46NISR Research GRANT
2008 Research Grant

A research on mitochondria-targeted breeding of a novel
brewery yeast
Hiroshi KITAGAKISaga University

Research aims
Since mitochondria have been described to be responsible for oxidative respiration and brewery yeast is exposed toan environment in which oxidative respiration does notoccur during alcoholic fermentation, the role of yeast mitochondria during alcoholic fermentation has not been described in detail. However, I have demonstrated that yeast
mitochondria do exist throughout brewing of sake, theJapanese traditional rice wine, and that their structures undergo fragmentation during sake brewing1). I was inspired
to apply these novel findings to the brewing technologies.
Methods
1. Comparison of gene expression change across diauxic
shift in WT cells and mitochondria-defective mutant cells
Yeast strain JK93d-a was used as the parental strain.
Yeast cells were routinely grown in Difco YPD broth withshaking at 30°C. RNA was isolated from cell pellets using
the RNeasy RNA isolation kit (Qiagen) following the enzymatic lysis protocol. Microarray analysis was carried out
essentially as described in the Affymetrix Expression
Analysis Technical Manual. Microarray data were derived
from two independent experiments for WT cells and one
experiment for respiration-defective petite cells. Absoluteanalysis was conducted for each analysis with scaling to atarget value of 2500 to facilitate comparison between experiments. In experiments shown in Fig. 1, genes whose expression at 24 h in WT was greater than 300 were selected.
Among the selected genes, the genes whose expression
ratio of WT-24 h/WT-4 h was more than 2-fold were furtherselected. Mitochondrial DNA was depleted as follows. Logarithmically grown yeast cells were diluted to 2106cells/ml in 1% yeast extract, 2% peptone, 2% dextrose, 50mM potassium phosphate, pH 6.24. Ethidium bromide wasadded to a final concentration of 50 m M, and the cultures
were incubated at 30°C in the dark for 8 h with shaking andthen plated onto YPD or YPGE medium to confirm inability to grow on nonfermentable medium.
2. Mitochondrial-morphology-targeted breeding of brewery
yeast strains
Sake yeast Kyokai No. 7 was purchased from the Brewing Society of Japan, Tokyo. The haploid strain K7H868
was obtained by sporulating Kyokai No. 7 and screening fora haploid strain with a fermenting ability similar to theparental diploid strain. The yeast cells were incubated in2% Bactopeptone, 1% Bacto-yeast extract (Beckton Dickinson, USA) and 2% glucose at 30°C with shaking. Cells
harboring mitochondria-targeted GFP were grown in a synthetic medium containing a 0.67% yeast nitrogen base without amino acids (Beckton Dickinson), a 0.2% complete
supplement mixture without leucine (MP Biomedicals,USA) and 2% glucose at 30°C with shaking. The content oforganic acids was determined using a high-performanceliquid chromatograph equipped with a conductivity detector
and a column Shim-pack SPR-H (Shimadzu). Disruption ofFIS1 gene in K7 haploid strain was performed by transformation using a disruption cassette amplified with suitableprimers, and selection on 100 m g/ml nourseothricin-containing plates by standard techniques. Disruption of FIS1
was confirmed by PCR analysis and staining with a mitochondrial dye (50 nM rhodamine 123, Invitrogen, USA). In
order to transform mitochondria-targeted GFP, LEU2 wasdisrupted using a disruption cassette amplified with suitableprimers. The transformation was performed by standard
techniques and colonies were plated onto YPD plates containing 500 m g/ml G418. For visualization of mitochondria,strain RAK1536, pYX142-mitoGFP, and pRS413GPDmtGFP were used. They were kind gifts from Professor
Rinji Akada of Yamaguchi University, Dr. Koji Okamoto ofFig. 1. Comparison of gene expression change across the diauxic shift
between respiration-competent WT (grande) cells and respiration-defective petite cells.
The genes that were up-regulated more than 2-fold in WT experiment 1 at
24 h relative to 4 h were selected. The log10 value of induction ratio of gene
expression at 24 h to that at 4 h in WT experiment 2 and petite experimentwere plotted against that in WT experiment 1.

