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Polyploid engineering by increasing mutant gene dosage in yeasts

FAS2 encoding α subunit of fatty acid synthetase is one of essential genes in S. cerevisiae, and theFAS2‐G1250S mutation decreases carbon chain elongation activity during fatty acid synthesis, resulting in high productivity of ethyl caproate. Evaluation for mutant gene dosage using polyploids with isogenic background revealed the trade‐off relationship between ethyl caproate productivity and cell growth rate. We further demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains.


Introduction
Yeast has been implemented in the production of various useful compounds. Saccharomyces cerevisiae, commonly referred to as budding yeast is employed in the production of ethanol via fermentation (Yamada et al., 2010;. S. cerevisiae cells primarily exist in two forms: haploid and diploid (Zhang et al., 2017). Although haploid cells cannot endure high-stress conditions such as nutrient starvation, diploid cells survive harsh environmental conditions via sporulation process in sexual reproduction (Fukuda and Honda, 2019). Diploidization has been considered as an evolutionary phenomenon that develops adaptation in response to drastic changes in growth conditions (Mangado et al., 2018). Diploid strains of S. cerevisiae are commonly used in brewing industries due to the better fermentation efficiency in terms of vitality and endurance as compared to those of haploid strains. However, strenuous efforts have been devoted to develop custom-engineered strains of S. cerevisiae to improve the production of ethanol and other compounds such as organic acids.
Mutagenesis is one of the conventional and effective approaches utilized for modifying yeast traits. Ethyl methanesulfonate (EMS) and ultraviolet (UV) light are commonly used mutagens. Mutagenic treatments can induce 100-fold increase in mutations per gene. The mutagenized cells can then be screened for any specific phenotype of interest, such as, auxotrophy and antibiotic sensitivity (Winston, 2008). Due to heterozygosity at one or more loci in diploid cells, genetic alterations that contribute to phenotypic changes as a result of mutagenesis are more complex compared to those in haploid cells. An exception to this would be in the case of recessive mutations, where modified diploid cells carry two alleles of the same gene on homologous chromosomes.
Dominance and recessiveness are fundamental principles of modern genetics. In the case of complete dominance, a phenotype of the heterozygous offspring is identical to one of the homozygous parents. Partial dominance is another type of dominance in which a heterozygote displays a phenotype that is an intermediate between the phenotypes of both homozygote parents. Cell morphology is one of attractive targets for phenotype analysis because it reflects a wide variety of cellular events (Ohya et al., 2005). A recent morphological study on heterozygous diploids of S. cerevisiae revealed that majority of essential genes are haploinsufficient (Ohnuki and Ohya, 2018), which indicates the contribution of gene dosage effects on phenotypes. Furthermore, foreign genes were downregulated in diploid cells generated through mating of haploid cells containing various foreign genes (Fukuda and Honda, 2018). Thus, gene dosage (the number of copies of a particular gene present in a genome) is an important factor in yeast trait modification.
S. cerevisiae has also been used as an excellent model organism for studying genome evolution (Lu et al., 2016). Polyploid (especially tetraploid) yeast cells are employed in several human-activities such as, bread-making (Albertin et al., 2009) and lager-brewing (Casaregola et al., 2001;Dunn and Sherlock, 2008;Nakao et al., 2009;Libkind et al., 2011). Due to multiple alleles of the same gene on homologous chromosomes, many polyploid species display higher heterozygosity levels compared to their diploid counterparts as in yeasts (Albertin et al., 2009). Although polyploid strains have been extensively used as models for cancer or cell cycle defects (Thorpe et al., 2007), there is only few scientific knowledges on polyploid yeasts industrially used in bakery, brewery, etc (Albertin and Marullo, 2012). Additionally, complex heterozygosity associated with diploid yeasts contributes to the difficulty in understanding the gene dosage effect in food industry, where it is quite important to balance out various components to adjust the flavour.
