Abstract Background:Eukaryotic organisms, like the model yeast S. cerevisiae, have linear chromosomes that facilitate organization and protection of nuclear DNA. A recent work described a stepwise break/repair method that enabled fusion of the sixteen chromosomes of S. cerevisiae into a single large chromosome. Construction of this strain resulted in the removal of 30 of 32 telomeres, over 300kb of subtelomeric DNA, and 107 subtelomeric ORFs. Despite these changes, characterization of the single chromosome strain uncovered modest phenotypes compared to a reference strain.Results:This study further characterized the single chromosome strain and found that it exhibited a longer lag phase, increased doubling time, and lower final biomass concentration compared with a reference strain when grown on YPD. These phenotypes were amplified when ethanol was added to the medium or used as the sole carbon source. RNAseq analysis showed poor induction of genes involved in diauxic shift, ethanol metabolism, and fatty-acid ß-oxidation during growth on ethanol compared to the reference strain. Enzyme-constrained metabolic modeling identified decreased flux through the enzymes that are encoded by these poorly induced genes as a likely cause of diminished biomass accumulation. The diminished growth on ethanol for the single chromosome strain was rescued by nicotinamide, an inhibitor of sirtuin family deacetylases, which have been shown to silence gene expression in heterochromatic regions. Conclusions:Our results indicate that sirtuin-mediated silencing in the single chromosome strain interferes with growth on non-fermentable carbon sources. We propose that the removal of subtelomeric DNA that would otherwise be bound by sirtuins leads to silencing at other loci in the single chromosome strain. Further, we hypothesize that the poorly induced genes in the single chromosome strain during ethanol growth could be silenced by sirtuins in wildtype S. cerevisiae during growth on glucose.