Transcriptome Analysis of Sake Yeast in Co-Culture with kuratsuki Kocuria

: Kuratsuki bacteria enter the sake production process and affect the flavor and taste of sake. This study compared gene expression in the sake yeast Saccharomyces cerevisiae in co-culture with kuratsuki Kocuria to that in monoculture. Among the 5922 genes of S. cerevisiae , 71 genes were upregulated more than 2-fold, and 61 genes were downregulated less than 0.5-fold in co-culture with kuratsuki Kocuria . Among the stress-induced genes, fourteen were upregulated, and six were downregulated. Among the fourteen upregulated genes, six were induced in response to replication stress. Although the G1 cyclin gene CLN3 was upregulated by more than 2-fold, eight genes that were induced in response to meiosis and/or sporulation were also upregulated. Fourteen metabolism-related genes, for example, the glyceraldehyde-3-phosphate dehydrogenase genes TDH1 , TDH2 , and TDH3 , were downregulated by less than 0.5-fold in co-culture with kuratsuki Kocuria . The gene expression patterns of S. cerevisiae co-cultured with kuratsuki Kocuria differed from those co-cultured with lactic acid bacteria. Therefore, S. cerevisiae responded differently to different bacterial species. This strongly suggests that kuratsuki bacteria affect gene expression in sake yeast, thereby affecting the flavor and taste of sake.


Introduction
Sake is a traditional Japanese alcoholic beverage composed of koji, moto, rice, and water.Koji is produced by growing the koji mold Aspergillus oryzae on steamed rice.The enzymes of A. oryzae digest rice and produce sugars and amino acids; however, they cannot produce ethanol.Moto is a fermentation starter made from a mixture of koji, rice, water, and sake yeast Saccharomyces cerevisiae.S. cerevisiae converts sugar into ethanol, which is used in beer, sake, and wine production [1].The flavor and taste of sake are mainly determined by the composition of esters and organic acids that are produced by the sake yeast [2][3][4][5][6].Thus, different strains of sake yeast produce different flavors and determine the taste of the sake [7].Some lactic acid bacteria interact with S. cerevisiae to change its metabolism, which results in the specific flavor and taste of the alcoholic beverage [8][9][10].The effects of co-culture of S. cerevisiae with bacteria were reported in the industrial ethanol production process [11].
Bacteria that live in a sake brewery are called kuratsuki bacteria [12,13]; these bacteria inevitably enter the sake production process.Kuratsuki bacteria have evolved to enable adaptation to the sake-making environment through the modification of their genomes [13][14][15].Although research on kuratsuki yeasts has been performed, research on kuratsuki bacteria has been considered unnecessary.As a result, there was no information on the difference in kuratsuki bacteria among different sake breweries.To elucidate the diversity of kuratsuki bacteria, we isolated and identified bacteria from the sake production process and subsequently characterized them [14,16].
Strains belonging to the genus Kocuria were isolated and identified as kuratsuki bacteria from the Narimasa Sake Brewery in Toyama, Japan [15,17].Kocuria belongs to the phylum Actinobacteria and is not a lactic acid bacterium.We isolated and identified two different strains belonging to the genus Kocuria, which were K. koreensis strain TGY1120_3 and K. uropygioeca strain TGY1127_2 [15].We have used these two strains as representative kuratsuki bacteria in our experiments, especially K. uropygioeca strain TGY1127_2 [18][19][20].
Although this strongly suggests that kuratsuki bacteria may interact with sake yeast to change the metabolism as well as lactic acid bacteria, which results in the taste of sake [14,[18][19][20][21], the function of kuratsuki bacteria remains uncertain.We used the taste recognition device to confirm whether the co-culture would change the taste.Different effects of kuratsuki bacteria were found on different types of koji and different strains of sake yeast [13,18,19].Thus, the taste of sake with kuratsuki bacteria differed from that without kuratsuki bacteria.We are performing research focusing on microbial interaction.
Comparison of gene expression is one of the most effective methods for elucidating the interactions between sake yeast and bacteria.There have been no reports on the genomewide gene expression analysis of sake yeast in response to kuratsuki bacteria.In this study, we compared the gene expression of sake yeast in monoculture and co-culture with that of kuratsuki Kocuria.

Culture of Sake Yeast and kuratsuki Kocuria
The sake yeast S. cerevisiae strain K1401 [7] and Kocuria uropygioeca strain TGY1127_2 [15] were used in this study.Each strain was pre-cultivated in TGY medium (5 g/L tryptone, 1 g/L glucose, and 3 g/L yeast extract) at 25 • C for 12 h.One milliliter of pre-cultivated cell suspension of K1401 (2.25 × 10 6 cells/mL) was added to 200 mL of modified TGY medium (20 g/L tryptone, 150 g/L glucose, and 10 g/L yeast extract).In addition, 1 mL of the pre-cultivated cell suspension of K1401 and 1 mL of the pre-cultivated cell suspension of TGY1127_2 (>1 × 10 8 cells/mL) were mixed with 200 mL of modified TGY medium.Thus, monoculture and co-culture were performed in the artificial medium, which differs from the fermentation environment during sake making.The two solutions were incubated at 14 • C for seven days.