Therefore, in this study, we investigated the dosage effect of the mutant gene of interest using haploids, diploids, triploids and tetraploids with isogenic background (Table 1). FAS2 encodes a subunit of fatty acid synthetase and the FAS2-G1250S mutation leads to an increase in the amount of caproic acid (Ichikawa et al., 1991;Aritomo et al., 2004), a precursor of ethyl caproate, which is one of the major flavour components in the Japanese alcoholic beverage called sake (Tamura et al., 2015). To trace back to its origin, yeast strains with high ethyl caproate-producing ability were isolated based on its resistance to cerulenin (Ichikawa et al., 1991), an inhibitor of fatty acid synthesis. We performed quantitative evaluation of the phenotypic change caused due to FAS2-G1250S mutation using the cerulenin resistance as an indicator. We also increased the mutant gene dosage within yeast cells via regulating cerulenin concentration in the cultivation media.

Confirmation of the FAS2 mutation and cerulenin resistance
Haploid strains BY4741C (MATa) and BY4742C (MATa) containing the FAS2-G1250S mutation (Table 1) were previously generated via homologous recombination (Fukuda, 2020). The difference in nucleotide sequence between the wild-type FAS2 and FAS2-G1250S allele was confirmed using Sanger sequencing (Fig. S1). To easily distinguish the FAS2-G1250S mutant from the wild-type gene, we amplified a region of the FAS2 gene (1424-bp in length) using PCR, cleaved the DNA fragments with the restriction enzyme BfaI (Fig. 1A), and analysed the final products using electrophoresis pattern (Fig. 1B), referring to a past report (Tamura et al., 2015). Since haploid strains have a single set of chromosomes, the FAS2-G1250S mutant (BY4741C and BY4742C) was distinguished from the FAS2 gene (BY4741 and BY4742) by the absence of a 645 bp DNA fragment, and the presence of 420 bp and 225bp DNA fragments. In some cases, there was a small amount of undigested DNA fragments were observed even after digestion with BfaI (lanes 2 and 6) as shown in Figure 1B, indicating incomplete digestion. These undigested DNA fragments completely disappeared by modulating the ratio of enzyme to DNA (Fig. S2).
For the quantitative evaluation of the effect of FAS2-G1250S mutation, dose responsive curves for cerulenin were compared between MATa haploid strains, BY4741 and BY4741C. We observed that strain BY4741C was able to grow in cultivation media with approximately 10fold higher concentration of cerulenin than strain BY4741 ( Fig. 1C and Table 2). With > 50 lM cerulenin, there was no significant increase in optical density measured at 620 nm (OD 620 ) in case of BY4741C strain. Since fatty acids are essential components of eukaryotic and bacterial cells, a large amount of cerulenin supplemented via extracellular sources would prevent cell growth via inhibition of fatty acid synthesis. The inhibition of fatty acid synthesis using cerulenin is based on its covalent binding to the cysteine residue at the active site of the condensing enzyme component (Kawaguchi et al., 1982;Tomoda et al., 1987). Although the FAS2-G1250S mutation decreases carbon chain elongation during fatty acid synthesis, substitution of the glycine residue at position 1250 causes steric hindrance to cerulenin binding, thereby inducing cerulenin resistance in yeast mutant strains (Aritomo et al., 2004).

Generation of diploid strains for evaluation of the homozygous or heterozygous mutations
Subsequently, we generated MATa/a diploid strains through yeast mating between MATa and MATa haploid strains ( Fig. 2A). We performed DNA fragment analysis using electrophoresis pattern similar to the haploid strains (Fig. 2B). Due to the homozygosity of the FAS2 gene, diploid strains BY4743 and BY4743CC exhibited the same electrophoresis patterns as haploid strains BY4741 and BY4741C respectively. In the case of the heterozygous diploid strain BY4743C, there we observed four DNA fragments of varying lengths (779, 645, 420 and 225 bp) due to the presence of two alleles, FAS2 and FAS2-G1250S. Next, we compared dose responsive curves for cerulenin among the three diploid strains. Similar to relationship between haploids, diploid strain BY4743CC was able to grow in cultivation media with approximately 10-fold higher concentration of cerulenin than strain BY4743 ( Fig. 2C and Table 2). Although homozygous diploid strains BY4743 and BY4743CC exhibited similar dose responsive curves to those of strains BY4741 and BY4741C, respectively, heterozygous diploid strain BY4743C exhibited intermediate behaviour, indicating partial dominance of the FAS2-G1250S mutation.
