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Noda Institute for Scientiﬁc Research GRANT
2011 Young Investigator Research Grant

Study of the High Ethanol Productivity of Sake Yeast byComprehensive Cellular Morphological Analysis
Daisuke WATANABE
National Research Institute of Brewing

Research aims
Sake yeast strains, which belong to the budding yeast
species Saccharomyces cerevisiae, produce much more
ethanol during sake fermentation than any other type of S.
cerevisiae strain. To elucidate the molecular mechanisms
responsible for their excellent fermentation properties, wehere investigated sake yeast-speciﬁc morphological traits
using the CalMorph system1), which has been developed forhigh-dimensional and quantitative morphological analysis
of yeast cell populations. This analysis led to identiﬁcationof a novel genetic determinant for ethanol production in S.
cerevisiae.
Methods
The sake yeast strains Kyokai no. 6, 7, 9, and 10 and alaboratory strain X2180 were grown to the logarithmic
phase in YPD medium and were then ﬁxed with formaldehyde. After triple staining of yeast cell wall mannoproteins,
actin, and nuclear DNA, cells were observed under the ﬂuorescent microscope, and the microscopic cellular images
were automatically processed using the CalMorph system.
For fermentation tests, yeast cells were inoculated into
20% glucose-containing YPD medium at a ﬁnal OD660 of0.1, and were then further incubated at 30°C without shaking. For sake fermentation assays, a sake mash was prepared by mixing 40 g pre-gelatinized rice, 10 g dried koji
(rice cultivated with A. oryzae), 20 m l 90% lactic acid, and80 ml water containing yeast cells at a ﬁnal OD660 of 1.0,
and was then incubated at 15°C without shaking. Fermentation was monitored by measuring the volume of evolved
carbon dioxide using Fermograph2).
To determine cellular DNA contents, yeast cells wereﬁxed in ethanol, suspended in a propidium iodide solution,
and analyzed using a ﬂow cytometer. Gene-speciﬁc quantitative real-time PCR (qRT-PCR) was performed to quantify
CLN3 mRNA. The actin-encoding gene ACT1 was used as
an internal control.
Results
Our high-throughput cytological analysis using CalMorph revealed that sake yeast cells exhibit speciﬁc morphological features in the logarithmic phase: Both the ratioof-no-bud (C119) and long-axis-length-of-mother-cell-on-

nuclear-A1B (C103_A1B) parameters were signiﬁcantly
lower in all of the sake strains than in X2180 (Fig. 1). Since
these morphological traits have also been typically observed in the mutants that cannot effectively repress theG1/S transition, we hypothesized that the G1 progression
might be accelerated in sake yeast.
Under fermentative conditions, is sake yeast defective incell cycle regulation? To test this, we performed fermentation tests using the sake and laboratory strains in 20%
glucose-containing YPD medium to analyze the cellular
DNA contents during fermentation. We found that the cell
population of sake yeast with a G1 DNA content wassmaller than that of laboratory yeast throughout the fermentative phase, as well as in the logarithmic growth phase(Fig. 2A). We also discovered elevated expression of the G1
cyclin gene CLN3 in sake yeast under the fermentation condition (Fig. 2B). These results indicate that laboratory yeast
exhibited efﬁcient G1 arrest during the fermentative phase,whereas some of the sake yeast cells failed to maintain G1arrest until fermentation termination3).
To elucidate the mechanism causing the defects of sakeyeast in cell cycle control, the mutations in the genes related to G1 progression were thoroughly searched by utilizing the genome sequence data of a sake strain Kyokai no. 7
that we recently reported4). Consequently, we identiﬁed anovel frameshift mutation, rim155055insA, causing dysfunction of the Rim15p protein kinase, which is responsible for
effective G1 arrest upon growth termination5). Among various industrial yeast strains (e.g., wine and beer yeast), sakestrains including Kyokai no. 6, 7, 9, and 10 speciﬁcally contained this mutation. Furthermore, expression of a functional RIM15 gene recovered an effective G1 arrest in sakeyeast (data not shown), demonstrating that the rim155055insA
mutation accounts for the defective cell cycle control insake yeast.
Fermentation tests using a Drim15 gene disruptant and arim155055insA mutant in the laboratory strain BY4743 background revealed how Rim15p-mediated G1 progression
control affects the fermentation property. Loss of theRim15p functions signiﬁcantly enhanced the fermentation
rate of the parental strain (Fig. 3)5). These rim15 mutants
exhibited markedly improved fermentation rates also insmall-scale sake brewing tests: The ethanol concentration
after 20 days of sake fermentation was higher in the ﬁnished sake made from the Drim15 disruptant (17.03⫾
0.44%) and that from the rim155055insA mutant (16.77⫾

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Fig. 1. Morphological traits of the sake yeast strains. (A) Three-dimensional PCA mapping of the CalMorph data. Colored dots correspond to data fromindividual experiments (n⬎200 cells). Sake yeast strains Kyokai no. 6, 7, 9, and 10 (K6, K7, K9, and K10) and a laboratory strain X2180 were used. (B)
Representative cell morphology of a sake and a laboratory yeast. Bar; 3 mm. (C) Morphological parameters that showed signiﬁcant differences between sakeand laboratory yeast.

Fig. 2. Cell cycle-related analysis under a fermentative condition. Phase I: logarithmic growth phase, Phases II–IV: fermentative phases; Phase V: fermentation termination. (A) DNA contents. (B) Analysis of CLN3 mRNA levels using qRT-PCR. Relative expression levels are displayed as fold differences compared to the levels induced in X2180 (phase I), using ACT1 as a reference gene.

Fig. 3. Improvement of the fermentation rate by dysfunction of Rim15p. (A) Fermentation tests in 20% glucose-containing YPD medium. (B) Small-scale
sake fermentation tests.

610.18%) than that from wild-type BY4743 (11.19⫾0.17%).
Taken together, these results clearly demonstrated that dysfunction of Rim15p caused by the sake yeast-speciﬁc
rim155055insA mutation leads to a striking increase of ethanol
production.
Conclusion
The cellular morphological analysis of sake yeast enabled us to ﬁnd its novel phenotype related to defective cell
cycle control. Furthermore, we revealed that therim155055insA mutation have played a pivotal inﬂuential rolein accelerated G1 progression and increased fermentation
rate that are both characteristic of sake yeast strains. Identiﬁcation of multiple genetic determinants for the fermentation properties of sake yeast5–7) will provide signiﬁcant insights into artiﬁcial modiﬁcation of the yeast metabolic proﬁles. In future studies, the molecular mechanism on how
the Rim15p-mediated cell cycle regulatory system controls
fermentation and ethanol production should be intensively
investigated.

References
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