We sought to breed an industrially useful yeast strain, specifically an ethanol-tolerant yeast strain that would be optimal for ethanol production, using a novel breeding method, called genome reconstruction, based on chromosome splitting technology. To induce genome reconstruction, Saccharomyces cerevisiae strain SH6310, which contains 31 chromosomes including 12 artificial mini-chromosomes, was continuously cultivated in YPD medium containing 6% to 10% ethanol for 33 days. The 12 mini-chromosomes can be randomly or specifically lost because they do not contain any genes that are essential under high-level ethanol conditions. The strains selected by inducing genome reconstruction grew about ten times more than SH6310 in 8% ethanol. To determine the effect of minichromosome loss on the ethanol tolerance phenotype, PCR and Southern hybridization were performed to detect the remaining mini-chromosomes. These analyses revealed the loss of mini-chromosomes no. 11 and no. 12. Mini-chromosome no. 11 contains ten genes (YKL225W,
To improve yeast strains for bioethanol production, yeasts with ethanol tolerance, thermotolerance, and β-1,3-glucanase activity were bred using yeast genome shuffling. Saccharomyces cerevisiae BY4742Δ exg1/pAInu-exgA, which has extracellular β-1,3-glucanase activity, and the Aspergillus oryzae and S. cerevisiae YKY020 strains, which exhibit ethanol tolerance and thermotolerance, were fused by yeast protoplast fusion. Following cell fusion, four candidate cells (No. 3,9,11, and 12 strains) showing thermotolerance at 40℃ were selected, and their ethanol tolerance (7% ethanol concentration) and β-1,3-glucanase activity were subsequently analyzed. All the phenotypes of the two parent cells were simultaneously expressed in one (No. 11) of the four candidate cells, and this strain was called BYK-F11. The BYK-F11 fused cell showed enhanced cell growth, ethanol tolerance, β-1,3-glucanase activity, and ethanol productivity compared with the BY4742Δexg1/pAInu-exgA and YKY020 strains. The results prove that a new yeast strain with different characters and the same mating type can be easily bred by protoplast fusion of yeasts.
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