Based on the difference in cerulenin resistance among diploid strains described above, we introduced to a cerulenin-sensitive diploid strain (BY4743) with FAS2-G1250S mutation via homologous recombination and increased mutant gene dosage in diploid cells through loss of heterozygosity (LOH). As shown in Fig. 3A, homologous recombination could occur at either of the FAS2 loci due to its low frequency when strain BY4743 was transformed with PCR fragments with the FAS2-G1250S mutation. We then isolated heterozygous cells generated via recombination of the FAS2 using 10 lM cerulenin, which yielded diploid strain BY4743C1. DNA fragment analysis shows the heterozygosity at the FAS2 loci in the generated diploid strain (Fig. 3B). LOH is a natural event that generates homozygous loci via chromosomal rearrangement in heterozygous loci (Daigaku et al, 2004;Alvaro et al, 2006;Andersen et al., 2008;Takagi et al, 2008). Although the spontaneous frequency of LOH is below 1 9 10 À4 as reported in previous studies (Hiraoka et al, 2000;Fukuda et al, 2016), it is higher than that of a point mutation, approximately 1 9 10 À6 (Hayashi and Umezu, 2017). After cultivating strain BY4743C without cerulenin, we isolated homozygous cells generated from heterozygous cells via LOH of the FAS2 using 25 lM cerulenin (Fig. 3C). In replica plating, five out of eight clones were able to grow in the same condition (25 lM cerulenin). After confirming equivalency, one of the replicated clones was selected as diploid strain BY4743C2. Then, we confirmed the homozygosity at the FAS2 loci in the generated diploid strain using DNA fragment analysis (Fig. 3D). These results indicated that it is possible to regulate mutant gene dosage of FAS2-G1250S in diploid cells using cerulenin resistance as an indicator. Similarly, we introduced the FAS2-G1250S mutation to a sake yeast strain Kyokai No.9 (K9) via homologous recombination. Sake is a traditional Japanese alcoholic  beverage made from fermented rice, and sake yeast strains have many characteristics suitable for brewing, such as aromatic production and high ethanol tolerance (Katou et al, 2008). A previous study (Fukuda et al, 2016) has reported that K9 strain maintains a neardiploid DNA content, which was measured using FACS analysis after propidium iodide-staining. Unexpectedly, only homozygous cells were generated via recombination of the FAS2 gene in K9 strain (Fig. S3A). K9C-S01, K9C-S03 and K9C-S04 strains exhibited reduced cerulenin resistance as compared to the homozygous diploid strain BY4743CC (Fig. 4A). To investigate the copy number of the FAS2 in K9 strain, real-time PCR was carried out using the haploid strain BY4741 as the reference sample. The relative copy number of the RAD51 (locating at the right arm of chromosome V) and PGK1 (locating at the right arm of chromosome III) was calculated against the FAS2 (locating at the left arm of chromosome XVI). Unlike the RAD51, the relative copy number of the PGK1 gene was approximately double that of the FAS2 gene in the K9 strain (Fig. 4B). On the other hand, the relative copy number of the RAD51 and PGK1 was the identical to FAS2 in the diploid strain BY4743 (Fig. S3B). These results suggest that K9 strain is an aneuploid and might have lost one of the two FAS2 loci through spontaneous chromosomal aberration such as mitotic chromosome loss . Since chromosomal aneuploidy is frequently seen in other sake brewery yeasts (Kadowaki et al, 2017), we further investigated the dosage effect of the FAS2-G1250S mutation using laboratory-adapted triploid and tetraploid strains with isogenic background.
