Metabolic switching of sake yeast by kimoto lactic acid bacteria through the [GAR+] non-genetic element.

In traditional kimoto-type sake production, cells of Saccharomyces cerevisiae sake yeast are grown in a starter mash generated by lactate fermentation by lactic acid bacteria (LAB) such as Leuconostoc mesenteroides and Lactobacillus sakei. However, the microbial interactions between sake yeast and kimoto LAB have not been well analyzed. Since the formation of a prion-like element (designated [GAR+]) in yeast cells is promoted by bacteria, we here examined the associated phenotype (i.e., increased glucosamine resistance) in sake yeast strains K701 (a representative sake strain) and Km67 (a strain isolated from kimoto-type sake mash). Approximately 0.5% of K701 and Km67 cells, as well as 0.2% of laboratory strain X2180 cells, exhibited increased glucosamine resistance under pure culture conditions, and the frequency of this metabolic switching was further enhanced by coculture with kimoto LAB. The LAB-promoted emergence of the glucosamine-resistant cells was the most prominent in Km67, suggesting that this strain possesses an advanced mechanism for response to LAB. While the glucosamine-resistant clones of X2180 and K701 exhibited lower rates of alcoholic fermentation under high-glucose conditions than did the respective naive strains, glucosamine resistance did not severely affect alcoholic fermentation in Km67. The population of dead cells after alcoholic fermentation was decreased in the glucosamine-resistant clones of X2180, K701, and Km67. These results suggested that the formation of [GAR+] in Km67 may be beneficial in kimoto-type sake making, since [GAR+] may increase cell viability in the sake starter mash without impairing alcoholic fermentation performance.

Improved stress resistance and ethanol production by segmental haploidization of the diploid genome in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains from industrial and natural geographical environments are reported to show great variation in copy number of chromosomal regions. Such variation contributes to the mechanisms underlying adaptation to different environments. Here, we created and phenotypically analyzed segmentally haploidized strains, each harboring a deletion of one copy of approximately 100-300 kb of the left or right terminal region of 16 chromosomes in a diploid strain by using a PCR-mediated chromosomal deletion method. No haploidized strain of the 158-kb deleted right terminal region of chromosome III or the 172-kb deleted right terminal region of chromosome VI was produced; however, segmentally haploidized strains of the remaining 30 terminal regions were obtained. Among these 30 strains, two exhibited higher lactic acid resistance and two displayed higher thermo-tolerance at 41°C versus the host diploid strain. By contrast, four and two segmentally haploidized strains showed sensitivity to 6% lactic acid and low temperature at 13°C, respectively. The effect of the decreased copy number of the chromosomal terminal regions on ethanol production was analyzed. As compared with the host diploid strain, a 3.8% and 4.3% improvement in ethanol production in 10% glucose medium was observed for two strains in which one of two copies of the 197-kb left terminal region of chromosome V and one of two copies of the 195-kb left terminal region of chromosome X was deleted, respectively. These results indicate that artificial segmental haploidization might contribute to improvement of industrially important phenotypes and provide a new approach to breeding superior yeast strains.

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network.

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.