Measurement of Brix
Brix was measured daily (on days 0, 1, 2, 3, 4, 5, 6, and 7) using a PAL-BX/ACID digital refractometer (ATAGO, Tokyo, Japan).Brix is a measure of the percentage of soluble solids in syrups and products.Each degree of Brix is equal to 1% concentration of sugar in solution when measured at 20 • C (https://www.foodscience-avenue.com/ (accessed on 20 April 2024)).Each measurement was repeated thrice.The median of the three values was selected as the representative value.The Kolmogorov-Smirnov test was performed to compare the two samples using R software (The R Project for Statistical Computing, http://www.R-projet.org/(accessed on 25 January 2024)).The Kolmogorov-Smirnov test is sensitive to differences in both the location and the shape of the empirical cumulative distribution functions of the two samples, which is one of the most useful nonparametric methods for the comparison of two samples.

Gene Expression Comparison
We calculated the ratio of reads per kilobase of exon per million mapped reads (RPKM) [22] in sake yeast in co-culture with the K. uropygioeca TGY1127_2 per RPKM in the sake yeast in monoculture and selected a ratio of >2 for the upregulated genes and a ratio of <0.5 for the downregulated genes.

Brix of Culture
The Brix change pattern of the co-culture of sake yeast K1401 and K. uropygioeca TGY1127_2 was similar to that of the monoculture of sake yeast (Figure 1, Table S1).These patterns were not significantly (p > 0.05) different in the Kolmogorov-Smirnov test.This indicates that kuratsuki Kocuria did not inhibit alcohol fermentation by sake yeast, which is consistent with a previous study [18].The 7-day monoculture and co-culture system was used for RNA isolation and sequencing.Brix was decreasing at 7 days (Figure 1, Table S1), thereby indicating that ethanol fermentation by sake yeast is in progress.Kit (MGI Tech) were used.Sequencing was performed (2 × 150 bp) using a DNBSEQ-T7RS High-throughput Sequencing Kit (MGI Tech) and DNBSEQ-T7 (MGI Tech).The adapter sequence was removed using Cutadapt (ver.4.0); bases with a quality score of less than 20 and paired reads with a length smaller than 75 bases were removed using Sickle (ver.1.33).High-quality reads were mapped to the Saccharomyces cerevisiae S288C genome (NCBI RefSeq ID: GCF_000146045.2).

Gene Expression Comparison
We calculated the ratio of reads per kilobase of exon per million mapped reads (RPKM) [22] in sake yeast in co-culture with the K. uropygioeca TGY1127_2 per RPKM in the sake yeast in monoculture and selected a ratio of >2 for the upregulated genes and a ratio of <0.5 for the downregulated genes.

Brix of Culture
The Brix change pattern of the co-culture of sake yeast K1401 and K. uropygioeca TGY1127_2 was similar to that of the monoculture of sake yeast (Figure 1, Table S1).These patterns were not significantly (p > 0.05) different in the Kolmogorov-Smirnov test.This indicates that kuratsuki Kocuria did not inhibit alcohol fermentation by sake yeast, which is consistent with a previous study [18].The 7-day monoculture and co-culture system was used for RNA isolation and sequencing.Brix was decreasing at 7 days (Figure 1, Table S1), thereby indicating that ethanol fermentation by sake yeast is in progress.

RNA Mapping and Gene Expression
The number of high-quality paired reads was 26,629,768 and 33,522,652 in the monoculture of sake yeast K1401 and co-culture with K. uropygioeca TGY1127_2, respectively.Among these reads, 24,367,924 (91.59%) and 30,433,279 (90.87%) reads were mapped to a single locus in the sake yeast monoculture and co-culture with kuratsuki Kocuria, respectively.In this study, we did not use any reads that were mapped to multiple loci because their origins were unclear, but we did use the reads that were mapped to a single locus.
We mapped the RNA sequences of 6420 genes of Saccharomyces cerevisiae.The number of genes that mapped more than 10 sequences in both sake yeasts in monoculture and co-culture with kuratsuki Kocuria was 5922.For these 5922 genes, the ratio of RPKM (the ratio of reads per kilobase of exon per million mapped reads) in co-culture with kuratsuki Kocuria per RPKM in monoculture.The mean ratio was 0.955 ± 0.008.We compared the gene expression levels of 5922 genes in sake yeast K1401 in monoculture and co-culture with K. uropygioeca TGY1127_2.Among the 5922 genes, 71 genes were upregulated more than 2-fold, and 61 genes were downregulated less than 0.5-fold in co-culture with kuratsuki Kocuria (Figures 2 and 3, Tables S2 and S3, Figure S1).

