy has potential applications in optimizing cell factories, constructing synthetic genomes, and advancing evolutionary research.