Homozygous gene disruption in diploid yeast through a single transformation.

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5'- and 3'-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5'- and 3'-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae.

THI3 contributes to isoamyl alcohol biosynthesis through thiamine diphosphate homeostasis.

Isoamyl alcohol is a precursor of isoamyl acetate, an aromatic compound that imparts the ginjo aroma to sake. The isoamyl alcohol biosynthesis pathway in yeasts involves the genes PDC1, PDC5, PDC6, ARO10, and THI3 encoding enzymes that decarboxylate α-ketoisocaproic acid to isovaleraldehyde. Among these genes, THI3 is the main gene involved in isoamyl alcohol biosynthesis. Decreased production of isoamyl alcohol has been reported in yeast strains with disrupted THI3 (Δthi3). However, it has also been reported that high THI3 expression did not enhance decarboxylase activity. Therefore, the involvement of THI3 in isoamyl alcohol biosynthesis remains unclear. In this study, we investigated the role of THI3 in isoamyl alcohol biosynthesis. While reproducing previous reports of reduced isoamyl alcohol production by the Δthi3 strain, we observed that the decrease in isoamyl alcohol production occurred only at low yeast nitrogen base concentrations in the medium. Upon investigating individual yeast nitrogen base components, we found that the isoamyl alcohol production by the Δthi3 strain reduced when thiamine concentrations in the medium were low. Under low-thiamine conditions, both thiamine and thiamine diphosphate (TPP) levels decreased in Δthi3 cells. We also found that the decarboxylase activity of cell-free extracts of the Δthi3 strain cultured in a low-thiamine medium was lower than that of the wild-type strain, but was restored to the level of the wild-type strain when TPP was added. These results indicate that the loss of THI3 lowers the supply of TPP, a cofactor for decarboxylases, resulting in decreased isoamyl alcohol production.

Shochu Koji Microstructure and Starch Structure during Preparation.

In this study, we investigated the changes in composition, microstructure, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13-20 % of the total sugar content, with glucose representing over 70 % of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization (DP) 4-73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6-30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.