Expression Changes of Stress-Related Genes in Co-Culture with the kuratsuki Kocuria
Among the 71 more than 2-fold upregulated genes in the co-culture with K. uropygioeca TGY1127_2, the following fourteen were stress-induced genes: AHP1, CAT8, CSR2, DDR2, GEX1, HSP150, MOB1, MPC3, PAU5, PAU19, POM33, SRX1, TIP41, and RCN1 (Figure 2, Table S2).Ahp1 is involved in oxidative stress response [30].The transcription factor Cat8 is involved in the reprogramming of carbon metabolism [31,32].Csr2 is involved in osmotic stress response [33].Ddr2 is involved in response to multiple stresses [34,35].Gex1 is a glutathione exchanger that is induced in response to ion depletion [36].Hsp150 is involved in the response to aluminum and oxidative stress [37,38].Mob1 is involved in the regulation of mitotic exit [39,40].Mpc3 stimulates respiration, which is involved in response to oxidative stress [41,42].S. cerevisiae contains 24 Pau proteins [43].Pau5 and Pau19 are structurally different [43].Pau5 may be involved in the recombination of transposable elements [43,44].The upregulation of PAU5 may be related to retrotransposon activation.Pom33 is a membrane protein of the nuclear pore complex [45,46].Srx1 is involved in the oxidative stress response [47].Tip41 is involved in the negative regulation of the TOR signaling pathway [48].Rcn1 is involved in the response to ethanol stress [49].Among these fourteen stress-induced proteins, the abundance of six proteins, Cat8, Mob1, Mpc3, Pom33, Srx1, and Tip41, increased in response to DNA replication stress [50].
Replication stress inhibits transcription of histone genes [51].In our data, seven of ten histone genes were downregulated in the co-culture with kuratsuki Kocuria (Table 1).Among the histone genes, HHT2 was downregulated (Figure 3, Table 1 and S3).In addition, RTF1, SAP30, and SDS3 were downregulated by less than 0.5-fold in co-cultures K. uropygioeca TGY1127_2 (Figure 3, Table S3) [52][53][54].Therefore, kuratsuki Kocuria is strongly suggested to increase replication stress in sake yeast.In contrast, six stress-induced genes, ASR1, GSH2, HSP30, SSA2, SSA4, and YHB1, were downregulated by less than 0.5-fold in co-culture with kuratsuki Kocuria (Figure 3, Table S3).Asr1 is involved in the response to ethanol stress [55].Gsh2 is a glutathione synthase, which is involved in response to oxidative stress [56,57].Hsp30 is involved in the re-sponse to several stressors [58,59].Ssa2 and Ssa4 are a member of Hsp70 chaperones [57,60].Yhb1 is involved in the oxidative stress [61].Thus, the expression of some stress-induced genes was diametrically opposed; for example, ASR1 vs. RCN1 in response to ethanol stress and AHP1 vs. YHB1 in response to oxidative stress.These results suggest that the stress levels of sake yeast co-cultured with kuratsuki Kocuria may not be as high.

