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Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells

Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells Sake yeasts belong to the budding yeast species Saccharomyces cerevisiae and have high fermentation activity and ethanol production. Although the traditional crossbreeding of sake yeasts is a time-consuming and inefficient process due to the low sporulation rates and spore viability of these strains, considerable effort has been devoted to the development of hybrid strains with superior brewing characteristics. In the present work, we describe a growth selection system for a- and α-type cells aimed at the crossbreeding of industrial yeasts, and performed hybridiza- tions with sake yeast strains Kyokai No. 6, No. 7 and No. 9 to examine the feasibility of this approach. We successfully generated both a- and α-type strains from all parental strains, and acquired six types of hybrids by outcrossing. One of these hybrid strains was subjected to continuous crossbreeding, yielding the multi-hybrid strain, which inherited the genetic characteristics of Kyokai No. 6, No. 7 and No. 9. Notably, because all of the genetic modifications of the yeast cells were introduced using plasmids, these traits can be easily removed. The approach described here has the potential to markedly accelerate the crossbreeding of industrial yeast strains with desirable properties. Keywords: Sake yeast, Crossbreeding, Mating type, Growth selection, Hybrid strain 2001; Kishimoto 1994; Shinohara et  al. 1997). Common Introduction breeding strategies, such as backcrossing and multi- Sake is a traditional Japanese alcoholic beverage made hybridization, require cycles of hybridization (continu- from fermented rice. In the production of sake, rice ous crossbreeding). Most sake yeasts are MATa/α diploid starch is first degraded by the koji fungus Aspergillus ory- strains and are unable to mate directly; therefore, the zae into glucose, which is then fermented to ethanol by isolation of MATa and MATα haploid strains via sporula- sake yeast, strains of the budding yeast species Saccha- tion is a prerequisite for crossbreeding. However, because romyces cerevisiae (Kitagaki and Kitamoto 2013; Shiroma industrially used sake yeast strains, such as Kyokai No. 7 et  al. 2014). Sake yeasts have many characteristics suit- and No. 9, have low sporulation rates (Suizu et al. 1996), able for sake brewing, such as aromatic production and the crossbreeding of sake yeast strains is inefficient and high ethanol tolerance (Katou et  al. 2008). Sake yeast technically challenging. strains have been selected through several hundred years To overcome the problem of poor sporulation, it is of brewing (Shiroma et  al. 2014); however, more rapid possible to select for strains that have undergone spon- methods for generating new and superior strains are taneous chromosomal aberrations, such as loss of hete- highly desirable. rozygosity (LOH) and mitotic chromosome loss, during Crossbreeding is an attractive approach to improve and mitotic division to obtain a- and α-type yeast cells that combine traits of different yeast strains (Higgins et  al. possesses similar mating abilities as MATa and MATα haploids generated via sporulation (Fukuda et al. 2013a). LOH is a natural genetic event that generates homozy- *Correspondence: nob-fukuda@aist.go.jp Biomedical Research Institute, National Institute of Advanced Industrial gous loci via chromosomal rearrangement of het- Science and Technology (AIST ), Higashi, Tsukuba, Ibaraki 305-8566, Japan erozygous loci (Alvaro et  al. 2006; Andersen et  al. 2008; Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fukuda et al. AMB Expr (2016) 6:45 Page 2 of 12 Daigaku et  al. 2004; Takagi et  al. 2008), whereas mitotic α-type derivative cells by removing unnecessary plasmids chromosome loss, which is also a naturally occurring and introducing new plasmids. In this work, the feasibil- event, involves the loss of single or multiple chromo- ity of this approach for sake yeasts was demonstrated by somes (Mayer and Aguilera 1990). Therefore, a LOH performing and generating modified strains through con - event at the mating-type (MAT) locus within MATa/α tinuous crossbreeding. cells produces either MATa/a or MATα/α cells, whereas yeast cells that lose one of both copies of chromosome Materials and methods III containing the MAT locus during mitotic division Strains and media become MATa or MATα cells (Fukuda et  al. 2013a). Detailed information about S. cerevisiae laboratory yeast However, because the spontaneous occurrence frequen- strain BY4742 (Brachmann et  al. 1998) and sake yeast cies of LOH and mitotic chromosome loss are less than strains Kyokai No. 6, No. 7 and No. 9 (provided by the −4 1  ×  10 , it is difficult to isolate the generated a- and Biological Resource Center, NITE, Japan), as well as the α-type cells from mixed cell populations (Hiraoka et  al. other strains used in this study, is shown in Table 1. Yeast 2000; Kumaran et al. 2013). cells were grown in YPD medium (1 % yeast extract, 2 % To isolate a- and α-type cells, we previously established peptone and 2  % glucose) or SD/MSG medium (0.17  % a growth selection system for laboratory yeast strains yeast nitrogen base without amino acids and ammo- using the auxotrophic marker URA3 (Fukuda et  al. nium sulfate [Becton–Dickinson and Company, Franklin 2013a). In this system, the expression of the marker gene Lakes, NJ, USA], 0.1  % monosodium glutamate and 2  % is induced in a mating-type-specific manner, thereby glucose). A final concentration of 2 % agar was added to permitting the efficient selection of a- and α-cells from both types of media to prepare solid media. within a mixed cell population. Using this approach, we succeeded in isolating a- and α-type derivative cells from Construction of plasmids a cell population of parental MATa/α laboratory yeasts The sequences of the oligonucleotides