The dosage effect of the FAS2-G1250S mutation in triploids and tetraploids Mating type conversion was carried out (Fig. 5A) according to a previously reported method (Fukuda and Honda, 2018). Triploid and tetraploid strains were generated via yeast mating. Haploid strains BY4741 (MATa) and BY4742 (MATa) are derived from S288C (Brachmann et al, 1998) strain, which is one of the most widely used laboratory-adapted yeast strains, with its genome sequence registered in the Saccharomyces Genome Database (http://www.yeastgenome.org/). All S288Cderived strains possess the stuck mutation (Ray et al, 1991) at the HMRa locus, which significantly reduces the Ho endonuclease cleavage (Herskowitz and Jensen, 1991) after it is transferred to the MAT locus via mating type conversion. As shown in Figure 5A, diploid strains generated in this study maintain both uncleavable MATa and cleavable MATa genes against the Ho endonuclease. After the introduction of pUY-Pa1G-HO (Table 1), the expression of HO in MATa/a diploid cells could induce unidirectional replacement of DNA sequences at the MAT loci from a-type to a-type. MATa/a diploid strains BY4743AA and BY4743CC-AA were generated using MATa/a diploid strains BY4743 and BY4743CC respectively (Table 1). Further, we generated triploid strains with varying numbers of the FAS2-G1250S mutation through yeast mating (Fig. 5B) and compared dose responsive curves for cerulenin among  of the four triploid strains (Fig. 5C). Like diploid strains, homozygous triploid strains BY4743-3 and BY4743-3CCC exhibited similar dose responsive curves to those of haploid strains BY4741 and BY4741C respectively. Furthermore, heterozygous triploid strains BY4743-3C and BY4743-3CC exhibited intermediate behaviours in response to the copy number of the FAS2-G1250S mutation (Table 2). These results suggest that phenotypic change in cerulenin resistance is determined by the ratio of partially dominant mutations at the FAS2 loci.
Triploid strains described above maintain two copies of uncleavable MATa and one copy of cleavable MATa genes against the Ho endonuclease (Fig. 5A). Using mating type conversion and yeast mating, we generated tetraploid strains with varying numbers of the FAS2-G1250S mutation via yeast mating (Table 1) and compared dose responsive curves for cerulenin among the five tetraploid strains (Fig. 5D). Similar to the triploid strains, both homozygous and heterozygous tetraploid strains exhibited gradual phenotypic change in cerulenin resistance according to the ratio of partially dominant mutations at the FAS2 loci (Table 2). From haploid to tetraploid, homozygous strains (containing either FAS2 or FAS2-G1250S) exhibited similar level of antibiotic resistance regardless of yeast ploidy.
Additionally, we investigated the effect of a single copy of the FAS2-G1250S mutation on the antibiotic resistance by comparing the OD 620 values of yeast cultures with initial cerulenin concentration set at 5 lM (Fig. S4). The antibiotic resistance induced by a single copy of mutation obviously declined as yeast ploidy increased. A similar phenomenon was also observed in the previous report, which used foreign genes encoding fluorescent proteins (Fukuda and Honda, 2018). In this case, the amount of intracellular Fas2 G1250S would have declined as yeast ploidy increased, whereas wild-type Fas2 would have increased. Comparing the step size of phenotypic changes, a fine adjustment could be achieved using polyploid strains (polyploid engineering) instead of diploid strains via regulation of cellular functions involved in partially dominant mutations.

Polyploid engineering by increasing the dosage of the FAS2-G1250S mutation
Similar to the diploid strains, we introduced the FAS2-G1250S mutation in a cerulenin-sensitive tetraploid strain (BY4743-4) via homologous recombination, and increased mutant gene dosage in tetraploid cells. As shown in Fig. 6A, homologous recombination could occur at one of four FAS2 loci. Setting the cerulenin concentration at 5 lM, we isolated heterozygous cells generated via recombination of the FAS2 and yielded tetraploid strain BY4743-4C1. DNA fragment analysis shows the existence of the FAS2-G1250S mutation in the generated tetraploid strain (Fig. 6B).
Unlike diploids, multicycle homologous recombination is required for generation of homozygous polyploids containing the same number of mutant alleles as the ploidy from heterozygous polyploids containing one copy of mutant allele. Setting cerulenin concentration at 25 lM, we increased the number of mutant alleles in tetraploid cells via homologous recombination of the FAS2 (Fig. 6C). In replica plating, two out of four clones were able to grow in the same condition (25 lM cerulenin). After confirming equivalency, one of the replicated clones was selected as tetraploid strain BY4743-4CX. Furthermore, we isolated homozygous cells generated through LOH by increasing cerulenin concentration to 100 lM. In replica plating, three out of three clones were able to grow in the same condition (100 lM cerulenin), and one of them was selected as tetraploid strain BY4743-4C4. Then, we confirmed the homozygosity at the FAS2 loci in the generated tetraploid strain by DNA fragment analysis (Fig. 6D). These results indicate that it is possible to regulate mutant gene dosage even in polyploid cells using cerulenin resistance as an indicator.
