Sake is a popular Japanese alcoholic beverage that is made by fermenting steamed rice with Saccharomyces cerevisiae (Desm.) Meyen and koji, which is a culture of Aspergillus oryzae (Ahlb.) Cohn, that is grown on steamed rice (Yoshizawa, 1999). During sake brewing, S. cerevisiae produces alcohol and a wide range of flavor compounds.
Historically, sake brewing relied on wild-house sake yeasts, which naturally develop in the fermentation environments of individual breweries. This led to a diverse array of sake varieties, as each brewery utilized its own distinct sake yeast strains. Over the past 80 y, a group of genetically similar sake yeast strains, known as the Kyokai no.7 (K7) group strains (Kyokai no. 6, 7, 9, and 10 series), have been isolated showing high fermentation rates in sake mash (Azumi & Goto, 2001). These K7 group strains have become the predominant choice for sake brewing over the past few decades, due to their effectiveness in enhancing the quality of sake and producing high-quality premium sake. However, the widespread use of these industrial yeasts may have reduced the diversity of its gustatory properties compared to when unique house sake yeast strains were used in each brewery. Consequently, interest has grown in brewing sake using alternative yeasts other than the K7 group strains to diversify the taste and flavor of sake.
The house sake yeast S. cerevisiae strain YS4 was isolated from the Sake Brewery “Yoshinogawa” (Settaya, Nagaoka, Japan), one of the oldest sake breweries in Niigata, established in 1548. The brewing properties of the YS4 strain differ from those of the K901 strain in the K7 group (Hatakeyama et al., 2020). Despite significant attention paid to house sake yeasts and their sake production, few reports have focused on the brewing characteristics of these house strains. In contemporary Japan, K7 group strains, which are characterized by superior brewing properties, have been extensively utilized to enhance the stability of sake production and improve the quality of the final product. Consequently, the isolation of house yeast strains that are disappearing from sake breweries has become challenging, and their biology is not well understood. Moreover, discriminating house sake yeasts from K7 group strains is difficult due to their mostly analogous physiological/genetic characteristics because both strains belong to the same species, S. cerevisiae (Azumi & Goto, 2001).
Recent studies have identified loss-of-function mutations in genes common to the K7 group strains, specifically RIM15, MSN4, and PPT1 loci (Watanabe, 2012). These mutations, found only in the K7 strains, offer a valuable tool for identifying them (Akao et al., 2018). Of these genes, RIM15, which is encoded by a protein kinase that plays a role in cell proliferation in response to nutrients, possesses a frameshift mutation that may serve as a critical genetic determinant for the increased production of ethanol in modern sake yeast strains (Watanabe & Takagi, 2016). The frameshift mutation at nucleotide position 5068 (RIM15ins5067A; Fig. 1) was predicted to cause a premature stop codon, which would lead to a reduction of 75 amino acids in the C-terminal region of the RIM15 gene product (Watanabe et al., 2012). While high-resolution melting analysis can detect RIM15ins5067A (Akao et al., 2018), it requires costly equipment, which is impractical for routine use by brewers.
Fig. 1. Mismatch PCR-RFLP assay for the identification of the RIM15ins5067A mutation. A: Single nucleotide insertion (A) at position 5067 of the RIM15 gene of the Kyokai no. 7 strain (highlighted in black). The PCR-RFLP method uses a reverse primer (P2) with two mismatches (indicated by asterisks [*]) that create a CspCI restriction site (displayed in a gray box and highlighted in gray in the RIM15 sequence) when the RIM15ins5067A mutation is present. At bottom right, gel electrophoresis analysis of PCR-RFLP; lane 1, PCR amplicons of laboratory yeast strain S288C; lane 2, PCR products from lane 1 after digestion with CspCI; lane 3, PCR amplicons of the sake yeast strain Kyokai no. 7; lane 4, PCR products from lane 3 after fragmentation with CspCI; M represents the Gene Ladder 100 (Nippon Gene, Tokyo, Japan).
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is useful for quickly and precisely identifying single nucleotide polymorphisms (SNPs) that create distinctive restriction sites (Zhang et al., 2005). PCR-RFLP is widely used to identify sake yeast mutants that produce high levels of fatty acid esters and have mutations FAS2-G1250S (G3748A) (Tanaka et al., 2023) and FAS2-F1279Y (T3836A) (Kuribayashi et al., 2022). This study presents a simple molecular assay designed to genotype RIM15ins5067A polymorphisms to differentiate K7 group strains from other sake yeast strains.
The nucleotide insertion at position 5067 in the RIM15 gene of K7 group strains did not situate near any restriction sites; therefore, we developed the reverse primer P2 with a mismatch (AA>TG) at the second and third positions from the 3′ end (DDBJ/EMBL/GenBank database accession no. U83459 as the reference sequence for primer construction; Fig. 1). This mismatch was designed to incorporate a restriction site for CspCI within the PCR amplicon when the RIM15ins5067A mutation was present at position 5067, resulting in 240-, 35-, and 10-bp fragments. However, in the wild-type RIM15 gene, lacking insert A at position 5067, the CspCI restriction site was not incorporated, resulting in an undigested 284 bp product.