used in this study without any false positives, and confirmed that these cells are listed in Table  2. The plasmids used in this study were able to mate and produce new hybrid yeasts. (Table  1) were constructed as follows. Using pTriEx-2 Unlike auxotrophic laboratory yeasts, however, indus- Hygro (Novagen, Inc., Madison, WI) as a template, the trially used yeasts, including sake yeasts, are generally hygro gene was amplified with oligonucleotide pair o1 prototrophic, which prevents the use of auxotrophic and o2. P was also amplified from genomic DNA TDH3 markers for the selection of strains following mating. In derived from strain BY4742 using oligonucleotide pair addition, industrial yeast strains have remarkably low o3 and o4. The two amplified DNA fragments, P TDH3 genetic transformation efficiencies compared to labora - and hygro, were combined using the In-Fusion HD Clon- 1 2 tory strains (sake yeast, 10 ~10  cfu/μg-DNA; laboratory ing kit (Takara Bio, Inc., Shiga, Japan), and the resulting 4 5 yeast, 10 ~10   cfu/μg-DNA) (Ogata et  al. 1993). There - DNA fragment, P -hygro, was amplified with oligonu - TDH3 fore, a complete method for the transformation, isolation, cleotide pair o3 and o2, and then digested with SacI and and evaluation of a- and α-type derivative and hybrid BamHI. In addition, T was amplified from genomic PGK1 cells, is required for the efficient crossbreeding of sake DNA derived from strain BY4742 using oligonucleotide yeasts. pair o5 and o6, and the obtained fragment was digested Here, we designed and constructed two types of plas- with BamHI and XhoI. The two digested DNA fragments, mids for isolating a- and α-type sake yeasts from mixed P -hygro and T , were inserted at the SacI-XhoI TDH3 PGK1 cell populations (Fig.  1). Although drug sensitivity var- sites of pLY-3U to replace the P -URA3-T cas- STE3 CYC1 ies from strain to strain, the hygro (hygromycin B-resist- sette (Fukuda et al. 2013a), yielding a plasmid designated ance) and kanMX4 genes (G418-resistance) have been pLY-hygro. commonly used for the selection of prototrophic yeasts Using pK6 (Fukuda et  al. 2013b) as a template, the (Murakami et al. 2012). To develop a versatile method for expression cassette of the kanMX4 marker (consisting crossbreeding of sake yeasts, we used the hygro gene as a of P , ORF and terminator) was amplified with oligo - TEF1 transformation marker and the kanMX4 gene as a marker nucleotide pair o7 and o8, and inserted in place of the for isolation of a- or α-type derivative cells. In this P -EGFP-T cassette at the SacII-XhoI sites of PGK1 ADH1 approach, the a- and α-type derivative cells must have pHY-PGA (Fukuda et  al. 2013a), yielding a plasmid des- different marker genes. Because yeast transformation ignated pHY-kan. is performed using plasmids in our system, the marker Using pLY-hygro as a template, a DNA fragment con- genes are lost in the absence of selection pressure. There - taining the P -hygro-T cassette was amplified TDH3 PGK1 fore, through plasmid exchange, different marker genes with oligonucleotide pair o3 and o9. Subsequently, a for hybridization can be introduced into the target a- and DNA fragment containing the P -a1-T cassette PGK1 ADH1 Fukuda et al. AMB Expr (2016) 6:45 Page 3 of 12 ac CEN6/ CEN6/ ARSH4 ARSH4 r r Amp Amp PTDH3 PTDH3 pLhyS-2K-Pa1 pHhyS-3K-2α TCYC1 TADH1 hygro hygro kanMX4 kanMX4 TPGK1 TPGK1 TADH1 TADH1 PSTE2 PSTE3 a1 α2 PPGK1 PSTE2 b a-type of cell d a-type of cell a1 a1 α2 G418 resistance kanMX4 kanMX4 α-type of cell α-type of cell α2 a1 α2 G418 resistance kanMX4 kanMX4 a/α-type of cell a/ -type of cell a1 a1 α2 α2 kanMX4 kanMX4 Fig. 1 Schematic outline of the strategy used for isolation of a- and α-type yeast cells. a Plasmid map of pLhyS-2 K-Pa1, which was used for the isolation of a-type yeast cells. b Isolation of a-type yeast cells using G418 selection. Formation of the a1-α2 complex in α-type and a/α-type cells represses expression of the kanMX4 marker gene. Only a-type cells are able to survive in culture medium containing G418 by expressing the kanMX4 marker gene. c Plasmid map of pHhyS-3 K-2α, which was used for the isolation of α-type yeast cells. d When introduced into a-type and a/α-type cells, the α2 protein represses expression of the kanMX4 marker gene. Only α-type cells are able to survive in culture medium containing G418 by expressing the kanMX4 marker gene was amplified with oligonucleotide pair o10 and o11 from sites of pLS-2 K (Fukuda et al. 2013a), yielding a plasmid pHPY-a1 (Fukuda et  al. 2013b). Using the In-Fusion kit, designated pLhyS-2 K-Pa1. the two amplified DNA fragments, P -hygro-T Similarly, a DNA fragment containing the P -α2- TDH3 PGK1 STE2 and P -a1-T , were inserted into the SacI-SacII T cassette was amplified with oligonucleotide pair PGK1 ADH1 ADH1 Fukuda et al. AMB Expr (2016) 6:45 Page 4 of 12 Table 1 Yeast strains and plasmids used in this study Name description Description Reference source Yeast strains BY4742 MATα his3∆1 ura3∆0 leu2∆0 lys2∆0 Brachmann et al. (1998) MCF4741 MATa his3∆1 ura3∆0 leu2∆0 met15∆0 Fig. 1::Fig. 1-EGFP-loxP-kanMX4-loxP Fukuda et al. (2013b) HR42-11T MATα his3∆1 ura3∆0 leu2∆0 lys2∆0 hmra ::HMRa -loxP-kanMX4-loxP Fukuda et al. (2013b) inc cut K6 Sake yeast; Kyokai No. 6; MATa/α NBRC2346 K7 Sake yeast; Kyokai No. 7; MATa/α NBRC2347 K9 Sake yeast; Kyokai No. 9; MATa/α NBRC2377 K6A a-type of strain derived from Kyokai No. 6 Present study K6AL α-type of strain derived from Kyokai No. 6 Present study K7A a-type of strain derived from Kyokai No. 7 Present study K7AL α-type of strain derived from Kyokai No. 7 Present study K9A a-type of strain derived from Kyokai No. 9 Present study K9AL α-type of strain derived from Kyokai No. 9 Present study K67 Hybrid strain generated by zygosis of K6A and K7AL Present study K69 Hybrid strain generated by zygosis of K6A and K9AL Present study K76 Hybrid strain generated by zygosis of K7A and K6AL Present study K79 Hybrid strain generated by zygosis