As described above, we revealed the relationship between cerulenin resistance and the FAS2-G1250S mutant gene dosage. To confirm whether the yeast cells that survived in cerulenin-rich environments can produce ethyl caproate at high concentration, we cultivated tetraploid strains at low temperature (15°C) appropriate for Japanese sake brewing (Fig. 7). The FAS2-G1250S mutation decreases carbon chain elongation activity during fatty acid synthesis, resulting in high productivity of ethyl caproate. Since fatty acids are essential components, an increase in the FAS2-G1250S mutant gene dosage caused declined cell growth rates (Fig. 7A). Tetraploid strains BY4743-4CX and BY4743-4C4 were able to grow to the same extent as BY4743-4CC (harbouring 2 copies of the FAS2-G1250S mutant allele) and BY4743-4CCCC (harbouring 4 copies of the mutant allele) respectively (Fig. S5A). At the end of cultivation, we evaluated and compared the productivity of ethyl caproate among tetraploid strains. Similar to cerulenin resistance, the productivity of ethyl caproate was improved with the FAS2-G1250S mutant gene dosage (Fig. 7B). There was no significant difference in the productivity of ethyl caproate between the strains, BY4743-4CCC (harbouring 3 copies of the mutant allele) and BY4743-4CCCC (harbouring 4 copies of the mutant allele). Considering that ethyl caproate concentration continued to increase till the end of cultivation of strain BY4743-4CCCC (Fig. S5B), longer cultivation period might be required to reach a plateau. By evaluating mutant gene dosage effect in tetraploids with isogenic background, it was also found that the trade-off relationship between ethyl caproate productivity and cell growth rate was caused by the FAS2-G1250S mutation (Fig. 7C). In addition, we successfully generated yeast strains with high ethyl caproate-producing ability (BY4743-4CX and BY4743-4C4) from heterozygous strain containing one copy of mutant allele (BY4743-4C) via cerulenin screening.
To evaluate the genome stability in the isolated homozygous yeast cells, we carried out passage culture (without cerulenin) of tetraploid strain BY4743-4C4 containing 4 copies of the FAS2-G1250S mutation. DNA fragment analysis demonstrated that yeast cells maintained the FAS2-G1250S mutation even after 10 passages of culture (Fig. S6A). Mutant gene dosages of yeast cells were estimated by evaluating the cell growth in cultivation medium containing 25 lM cerulenin (Fig. S6B). Compared to the tetraploid strain containing 3 copies of the FAS2-G1250S mutation (BY4743-4CCC), passaged cells exhibited higher cerulenin resistance, indicating the existence of 4 copies of the FAS2-G1250S mutation. These results indicate that the FAS2-G1250S mutation could be stably maintained in yeast cells even in the absence of cerulenin, and the generated yeast cells would exhibit stable performance in the industrial use such as brewing.

Challenge in yeast trait modifications using polyploid engineering
A variety of antibiotics are utilized for selection of yeast cells of interest from mutagenized populations. In this study, we utilized cerulenin to distinguish the activity of the FAS2-G1250S. Unfortunately, however, it is not rare to isolate yeast cells having undergone off-target mutations instead of the on-target mutation in antibiotic screening. A previous study (Tamura et al, 2015) showed that less than 0.5% of cerulenin resistant mutants exhibited high ethyl caproate-producing ability. Similar to tumour cells, parasites, fungal pathogens or even bacteria, S cerevisiae exhibits multidrug resistance to unrelated chemicals, as a result of functional enhancement of ATP-binding cassette (ABC) transporters (Jungwirth and Kuchler, 2006). It is known that the genome of S. cerevisiae harbours 30 distinct genes encoding ABC transporters, several of which are involved in cellular detoxification (Nourani et al, 1997). As described above, there is a possibility of including off-target mutations in case of antibiotic screening. On the other hand, homologous recombination between intracellular homologous chromosomes occurs with higher frequencies than point mutations in polyploid cells (Hayashi and Umezu, 2017). Therefore, mutant gene dosage in polyploid cells can be increased via antibiotic screening without inducing undesirable off-target mutations. Additionally, excess amount of antibiotics causes annihilation of yeast cells, and shortage of antibiotics makes it difficult to isolate yeast cells with increased mutant gene dosage in polyploid engineering. In this study, we revealed the difference in cerulenin resistance provided by the FAS2-G1250S mutant gene dosage in haploids, diploids, triploids, and tetraploids. According to the quantitative analysis of cerulenin resistance, we successfully achieved an increased FAS2-G1250S mutant gene dosage via antibiotic screening with appropriate concentration.