To validate the custom-designed primers and PCR-RFLP assay, we used the Kyokai no.1-10, shochu yeast strains S-2 and SH-4, wine yeast strain KW-1 (provided by National Research Institute of Brewing) and the wild-type strain S288C (purchased from Open Biosystems, Huntsville, AL, USA). PCR amplification was carried out in reaction volumes of 50 μL, which included 25 μL of Takara Taq HS Perfect MIX (Takara Bio, Shiga, Japan), 0.4 μM of each primer, and 20 ng of genomic DNA from yeast cells. The PCR cycling conditions included 35 cycles of denaturation at 94 °C for 5 s, annealing at 55 °C for 1 s, and extension at 68 °C for 10 s. PCR products were extracted using a FastGene Gel/PCR Extraction Kit (Nippon Genetics, Osaka, Japan), purified (18 μL) using 10× rCutSmart Buffer (2 μL) and restriction enzyme CspCI (1 μL) from New England Biolabs (Ipswich, MA, USA), and incubated for one hour at 37 °C before purification with the FastGene Gel/PCR Extraction Kit (Nippon Genetics). The purified samples were then run on 4% agarose gels (Agarose L03, Takara Bio) for 40 min at 100 V and visualized with Midori Green Advance (Nippon Genetics) staining.
To assess the accuracy of the mismatch PCR-RFLP assay for detecting the RIM15ins5067A polymorphism (with the insertion) and S288C (wild-type, lacking the insertion). As shown in Fig. 1, the PCR product from S288C was 284 bp and remained undigested by CspCI, while the K7 amplicon was cleaved by CspCI into a 240 bp fragment. DNA sequence analysis confirmed the intended mismatched nucleotides (Supplementary Fig. 1).
Among the 14 yeast strains examined, the PCR-RFLP assay detected the mutation in Kyokai no. 6, 7, 9, and 10 strains (Table 1; Supplementary Fig. S1), consistent with a previous study identifying the RIM15ins5067A in these K7 strains (Watanabe et al., 2012). We also confirmed this mutation using DNA sequence analysis of RIM15 (Table 1). These findings indicate that this assay identify sake yeasts possessing the RIM15ins5067A gene but cannot distinguish between the individual strains, such as Kyokai no. 9 and Kyokai no. 10 or SH-4 and S288C. Despite this methodological limitation, the results obtained showed strong concordance with our data (Table 1) and previously reported mutation sequences (Watanabe et al., 2012). Therefore, the PCR-RFLP is effective for distinguishing K7 group strains from other S. cerevisiae strains.
Table 1. Specificity of PCR-RLFP analysis to detect
RIM15 gene and a single nucleotide insertion in
RIM15 across different yeast strains.
| No. | Yeast strain | PCR-RFLP methoda | Insertion of a single nucleotide in RIM15 as the target for PCR-RFLPb |
| Sake yeast |
| 1 | Kyokai no. 1 | - | - |
| 2 | Kyokai no. 2 | - | - |
| 3 | Kyokai no. 3 | - | - |
| 4 | Kyokai no. 4 | - | - |
| 5 | Kyokai no. 5 | - | - |
| 6 | Kyokai no. 6 | + | A |
| 7 | Kyokai no. 7 | + | A |
| 8 | Kyokai no. 8 | - | - |
| 9 | Kyokai no. 9 | + | A |
| 10 | Kyokai no. 10 | + | A |
| Shochu yeast |
| 11 | S-2 | - | - |
| 12 | SH-4 | - | - |
| Wine yeast |
| 13 | KW-1 | - | - |
| Laboratory yeast |
| 14 | S288C | - | - |
a Marks: +, cutting of PCR product with CspCI; -, no-cutting of PCR product CspCI.
b Marks: A, adenine; -, no insertion.
The RIM15ins5067A mutation, found exclusively in K7 group strains was used to differentiate specific yeast strains, including the house yeast strain. A previous study indicated that the house yeast strain Km67 from the Kiku-masamune brewery lacked this mutation (Takao et al., 2018). We employed the PCR-RFLP method on the house yeast strain YS4 from Yoshinnogawa (Fig. 2). The PCR product from the house yeast strain YS4 was 284 bp in length and was not digested by CspCI; therefore, the mismatch PCR-RFLP assay effectively distinguished between K7 and the house yeast strain YS4. We also confirmed that YS4 does not harbor this mutation in RIM15 using direct sequence analysis (Sanger sequencing using a capillary electrophoresis sequencer; FASMAC, Kanagawa, Japan) of both the PCR products before restriction enzyme treatment in this assay (Supplementary Fig. S2). These results suggest that our PCR-RFLP assay is helpful for the isolation of a house sake yeast strain from a sake brewery that usually produces sake using K7 group strains carrying RIM15ins5067A.
Fig. 2. Mismatch PCR-RFLP pattern for the house yeast strain YS4 isolated from Yoshinogawa in Niigata prefecture, Japan. Agarose gel electrophoresis analysis of PCR-RFLP: lane 1, PCR amplicons of sake yeast Kyokai no. 7; lane 2, PCR products from lane 1 after fragmentation with CspCI; lane 3, PCR amplicons of the house yeast strain YS4; lane 4, PCR fragments from lane 3 after digestion with CspCI; M represents the Gene Ladder 100 (Nippon Gene, Tokyo, Japan).
In recent years, sake manufacturers and research institutions in Japan have developed a variety of sake yeast strains. This trend may have increased the diversity of the flavor profile of sake, similar to that of sake fermented with unique house yeast strains specific to each brewery in the past. The use of non- K7 group strains has also gained interest for diversifying the taste and flavor of sake. House yeasts offer potential for creating novel sake varieties with distinct flavors (Hatakeyama et al., 2020; Takao et al., 2018). Although it has been reported that the K7_02212, PPT1, and PHO3 genes have been targeted for the facile identification of industrial yeast strains (Hatakeyama et al., 2020; Kuribayashi et al., 2014), the addition of RIM15 to these targets provides a more meticulous approach to distinguish K7 group strains and screen for house yeast strains.
This study demonstrates the usefulness of PCR-RFLP for identifying the RIM15ins5067A mutation in K7 group of yeast. This simple and effective method is valuable for distinguishing specific yeasts in sake breweries.