of K7A and K9AL Present study K96 Hybrid strain generated by zygosis of K9A and K6AL Present study K97 Hybrid strain generated by zygosis of K9A and K7AL Present study K76A a-type of strain derived from K76 Present study K76AL α-type of strain derived from K76 Present study K76x9 Hybrid strain generated by zygosis of K76A and K9AL Present study Plasmids pLY-hygro 2μ ori, LEU2 marker and P -hygro Present study TDH3 pHY-kan 2μ ori, HIS3 marker and P -kanMX4 Present study TEF1 pAUR112 CEN4/ARS1 ori, URA3, AUR1-C Takara Bio, Inc., Shiga, Japan pLhyS-2 K-Pa1 CEN6/ARSH4 ori, LEU2, P -hygro, P -kanMX4 and P -a1 Present study TDH3 STE2 PGK1 pHhyS-3 K-2α CEN6/ARSH4 ori, HIS3, P -hygro, P -kanMX4 and P -α2 Present study TDH3 STE3 STE2 Resources were provided by Biological Resource Center (NBRC), NITE, Japan Table 2 Sequences of  oligonucleotides used to  construct o12 and o13 from pL3G-2α (Fukuda et al. 2013b). Using plasmids the In-Fusion kit, the two DNA fragments P -hygro- TDH3 T and P -α2-T were inserted into the SacI- PGK1 STE2 ADH1 Number Sequence SacII sites of pHS-3  K (Fukuda et  al. 2013a), yielding a 1 5′-CAAAgcggccgcATGGATAGATCCGGAAAGCC-3′ plasmid designated pHhyS-3 K-2α. 2 5′-AATTTATTTCggatccCTATTCCTTTGCCCTCGGAC-3′ Each constructed plasmid was introduced into yeast 5′-TATAGGGCGAATTGgagctcGAATAAAAAACACGCTTTTT-3′ cells using the lithium acetate method (Gietz et al. 1992). 4 5′-CATgcggccgcTTTGTTTGTTTATGTGTGTT-3′ 5 5′-GCTTATGTAAggatccGAAATAAATTGAATTGAAT-3′ Investigation of cell growth characteristics 6 5′-CGGGCCCCCCctcgagAGCTTTAACGAACGCAGAA-3′ Each yeast strain was grown at 30  °C in 500  μL YPD 7 5′-AATTGGAGCTCCAccgcggATCTGTTTAGCTTGCCTCGT-3′ medium supplemented with or without the antibiotics 5′-CGGGCCCCCCctcgagCTCGTTTTCGACACTGGAT-3′ hygromycin B (HYG), geneticin (G418) or aureobasidin 9 5′-CTCGAGGGGGGGCCCGgagctcAGCTTTAACGAACGCA- A (AUR; Takara Bio, Inc., Shiga, Japan) at the indicated GAA-3′ concentrations. The initial optical density at 600  nm 5′-GGTGATATTGGATaccgcggAGATGCCGATTTGGGC-3′ (OD ) values of the cultures were 0.03, and changes in 11 5′-CGGGCCCCCCctcgag-3′ the OD were monitored using a UV/visible spectro- 12 5′-TTTTCAACAAAATccgcgg-3′ photometer (Ultrospec 3100 pro; GE Healthcare Japan 13 5′-CGGGCCCCCCctcgagGAGCGACCTCATGCTATA-3′ Corp., Tokyo, Japan). Fukuda et al. AMB Expr (2016) 6:45 Page 5 of 12 Isolation of yeast cells with target mating‑type mating responses in both cell types. In contrast, a/α-type Parental a/α-type cells were grown in 500  μL YPD cells express both the a1 and α2 genes from MAT loci, medium containing 500 μg/mL HYG at 30 °C for 2 days, resulting in the formation of the a1-α2 complex, which and were passaged daily with 10,000-fold dilution. Yeast represses the expression of hsg (Fukuda et  al. 2013b). cells were then harvested and washed and resuspended Here, using machinery for mating-type-dependent gene in distilled water. Cell suspensions were spread on expression (Fukuda et  al. 2013a), the kanMX4 marker YPD + G418 (500 μg/mL for the isolation of a-type cells, gene was expressed in a- and α-type derivative cells. and 1.0  mg/mL for α-type cells) plates to isolate a- and The plasmid pLhyS-2  K-Pa1 (Fig.  1a) was constructed α-type yeast cells. for the selection of a-type yeast cells and contains the hygro gene as a transformation marker (hygromycin Mating assay B-resistance), the a1 gene for preventing undesirable Evaluation of mating ability was performed by cultivating mating between derivatives from the same parent by yeast cells with a mating partner in 1  ml YPD medium formation of the a1-α2 complex (Fukuda et al. 2013a, b), at 30  °C for 1.5  h. The initial OD of each strain was and the kanMX4 marker gene (repressed by α2 alone) 0.1. After cultivation, yeast cells were harvested, washed, (Fig. 1b). For the selection of α-type yeast cells, the plas- and resuspended in distilled water to give cell suspen- mid pHhyS-3  K-2α (Fig.  1c), which contains the hygro sions with OD values of 1, 0.1, and 0.01. A total of gene as a transformation marker, the α2 gene for the pre- 10  μl of each cell suspensions was spotted on SD/MSG venting undesirable mating by formation of the a1-α2 solid medium (without amino acids) containing 500  μg/ complex, and the kanMX4 marker gene (repressed by the mL G418 for the growth selection of zygotes. After incu- a1-α2 complex), was constructed (Fig.  1d). More details bation of the plates at 30  °C for 2  days, image data was of the gene expression regulation system used in these recorded for colonies that formed on the solid medium. constructs are described in previous reports (Fukuda et al. 2013a, b). Ploidy analysis using FACS The basic experimental design for the generation of Each yeast strain was grown overnight in 500  μL YPD a- and α-type sake yeast cells is schematically illustrated medium at 30 °C. The cells then were harvested, washed in Fig.  2a. Briefly, the plasmid pLhyS-2 K-Pa1 was intro - with 500 μL distilled water, and resuspended in 500 μL of duced into parental a/α-type sake yeast strains to isolate 70  % ethanol. After a 1-h incubation at room tempera- a-type of derivatives, whereas the plasmid pHhyS-3 K-2α ture, the ethanol-treated cells were harvested, washed was used to transform other a/α-type strains to iso- with 500  μL phosphate buffered saline (PBS), and resus - late α-type derivatives. In both cases, positive transfor- pended in 90  μL PBS containing 0.5  mg/mL RNase A. mants were selected on solid medium containing HYG After 1 h of incubation at 37 °C, 10 μL of 1 mg/mL pro- (YPD + HYG plates). After several passages of the trans- pidium iodide (PI) solution was added to the cell suspen- formant cultures, each cell mixture was individually sion, which was further incubated at 37  °C for 30  min spread on solid medium containing G418 (YPD +  G418 to stain DNA. The stained cells were harvested, washed plates) for the selection of a- or α-type cells expressing with 100 μL PBS, and resuspended in 1 mL sheath