Expression Changes of Cell Cycle-Related Genes in Co-Culture with the kuratsuki Kocuria
The following eight genes, HFM1, IRC18, LDS2, LOH1, MOB1, SPO24, SPS19, and SSP1, are involved in meiosis and/or sporulation, and were upregulated more than 2fold in co-culture with K. uropygioeca TGY1127_2 (Figure 2, Table S2).Hfm1 is a DNA helicase involved in meiotic crossing over [62].Irc18 and Lds2 are involved in spore wall assembly [63,64].Loh1 is involved in the loss of heterozygosity [65].As previously mentioned, stress-induced Mob1 is involved in the regulation of mitotic exit [40].SPO24 produces two mRNA forms, which appear during meiosis and mid-late meiosis [66].Sps19 is a 2, 4-dienoyl-CoA reductase involved in sporulation [67].Ssp1 is involved in meiotic division and spore formation [68].
In addition, four genes, CLG1, CLN3, GSP1, and VHS1, are involved in the cell cycle and/or maintenance of nucleolar organization and were also upregulated in co-culture with kuratsuki Kocuria (Figure 2, Table S2).Clg1 is a cyclin-like protein, which interacts with the cyclin-dependent kinase Pho85 [69,70].Cln3 is activated in response to nutrient repletion induced by acetyl-CoA [71].Gsp1 is a GTP-binding protein involved in the maintenance of nuclear organization [72].Vhs1 suppresses cell cycle arrest during the G1-S transition [73].
Meanwhile, TEM1, which is involved in the termination of the M phase [74], was downregulated by less than 0.5-fold (Figure 3, Table S3); this is inconsistent with the upregulation of genes related to the meiosis/sporulation/cell cycle.In addition, the upregulation (Figure 2, Table S2) of CLN3 is also inconsistent with the upregulation of meiosis-induced genes because G1 cyclin Cln3 blocks meiosis [75,76].Therefore, it is uncertain whether sake yeast was about to undergo meiosis and enter the spore formation stage in co-culture with kuratsuki Kocuria.It is possible that various cells at different cell cycle stages existed.
The two upregulated genes, PAU19 and SRX1, and the two downregulated genes, TEM1 and THI4, are listed in Tables S2 and S3, respectively.As previously mentioned, PAU19 and SRX1 are stress-inducing genes.TEM1 and THI4 are involved in the termination of the M phase and thiamine biosynthesis, respectively.The other upregulated and downregulated genes differed between co-cultures with K. uropygioeca TGY1127_2 and Lactiplantibacillus plantarum.
Although no genes with similar expression changes were found between co-cultures with K. uropygioeca TGY1127_2 and Lactobacillus delbrueckii subsp.bulgaricus, four genes were found between co-cultures with K. uropygioeca TGY1127_2 and Lactiplantibacillus plantarum.In addition, although both L. delbruechii subsp.bulgaricus and Lactiplantibacillus plantarum are lactic acid bacteria, a similar expression pattern was observed only for CIN5.These results indicate that S. cerevisiae has different gene expression responses in the coculture of different bacterial species.

Conclusions
We showed that K. uropygioeca TGY1127_2 interacted with sake yeast and altered its gene expression patterns.The genes involved in cell cycle/sporulation trended to be upregulated and those involved in metabolism trended to be downregulated in response to co-culture with K. uropygioeca TGY1127_2.The gene expression of yeast in co-culture with kuratsuki Kocuria differed from that in co-culture with lactic acid bacteria.This strongly suggests that S. cerevisiae exhibits different gene expression patterns in response to different bacteria during sake production.Thus, a novel combination of sake yeast and kuratsuki bacteria can lead to a unique sake that has never existed before.
Although our findings showed that the sake yeast changed its gene expression under co-existing with the kuratsuki Kocuria, these results cannot be directly applied to the relationship between sake yeast and kuratsuki bacteria in sake making.This is because the culture conditions are different.It is already known that the effect of kuratsuki bacteria differs by changing the type of koji, etc.In the future, it will be necessary to investigate how sake yeast is affected by kuratsuki bacteria in the sake-making environment.

Figure 1 .
Figure 1.Brix of monoculture of the sake yeast K1401 and co-culture with the kuratsuki Kocuria TGY1127_2.These change patterns were not significantly different (p > 0.05 in the Kolmogorov-Smirnov test).

Figure 1 .
Figure 1.Brix of monoculture of the sake yeast K1401 and co-culture with the kuratsuki Kocuria TGY1127_2.These change patterns were not significantly different (p > 0.05 in the Kolmogorov-Smirnov test).

Fermentation 2024 , 15 Figure 2 .
Figure 2.More than 2-fold upregulated genes of sake yeast in co-culture with the kuratsuki Kocuria.Blue, green, red, and yellow indicate the genes involved in stress response, cell cycle, and/or sporulation, retrotransposons, and metabolism, respectively.Purple indicates the gene involved in stress response and cell cycle.Grey indicates the other genes.

Figure 2 .
Figure 2.More than 2-fold upregulated genes of sake yeast in co-culture with the kuratsuki Kocuria.Blue, green, red, and yellow indicate the genes involved in stress response, cell cycle, and/or sporulation, retrotransposons, and metabolism, respectively.Purple indicates the gene involved in stress response and cell cycle.Grey indicates the other genes.

Figure 3 .
Figure3.Less than 0.5-fold downregulated genes of sake yeast in co-culture with the kuratsuki Kocuria.Blue, green, red, and yellow indicate the genes involved in stress response, cell cycle, and/or sporulation, retrotransposons, and metabolism, respectively.Pink indicates the gene involved in stress response and metabolism.Grey indicates the other genes.

Figure 3 .
Figure3.Less than 0.5-fold downregulated genes of sake yeast in co-culture with the kuratsuki Kocuria.Blue, green, red, and yellow indicate the genes involved in stress response, cell cycle, and/or sporulation, retrotransposons, and metabolism, respectively.Pink indicates the gene involved in stress response and metabolism.Grey indicates the other genes.

Table 1 .
Expression change of histone genes of sake yeast in co-culture with the kuratsuki Kocuria.