Conclusions
Mutant gene dosage plays a crucial role in yeast trait modification and can be achieved using the principle of partial dominance. In this study, we confirmed the dosage effect of the FAS2-G1250S mutation using haploids, diploids, triploids and tetraploids with isogenic laboratory-adapted yeast strains. In polyploid engineering, homozygous strains generated were successfully isolated at higher concentrations of cerulenin as compared to heterozygous strains. There was an improved productivity of ethyl caproate with increase in mutant gene dosage. Therefore, this approach can be unrestrictedly used for growth-based selections of yeast strains which have been generated through mutagenesis and are currently used in food industries, by taking advantage of partial dominance of mutant genes. It would also facilitate fine adjustments in yeast trait modifications, thereby improving productivity, flavour and taste of compounds processed using industrial yeasts that are used in bakery, brewery, etc.

Strains and media
Detailed information regarding the S. cerevisiae laboratory yeast strains and plasmids used is listed in Table 1. Yeast cells were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose), YPD10 medium (1% yeast extract, 2% peptone, and 10% glucose), SD medium (0.67% yeast nitrogen base without amino acids [Becton Dickinson and Company, Franklin Lakes, NJ, USA], 2% glucose) or SGR medium (0.67% yeast nitrogen base without amino acids, 2% galactose and 2% raffinose). A final concentration of 2% agar was added to the liquid media to prepare solid media.

Construction of yeast strains
The yeast strains constructed in this study are listed in Tables 1, and Table 2 shows the sequences of oligonucleotides used in this study.
Yeast mating was performed as follows. Two kinds of yeast strains were co-cultivated in 1 mL of YPD medium at 30°C for 1.5 h, with an initial OD 600 of 0.1 for each. After cocultivation, yeast cells were harvested, washed and resuspended in distilled water. Starting from an initial OD 600 of 1.0, cell suspensions (100 ll) were spread on SD solid medium with appropriate minimum nutrients for growth selection of zygotes. SD solid medium without methionine and lysine was used for diploid formation. On the other hand, SD solid medium without histidine and lysine was used for triploid and tetraploid formation, following introduction of plasmid pYO323 into haploid strains BY4742 and BY4742C. Plasmid removal after yeast mating was achieved through passage culture in YPD medium (Fukuda, 2020).
For mating type conversion, pUY-Pa1G-HO was used to transform yeast strains. Transformants were selected on solid SD medium lacking uracil. Mating-type-converted transformants (by the action of the Ho endonuclease) were then grown in SD medium lacking uracil following cultivation in SGR medium. The URA3 gene encodes orotidine 5'-phosphate decarboxylase (ODCase), which converts 5-FOA into a toxic compound within the cell (Boeke et al., 1984). Yeast cells that lacked plasmids containing the URA3 gene were isolated on solid medium supplemented with 5-fluoroorotic acid (5-FOA). To remove pUYG-HO, transformants were cultured in YPD medium and spread on solid SD medium containing 20 lg ml -1 uracil and 0.5 mg ml -1 5-FOA.

Investigation of cell growth characteristics
Each yeast strain was grown in 100 ll of YPD medium containing cerulenin at 30°C, with an initial OD 620 at 0.01. After 24 h of cultivation, the OD 620 values of cultures were measured using a photoelectric colorimeter (AE-15F; ERMA Inc., Tokyo, Japan).

DNA fragment analysis
DNA fragments of the partial region (1424 bp) of FAS2 were amplified using PCR with oligonucleotides 1 and 2 (described above) using genomic DNA as a template. Following DNA cleavage using restriction enzyme BfaI, separation of DNA fragments was carried out using agarose gel electrophoresis. Band patterns were detected using gel imager (FAS-IV; NIPPON Genetic Co, Ltd., Tokyo, Japan).