solu- the kanMX4 gene. tion (Becton, Dickinson and Co., Franklin Lakes, NJ, Hybrid cells cannot be generated via the mating of USA). After a 5-s sonication to reduce cell flocculation, derivative a- and α-type cells, because both cell types PI-fluorescence of yeast cells was detected using a BD express the same marker genes (hygro and kanMX4). To FACS Canto II flow cytometer (Becton, Dickinson and allow differentiation of the a - and α-type cells, plasmid Co., Franklin Lakes, NJ, USA) equipped with a 488-nm exchanges were performed to introduce unique selection blue laser, and the collected data were an analyzed using maker gene (Fig.  2a). The plasmids pLhyS-2  K-Pa1 and FlowJo software (Tree star, Ashland, Oregon, USA). The pHhyS-3  K-2α were removed by passaging cells in the fluorescence signal was collected through a 585/21  nm absence of selection pressure, and the plasmids pLY-hygro band-pass filter. (HYG-resistance) and pHY-kan (G418-resistance) were then introduced into derivative a-type and α-type cells, Results respectively. Hybrid a/α-type cells were selected on solid General isolation strategy for a‑ and α‑type yeast cells YPD medium containing HYG and G418 (YPD  +  HYG, An outline of the strategy used for the isolation of a- G418 plates) and were then isolated on YPD plates after and α-type sake yeast cells is shown in Fig.  1. Typically, the removal of both plasmids through passage cultures a-type cells express the a1 gene, whereas α-type hap- performed with YPD medium without antibiotics. loids express the α2 gene from their respective MAT loci. Although repetitive hybridizations using the newly Haploid-specific genes (hsg ) are expressed and induce generated a/α-type sake yeast strains as the parent Fukuda et al. AMB Expr (2016) 6:45 Page 6 of 12 backcrossing a/α-type of cell a-type of cell (parent) (derivative) without + G418 a/α-type of cell selection (hybrid) pLhyS- pLY- pressure hygro 2K-Pa1 Multi- hybridization mating without a/α-type of cell α-type of cell selection (parent) (derivative) pressure without + G418 selection pHY- pHhyS- pressure 3K-2 kan backcrossing backcrossing a/α-type of cell a-type of cell (parent) (derivative) + G418 a/α-type of cell a-type of cell (hybrid) (derivative) pLhyS- 2K-Pa1 Multi- hybridization Mating without a/α-type of cell α-type of cell + G418 AUR (parent) (derivative) without + G418 selection pAUR112 pHhyS- pressure 3K-2 a/α-type of cell a-type of cell (parent) (derivative) without + G418 a/ -type of cell α α-type of cell selection (hybrid) (derivative) pAUR112 pLhyS- pressure 2K-Pa1 Multi- hybridization Mating without a/α-type of cell α-type of cell + G418 AUR (parent) (derivative) + G418 backcrossing pHhyS- 3K-2α Fig. 2 Schematic outline of the experimental design for the crossbreeding of sake yeast strains. a Outline of the basic method used for the cross- breeding of sake yeast strains. Plasmids pLhyS-2 K-Pa1 and pHhyS-3 K-2α were used for isolation of a- and α-type derivative cells, respectively, and plasmids pLY-hygro and pHY-kan were used for the hybridization of derivatives. Unnecessary plasmids were removed from yeast cells by cultivat- ing cells in the absence of selection pressure. b Improved method for continuous crossbreeding without the requirement for removal of plasmid pLhyS-2 K-Pa1. c A second improved method for continuous crossbreeding without the requirement for removal of plasmid pHhyS-3 K-2α. Plasmid pAUR112 was used for the hybridization of obtained derivatives in (b) and (c) Fukuda et al. AMB Expr (2016) 6:45 Page 7 of 12 (continuous crossbreeding) can be performed using the or pHhyS-3  K-2α were selected on YPD plates con- scheme described in Fig. 2a, it is necessary to re-transform taining 300  μg/ml HYG, and after repeated passag- the strains with pLhyS-2 K-Pa1 or pHhyS-3 K-2α. To avoid ing, cell suspensions of each transformant were spread the labor required for plasmid exchange for continuous on YPD  +  G418 (>500 μg/mL) plates. The plasmids crossbreeding, a third selection marker gene, AUR1-C, was pLhyS-2  K-Pa1 and pHhyS-3  K-2α were removed from used to generate a/α-type hybrid strains (Fig. 2b, c). the isolated a- or α-type yeast cells, as shown in Fig.  2a, Figure  2b shows the procedure used to acquire a-type yielding strains K6A, K7A and K9A (a-type), and K6AL, cells derived from the generated hybrids. After isolation K7AL and K9AL (α-type) (Table  1). The removal of the of the derivatives from the parent a/α-strains, α-type cells plasmids containing the hygro and kanMX4 genes was were cultivated without selection pressure to remove the confirmed by PCR (Additional file  1: Table S3 and Fig. plasmid pHhyS-3  K-2α, and plasmid pAUR112 (Takara S1). Bio, Inc., Shiga, Japan), which contains the AUR1-C To verify the mating type and examine the mating marker gene, was then introduced into the α-type deriva- abilities of the derivative strains, a mating assay was per- tive cells. The mating of a- and α-type derivative cells was formed with the auxotrophic and G418-resistant labora- performed in mixed cultures in YPD medium, and a/α- tory haploid strains HR42-11T (α-type) and MCF4741 type hybrid cells were the isolated on solid YPD medium (a-type) (Fukuda et  al. 2013b) (Table  1). Although all of containing HYG and AUR (YPD  +  HYG, AUR plates). the derivative strains are prototrophs, they do not have After the removal of pAUR112 from cells by repeated resistance to G418, and therefore only zygotes can grow passage in medium containing only HYG (YPD  +  HYG on SD/MSG plates containing G418, but lacking any medium), a-type derivative cells expressing the kanMX4 amino acids (Fig.  3a). In the mating assay, the deriva- gene were selected on YPD  +  G418 plates. The isolated tive strains K6A, K7A and K9A mated with HR42-11T a-type cells can be directly utilized for hybridization, (α-type), whereas the parental strains (Kyokai No. 6, No. such as backcrossing and multi-hybridization, without 7 and No. 9) did not