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National Institute of Basic Biology, and Professor Shaw ofUtah University, respectively. Sake brewing was performedby mixing 2109 cells of yeast with 60 g of dried gelatinized rice, 23 g of dried gelatinized koji, 45 m l of lacticacid, 200 ml of water and incubating the mash at 15°C for
14 days.
Results
1. Mitochondrial function has a critical role during the diauxic shift of yeast.
Yeast has two energy-generating status: fermentation andrespiration. In industrial fermentation processes, yeast cells
undergo transitions between the two metabolic status. For
example, transferring yeast cells from a starter culture,
which is often incubated with aeration, to a batch culture
would accompany the transition from respiration to fermentation. Although it has been shown that yeast cells retain
their mitochondria during alcoholic fermentation, the roleof mitochondrial function(s) during such metabolic transitions has not been studied. In order to examine whether
yeast mitochondria play unknown roles in the transition, I
investigated the role of mitochondrial function during thediauxic shift from fermentation to respiration.
The genome-wide expression of genes was compared be-tween respiration-competent WT cells and respiration-defective petite cells, in which mitochondrial DNA was depleted. As a result, it turned out that the respiration-defective petit cells were defective in upregulation of certain
genes across the diauxic shift (Fig. 1). The genes that failed
to be upregulated during the diauxic shift in the respirationdefective petite cells did not exactly correspond to the glucose-repressed genes, indicating that mitochondrial function specifically regulates diauxic-associated genes.
Taken together, our results demonstrated for the first timethat mitochondrial function(s) is necessary for the diauxicshift2).
2. Breeding of a mitochondrial morphology-targeted brewery yeast.
I have previously demonstrated that mitochondrial structures of yeast undergo fragmentation during sake brewing(Fig. 2)1).
Based on this finding, I aimed to breed a novel breweryyeast. I came up with an idea of altering the flux of substances within yeast cells by altering the structure of mitochondria during sake brewing. To achieve this goal, I first
tried to determine the factor responsible for fragmentationof mitochondria during sake brewing. I first investigatedwhich factor is responsible for mitochondrial fragmentation

Fig. 2.

Mitochondrial structure of wild type sake yeast during sake brewing.

Fig. 3.

Mitochondrial structure of fis1-disrupted sake yeast during sake brewing.

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Fig. 4.

Organic acid content of sake brewed with the parent strain and fis1 disruptants of sake yeast.

upon ethanol treatment of yeast cells. It turned out that
FIS1 is responsible for mitochondrial fragmentation uponethanol treatment3). Fis1 has been reported to be the factor
responsible for mitochondrial fragmentation during vegetative growth4). Thus, I disrupted FIS1 gene in the sake yeast
background. I found that the fis1 disruptant obtained has a
networked mitochondrial structure.
I then brewed sake with this fis1 disruptant, and foundthat mitochondria of this strain keep the networked structure throughout sake brewing, indicating that Fis1 is responsible for mitochondrial fragmentation during sakebrewing.
As a result of these experiments, I obtained a sake yeast
strain with networked mitochondrial structure. I brewed
sake with this strain, and measured the organic content ofsake. It turned out that the amount of malate, which has acrispy taste, is increased by two folds. This result clearly indicates that mitochondrial structure in yeast cells during
sake brewing has a critical role in determining organic acidcontent of sake5–10).

References

Conclusion8)

I have demonstrated that (1) mitochondrial function has acritical role during the diauxic shift, and (2) mitochondrial
structure can be a target of breeding of brewery yeaststrains.

1)
2)

3)

4)

5)

6)

7)

9)
10)

Kitagaki H, Shimoi H. (2007) Mitochondrial dynamics of yeast during sake brewing. J. Biosci. Bioeng. 104, 227–230.
Kitagaki H, Cowart LA, Matmati N, Montefusco D, Gandy J, deAvalos SV, Novgorodov SA, Zheng J, Obeid LM, Hannun YA.
(2009) ISC1-dependent metabolic adaptation reveals an indispensable role for mitochondria in induction of nuclear genes during the diauxic shift in Saccharomyces cerevisiae. J. Biol. Chem. 284,10818–10830.
Kitagaki H, Araki Y, Funato K, Shimoi H. (2007) Ethanol-induced
death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway. FEBS Lett. 581, 2935–2942.
Okamoto K, Shaw JM. (2005) Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. Annu. Rev. Genet. 39,503–536.
Kitagaki H, Kato T, Isogai A, Mikami S, Shimoi H. (2008) Inhibition of mitochondrial fragmentation during sake brewing causes highmalate production in sake yeast. J. Biosci. Bioeng. 105, 675–678.
Kitagaki H, Mikami S, Shimoi H. (2007) A novel method to increase
the productivity of malate by yeast. Japan Official Patent Gazette
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Kitagaki H. (2009) Mitochondrial-morphology-targeted breeding ofindustrial yeast strains for alcohol fermentation. Biotechnol. Appl.
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Kitagaki H, Shimoi H (2008) The role of yeast mitochondria during
sake brewing and its application for breeding of a new brewery yeast.
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