Evaluation for ethyl caproate productivity
The optical density was measured at 600 nm (OD 600 ) using a UV-Visible spectrophotometer (UV-2550; Shimadzu Corporation, Kyoto, Japan). Setting the initial OD 600 at 0.02, the yeast cells were cultivated in 100 ml of YPD10 medium at 15°C for 8 days without shaking. At the end of cultivation, supernatants were obtained by centrifugation and then injected to gas chromatography mass spectrometer (GC/MS) (7890/5975C; Agilent Technologies, Santa Clara, CA, USA). The amount of ethyl caproate in culture supernatants was evaluated according to the standard method of National Research Institute of Brewing, Japan (Brewing Society of Japan, 2017). Additionally, daily sampling was carried out, and supernatants obtained by centrifugation were frozen and stored at À20°C. To avoid undesirable chemical reactions (caused by secreted enzymes or uneliminated yeasts) in supernatants used for multi-sample measurement, the frozen-thawed supernatants were incubated at 65°C for 10 min before injection to GC/MS. Although there must be partial loss in the concentrations of ethyl caproate, changing trends during cultivation can be indicated by relative comparison.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article. strain BY4742, and the second line indicates that of the FAS2-G1250S mutant derived from haploid strain BY4742C. Fig. S2. Cleavage pattern of the DNA fragments of the FAS2. BfaI-digested (+) and non-digested DNA fragments (À) were loaded onto an agarose gel. Lane M indicates the DNA size marker 1 and 2 indicate strain BY4741, and 3 and 4 indicate strain BY4742. Fig. S3. Generation and evaluation of sake yeasts with the FAS2-G1250S mutation. (A) Cleavage pattern of the DNA fragments of the FAS2. BfaI-digested (+) DNA fragments were loaded onto an agarose gel. Lane M indicates a DNA size marker. 1, 2 and 3 indicate the generated sake yeast strains K9C-S01, K9C-S03 and K9C-S04, respectively. (B) Ploidy analysis using real-time PCR. Blue columns indicate the relative copy number of the RAD51 against the FAS2, and red columns indicate that of the PGK1. Fig. S4. Comparison of antibiotic resistance among yeast strains containing a single copy of the FAS2-G1250S with different ploidy. Yeast cells were cultivated in YPD medium containing 5 lM cerulenin at 30°C for 24 hours, setting initial OD 620 at 0.01. Black columns indicate homozygous FAS2-G1250S mutant strains of haploid (BY4741C), diploid (BY4743CC), triploid (BY4743-3CCC) and tetraploid (BY4743-4CCCC) yeast cells, respectively. Blue columns indicate yeast strains containing a single copy of the FAS2-G1250S mutation with varying ploidy (haploid, diploid, triploid, and tetraploid indicate BY4741C, BY4743C, BY4743-3C and BY4743-4C, respectively). Values are presented as means AE standard deviations from three independent experiments. Fig. S5. Evaluation for growth and ethyl caproate producing-ability of tetraploid strains with different number of the FAS2-G1250S mutation. (A) The OD 600 values of yeast cultures (117 hours). (B) Changes in ethyl caproate concentrations. After incubating the collected and frozen-thawed supernatants at 65°C for 10 minutes, ethyl caproate concentrations were measured using GC/MS to trace the time courses (ND: not detected). Values are presented as means AE standard deviations from two independent experiments. Fig. S6. Evaluation for the genome stability of the homozygous tetraploid strain BY4743-4C4 generated through LOH.
(A) Cleavage pattern of the DNA fragments of the FAS2. Yeast cells were passaged daily with 100-fold dilution using YPD medium without cerulenin for 10 days. FAS2 was amplified using PCR, and BfaI-digested (+) and non-digested DNA fragments (À) were loaded onto an agarose gel. Lane M indicates a DNA size marker, 1 and 2 indicate passage 1 (P1), 3 and 4 indicate passage 5 (P5), and 5 and 6 indicate strain passage 10 (P10). (B) Comparison of the cerulenin resistance. Yeast cells were cultivated in YPD medium containing 25 lM cerulenin, and the OD 620 values were measured and compared. Values are presented as means AE standard deviations from three independent experiments. The red dotted line indicates the level of cerulenin resistance of heterozygous tetraploid strain BY4743-4CCC harbouring three copies of the FAS2-G1250S mutations.