mate (Fig. 3b). Similarly, the deriva- removing the plasmid pLhyS-2 K-Pa1. tive strains K6AL, K7AL and K9AL mated with MCF4741 Figure  2c illustrates the procedure used to acquire (a-type), whereas the parental strains again did not mate α-type cells from the a/α-type hybrids. After isolation (Fig.  3c). These results confirmed that the growth selec - of the derivatives from the parent a/α-type of strains, tion system designed here promoted the generation and a-type cells were cultivated without selection pressure allowed for the isolation of a- and α-type yeast derivative to remove the plasmid pLhyS-2  K-Pa1, and plasmid cells. pAUR112 was then introduced into the a-type deriva- Outcrossing of Kyokai No. 6, No. 7 and No. 9 was next tive cells. The a- and α-type derivative cells were mated conducted according to the scheme illustrated in Fig. 2a. in YPD medium, and a/α-type hybrid cells were then After transformation of a- and α-type derivative cells isolated on YPD  +  HYG, AUR plates. After removal with the plasmids pLY-hygro and pHY-kan, cells were of pAUR112 by repeated passage in YPD  +  HYG selected on YPD + HYG and YPD + G418 plates, respec- medium, a-type cells expressing the kanMX4 gene were tively. Zygotes (hybrid cells) were isolated from mixed selected on YPD  +  G418 plates. As described for the cultures of the derivative cells using YPD +  HYG, G418 a-type cells generated using this procedure, the plas- plates, and all plasmids were then removed from each mid pHhyS-3  K-2α does not need to be removed from hybrid cell, yielding hybrid strains K67, K69, K76, K79, the isolated α-type cells prior to performing subsequent K96 and K97 (Table 1). hybridizations. Improvement of the continuous crossbreeding procedure Outcrossing of Kyokai No. 6, No. 7 and No. 9 Although the feasibility of the basic hybrid selec- The basic method for generating strains for continuous tion method (Fig.  2a) was demonstrated in the above- crossbreeding (Fig. 2a) was utilized for the outcrossing of described outcrossing experiment, each stage of the the sake yeast strains Kyokai No. 6, No. 7 and No. 9. The continuous crossbreeding procedure requires the minimum inhibitory concentration (MIC) of HYG and exchange of plasmids. As plasmid exchange is a time- G418 was first determined for these three strains (Addi - and labor-consuming process, we attempted to improve tional file  1: Table S1 and S2). The MIC values of HYG the continuous crossbreeding method (Fig.  2b, c) using for strains Kyokai No. 6, No. 7 and No. 9 were 300, 200 a third marker gene (AUR1-C; AUR-resistance). Using and 300  μg/ml, respectively, and the MIC of G418 was this approach, a- and α-type derivative cells were isolated 200 μg/ml for all three strains. from hybrid strain K76. To isolate a- and α-type cells of Kyokai No. 6, No. In this modified selection system, the isolation of a- 7 and No. 9, cells transformed with pLhyS-2  K-Pa1 and α-type derivative cells based on transformation with Fukuda et al. AMB Expr (2016) 6:45 Page 8 of 12 pAUR112 on YPD  +  AUR plates. K6AL/pAUR112 cells Parents (a/α) Partner (a or α) Derivatives Partner (a or α) were cultured together with K7A/pLhyS-2  K-Pa1 cells, prototroph G418-resistance prototroph G418-resistance and hybrid K76/pLhyS-2 K-Pa1/pAUR112 cells were then isolated on YPD  +  HYG, AUR plates and further culti- vated in YPD  +  HYG medium (without AUR). The cells were transferred to YPD  +  G418 plates for the selec- Zygote tion of a-type derivative cells (K76A/pLhyS-2  K-Pa1). prototroph To obtain α-type derivative cells from hybrid strain K76 G418-resistance according to the scheme illustrated in Fig.  2c, plasmid No colonies on SD/MSG+G418 plates Colony formation on SD/MSG+G418 pAUR112 was introduced into strain K7A after removing (No zygotes are generated) plates (when zygosis occurs) plasmid pLhyS-2  K-Pa1, yielding K7A/pAUR112 trans- OD formants, which were selected on YPD  +  AUR plates. Hybrid K76/pHhyS-3  K-2α/pAUR112 cells were then isolated on YPD  +  HYG, AUR plates from mixed cul- tures of K7A/pAUR112 and K6AL/pHhyS-3  K-2α cells, and were further cultivated in YPD  +  HYG medium (without AUR). Finally, a-type derivative cells, K76AL/ Kyokai No.6 K6A Kyokai No.7 K7A Kyokai No.9 K9A pHhyS-3 K-2α, were isolated on YPD + G418 plates. × × × × × × HR42-11T HR42-11T HR42-11T HR42-11T For determination of mating type, all plasmids were HR42-11T HR42-11T removed via cultivation in YPD medium to yield strains OD600 K76A and K76AL (Table  1). In a mating assay, strain K76A successfully mated with HR42-11T (α-type) and K76AL mated with MCF4741 (a-type) (Fig.  4). These results suggest that a- and α-type derivative cells can be generated from hybrid strains (polyploids) as well as parental diploid strains. Notably, the generated K76A/ Kyokai No.7 K7AL Kyokai No.9 K9AL Kyokai No.6 K6AL pLhyS-2  K-Pa1and K76AL/pHhyS-3  K-2α derivative × × × × × × MCF4741 MCF4741 MCF4741 MCF4741 MCF4741 MCF4741 cells can be directly utilized for hybridization during Fig. 3 Evaluation of the mating abilities of derivative strains. a Sche- continuous crossbreeding without removing plasmids matic outline of the mating assay. Using haploid strains HR42-11T pLhyS-2 K-Pa1and pHhyS-3 K-2α, respectively. (α-cell) and MC4741 (a-cell) as the mating partners, zygotes were To verify the feasibility of performing continu- selected on solid medium containing G418, but lacking amino acids ous crossbreeding using the a- and α-type derivative (SD/MSG + G418 plates). The Parent strains were Kyokai No. 6, No. 7 and No. 9 (a/α-type cells), and the Derivative strains were K6A, K7A and K9A (isolated as a-type cells) and K6AL, K7AL and K9AL (isolated OD as α-type cells), respectively. b Images showing colony formation in the mating assay for strains K6A, K7A and K9A. c Images show- ing colony formation in the mating assay for strains K6AL, K7AL and K9AL. The OD values of 10-μL cell suspensions spotted on the solid media were set at 1.0, 0.1 and 0.01 the plasmids pLhyS-2  K-Pa1 and pHhyS-3  K-2α was performed as described in Fig.  2a. The obtained deriva - tive cells, K7A/pLhyS-2 K-Pa1 and K6AL/pHhyS-3 K-2α, were then subjected to plasmid exchange using one of K76 K76AL K76 K76A two methods. Because the MIC values of AUR against × × × × strains K7A and K6AL were 100 and 300  ng/ml, respec- MCF4741 MCF4741 HR42-11T HR42-11T tively (Additional file  1: Table S4), yeast transformation Fig. 4 Evaluation of mating abilities of the derivative strains K76A with plasmid pAUR112 was performed using YPD plates and K76AL. Images showing colony formation on SD/MSG + G418 containing 300 ng/ml AUR. plates in the mating assay are shown. The Parent strains were K76 To obtain a-type derivative cells from hybrid strain K76 (a/α-type cells), and the Derivative strains were K76A (isolated as a-type cells) and K76AL (isolated as α-type cells), respectively. The according to the scheme illustrated in Fig.  2b, plasmid OD values of 10-μL cell suspensions spotted on the solid media pAUR112 was introduced into strain K6AL after remov- were as in Fig. 3b ing plasmid pHhyS-3 K-2α, yielding transformants K6AL/ Fukuda et al. AMB Expr (2016) 6:45 Page 9 of 12 cells, multi-hybridization was performed with K76A/ hybrid strain K76 was much larger than that of strains pLhyS-2  K-Pa1 and K9AL/pHhyS-3  K-2α cells (isolated K7A and K6AL, suggesting that the DNA content within as described above) according to the scheme illustrated in strain K76 cells had increased. In contrast, the cell size of Fig. 2b. The plasmid pAUR112 was introduced into strain multi-hybrid strain K76x9 was clearly smaller than that K9AL after removing the plasmid pHhyS-3  K-2α, yield- of strains K76 and K76A, suggesting that a major loss ing K9AL/pAUR112 cells after selection on YPD +  AUR of DNA occurred after the hybridization of K76A and plates. Mixed cultures of K76A/pLhyS-2  K-Pa1 and K9AL. K9AL/pAUR112 were plated on YPD  +  HYG, AUR To more accurately estimate the DNA content of cells, medium to select for hybrid K76x9/pLhyS-2  K-Pa1/ FACS analysis was performed after propidium iodide pAUR112 cells, and all plasmids were then removed from (PI)-staining of each yeast strain (Fig.  5). In DNA con- the hybrid cells via cultivation in YPD medium without tent histograms, two PI-fluorescence signal peaks, cor - antibiotics to yield multi-hybrid strain K76x9 (Table 1). responding to the G0/G1 and G2/M phases, are typically observed (Qiu et  al. 2013). The DNA content of diploid Estimation of cellular DNA content strains Kyokai No. 6, No. 7 and No. 9 was measured, and To further characterize the derivative and hybrid cells, reference values of 2 N (two sets of all chromosomes) and microscopic observation of strains K7A, K6AL, K9AL, 4  N (four sets of all chromosomes) were defined as the K76, K76A and K76x9 was performed using BY4742 average of each peak value. The 3 N value was calculated and Kyokai No. 7 as control haploid and diploid strains, as the average of the 2 and 4 N values, and 1, 1.5, 2.5, 4.5, respectively (Additional file  1: Fig. S2). The diameter of 5, 6, 8 and 9 N values were then calculated from the 3 N a yeast cell increases with increasing amounts of nuclear value (Table  3). Notably, although haploid strain BY4742 DNA (Amodeo and Skotheim 2016). The cell size of is a laboratory yeast strain, the two peak values obtained Fig. 5 DNA content histogram of PI-stained yeast cells using FACS. PI-fluorescence data was collected from yeast cells within a gate drawn in the FSC-SSC dot plots (Additional file 1: Fig. S3) according to the data collected from control diploid strains. For relative comparison of DNA content, ref- erence values of 2 N (two sets of all chromosomes) and 4 N (four sets of all chromosomes) were defined as the average of each peak value of Kyokai No.6, No.7 and No.9. The 1 and 8 N values were calculated from the 2 and 4 N values Fukuda et al. AMB Expr (2016) 6:45 Page 10 of 12 for this strain were well accorded with the calculated 1 N isolation of a- and α-type derivative cells from a/α-type and averaged 2 N value. parental cells. The constructed plasmids required at least The DNA content of sake yeast strains generated in the two types of marker genes: one for the selection of yeast present study was measured and evaluated based on com- transformants and one for the selection of a- or α-type parison to the reference values (from 1 to 9  N) (Table  4; derivative cells. To this end, we employed two commonly Fig. 5). The results of FACS analysis (Additional file  1: Fig. used marker genes for industrial yeast strains, namely S3) and microscopic observation (Additional file  1: Fig. the hygro and kanMX4 genes, for the transformation and S4) indicate that generated strains have cell flocculation isolation of a- and α-type derivative cells, respectively ability, unlike the control strains. To exclude signals origi- (Murakami et al. 2012). nating from the flocculated cells, a data collection gate As a- and α-type yeast cells can be generated by spon- was drawn according to FSC-SSC dot plots from the con- taneous chromosomal mutations, such as LOH in the trol strains (Additional file  1: Fig. S3). As shown in Table 4 region containing the MAT locus or the mitotic loss and Fig.  5, strains K7A and K7AL had similar DNA con- of chromosome III (Alvaro et  al. 2006; Andersen et  al. tents to those of the parental strain (Kyokai No. 7). In 2008; Daigaku et  al. 2004; Mayer and Aguilera 1990; contrast, strains K6A, K9A and K9AL appeared to have Takagi et al. 2008), we examined the nuclear DNA of the partially lost chromosomal DNA due to unequal distribu- generated a- or α-type derivative strains. The DNA con - tion of DNA during mitotic cell division, whereas strain tent of cells after the removal of all plasmid DNA was K6AL acquired additional chromosomal DNA. evaluated qualitatively by the microscopic analysis of In the outcrossing experiments, the DNA content of cell size and quantitatively by the FACS measurement hybrid strains K67, K69, K76, K79, K96 and K97 was found of DNA content based on PI fluorescence signal inten - to be X ± 0.5 N, where X is the total DNA content of the sity. In cases of derivative strains K6A, K9A, K9AL and two parental strains. However, strain K76A appeared to K76A, the DNA content was clearly smaller compared to have lost an amount of DNA that was nearly equivalent to those of the parental strains, suggesting that the loss of one set of chromosomes, whereas strain K76AL acquired chromosomal DNA occurred. In contrast, other deriva- additional DNA that was almost equivalent to a half set tive strains had DNA contents that were nearly equiva- of chromosomes. It was also determined that the multi- lent to those of the parental strains. Notably, however, hybrid strain K76x9 lost a substantial amount of chromo- it is not possible to determine the rearrangement event somal DNA during the mating process and maintained a that occurred in the obtained derivative strains based on near-diploid DNA content. These results are consistent these results alone, because the MAT locus only occu- with the microscopic observations of cellular morphology pies 2 % of all chromosomes (Kumaran et al. 2013). u Th s, described above (Additional file 1 : Fig. S2). although these methods do not require complicated or time-consuming procedures, estimation of chromo- Discussion some III number within cells by real-time PCR (Fukuda The aim of this study was to establish a versatile method et  al. 2013a), array comparative genomic hybridization for the crossbreeding of sake yeasts, which have low spor- (Abunimer et  al. 2016), or whole-genome sequencing ulation rates. We constructed two new plasmids for the would be needed to investigate the underlying events in more detail. Although it was demonstrated that the basic method developed here (Fig.  2a) was applicable for the isola- Table 3 Reference values used in the FACS analysis tion of yeast cells with mating ability from common sake Peak Value of PI‑fluorescence yeast strains, due to the requirement for the repeated exchange of plasmids, this approach still required multi- 1 N 189.18 ple steps to perform the final hybridization and isolated 1.5 N 283.77 a- or α-type derivatives. To reduce the need for plasmid 2 N 379.28 ± 18.59 exchange steps, we introduced a third marker gene, which 2.5 N 472.95 was compatible with the hygro and kanMX4 marker 3 N 567.54 genes, into the selection system. Using the commercial 4 N 755.79 ± 35.58 plasmid pAUR112, which contains the AUR1-C marker 4.5 N 851.30 gene, we successfully isolated hybrid cells without the 5 N 945.89 need for the time-consuming plasmid exchange process. 6 N 1135.07 In a back-crossing comprising n cycles of hybridization, 8 N 1513.43 only a single plasmid exchange step would be required in 9 N 1702.61 the scheme illustrated in Fig.  2b and c, whereas (2n +  1) ±means standard deviations from three diploid strains Fukuda et al. AMB Expr (2016) 6:45 Page 11 of 12 Table 4 Evaluation of DNA content and ploidy assessment In general, crossbreeding induces genomic shuffling by FACS analysis based on mechanisms of sexual reproduction (Snoek et al. 2015), and the resulting derivative strains may therefore Strain Peak 1 Peak 2 Ploidy acquire phenotypes different from those of the parent Control strains. In the present approach, LOH and the mitotic BY4742 188.34 345.07 1 N loss of chromosomal DNA occur randomly, and the gen- Kyokai No.6 366.04 730.97 2 N erated hybrid yeast strains may undergo additional chro- Kyokai No.7 366.22 730.30 2 N mosomal loss after mating to reach stable genomic states. Kyokai No.9 405.57 806.11 2 N Such changes in ploidy are termed meiosis-like adaptation Tested (Storchova 2014) and can result in the formation of deriv- K6A 207.37 413.17 1 N ative and hybrid cells with diverse genotypes and pheno- K7A 379.61 806.97 2 N types, even from the same parental polyploid strain. The K9A 280.76 616.19 1.5 N genomic instability of polyploids is beneficial for cross - K6AL 433.52 865.21 2.5 N breeding because it has contributed to the adaptation and K7AL 331.98 728.50 2 N evolution of yeasts in nature (Snoek et al. 2015). K9AL 280.73 520.07 1.5 N In conclusion, we have established a complete proce- K67 717.23 1475.10 4 N dure for the crossbreeding of industrially used sake yeasts K69 547.61 1125.63 3 N possessing sporulation defects. The outcrossing of sake K76 718.22 1527.81 4 N yeasts was achieved in all examined combinations, and K79 739.99 1528.83 4 N the feasibility of continuous crossbreeding was demon- K96 717.00 1532.82 4 N strated by generating a multi-hybrid strain. The method K97 766.30 1637.93 4 N developed here may allow the numerous and valuable K76A 529.92 1022.16 3 N yeast resources, including sake yeasts, to be efficiently K76AL 821.08 1640.31 4.5 N used for generation of new strains with desirable proper- K76x9 367.34 756.46 2 N ties for industrial applications. Additional file plasmid exchange steps would be needed in the scheme Additional file 1. Supporting Information for Materials and Methods. described in Fig. 2a. To demonstrate the feasibility of the continuous cross- breeding approach developed in the present work, we Authors’ contributions NF designed the study; NF and MK conducted experiments; NF, JI, AK and SH generated a multi-hybrid strain, K76x9, from strains K76A analyzed data; and NF and SH co-wrote the manuscript. All authors read and and K9AL, with the expectation that the obtained strain approved the final manuscript. would inherit genetic characteristics from strains Kyokai Author details No. 6, No. 7 and No. 9. Microscopic observation and FACS Biomedical Research Institute, National Institute of Advanced Industrial analysis revealed that both strains K76A and K9AL under- Science and Technology (AIST ), Higashi, Tsukuba, Ibaraki 305-8566, Japan. went partial loss of chromosomal DNA, whereas the multi- Department of Chemical Science and Engineering, Graduate School of Engi- neering, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan. Organization hybrid strain K76x9 possessed a near-diploid DNA content, of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, having lost a major part of its DNA during the mating pro- Kobe, Japan. cess. There are two possible factors that lead to decreased Acknowledgements genomic stability in the generated strains. First, ane- The sake yeast strains Kyokai No. 6, No. 7, and No. 9 were provided by the uploidy (a chromosomal content differing from multiple Biological Resource Center (NBRC), NITE, Japan. This work was supported in sets of haploid chromosomes) induces genomic instability part by JSPS KAKENHI Grant Number 25820406 and 16K14497. (Skoneczna et  al. 2015). Strain K76x9 was generated from Competing interests aneuploid strain K9AL and suspected aneuploid strain N. Fukuda and S. Honda declare that they are inventors on a pending patent K76A, which appeared to have lost DNA), and hence, the using aspects of this work. M. Kaishima, J. Ishii and A. Kondo declare that they have no competing interests. zygotes must also be aneuploid, which might induce chro- mosomal loss. The other possible factor leading to genomic Compliance with ethical standards instability is an increase in the ploidy of yeast cells. Accord- This article does not contain any studies with human participants or animals performed by any of the authors. ing to a previous report (Kumaran et  al. 2013), chromo- somal stability is reduced as the ploidy of a cell increases. Received: 15 June 2016 Accepted: 25 June 2016 These two factors presumably had synergistics effects on the genomic instability of the multi-hybrid strain K76x9. Fukuda et al. AMB Expr (2016) 6:45 Page 12 of 12 References Kishimoto M. Fermentation characteristics of hybrids between the cryophilic Abunimer AN, Salazar J, Noursi DP, Abu-Asab MS. 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Suizu T, Tsutsumi H, Kawado A, Murata K, Suginami K, Imayasu S. Methods 2001;67:4346–8. for sporulation of industrially used sake yeasts. J Ferment Bioeng. Hiraoka M, Watanabe K, Umezu K, Maki H. Spontaneous loss of heterozygosity 1996;81:93–7. in diploid Saccharomyces cerevisiae cells. Genetics. 2000;156:1531–48. Takagi Y, Akada R, Kumagai H, Yamamoto K, Tamaki H. Loss of heterozygosity Katou T, Kitagaki H, Akao T, Shimoi H. Brewing characteristics of haploid strains is induced in Candida albicans by ultraviolet irradiation. Appl Microbiol isolated from sake yeast Kyokai No. 7. Yeast. 2008;25:799–807. Biotechnol. 2008;77:1073–82. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells

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Copyright © 2016 by The Author(s)
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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10.1186/s13568-016-0216-x
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Abstract

Sake yeasts belong to the budding yeast species Saccharomyces cerevisiae and have high fermentation activity and ethanol production. Although the traditional crossbreeding of sake yeasts is a time-consuming and inefficient process due to the low sporulation rates and spore viability of these strains, considerable effort has been devoted to the development of hybrid strains with superior brewing characteristics. In the present work, we describe a growth selection system for a- and α-type cells aimed at the crossbreeding of industrial yeasts, and performed hybridiza- tions with sake yeast strains Kyokai No. 6, No. 7 and No. 9 to examine the feasibility of this approach. We successfully generated both a- and α-type strains from all parental strains, and acquired six types of hybrids by outcrossing. One of these hybrid strains was subjected to continuous crossbreeding, yielding the multi-hybrid strain, which inherited the genetic characteristics of Kyokai No. 6, No. 7 and No. 9. Notably, because all of the genetic modifications of the yeast cells were introduced using plasmids, these traits can be easily removed. The approach described here has the potential to markedly accelerate the crossbreeding of industrial yeast strains with desirable properties. Keywords: Sake yeast, Crossbreeding, Mating type, Growth selection, Hybrid strain 2001; Kishimoto 1994; Shinohara et  al. 1997). Common Introduction breeding strategies, such as backcrossing and multi- Sake is a traditional Japanese alcoholic beverage made hybridization, require cycles of hybridization (continu- from fermented rice. In the production of sake, rice ous crossbreeding). Most sake yeasts are MATa/α diploid starch is first degraded by the koji fungus Aspergillus ory- strains and are unable to mate directly; therefore, the zae into glucose, which is then fermented to ethanol by isolation of MATa and MATα haploid strains via sporula- sake yeast, strains of the budding yeast species Saccha- tion is a prerequisite for crossbreeding. However, because romyces cerevisiae (Kitagaki and Kitamoto 2013; Shiroma industrially used sake yeast strains, such as Kyokai No. 7 et  al. 2014). Sake yeasts have many characteristics suit- and No. 9, have low sporulation rates (Suizu et al. 1996), able for sake brewing, such as aromatic production and the crossbreeding of sake yeast strains is inefficient and high ethanol tolerance (Katou et  al. 2008). Sake yeast technically challenging. strains have been selected through several hundred years To overcome the problem of poor sporulation, it is of brewing (Shiroma et  al. 2014); however, more rapid possible to select for strains that have undergone spon- methods for generating new and superior strains are taneous chromosomal aberrations, such as loss of hete- highly desirable. rozygosity (LOH) and mitotic chromosome loss, during Crossbreeding is an attractive approach to improve and mitotic division to obtain a- and α-type yeast cells that combine traits of different yeast strains (Higgins et  al. possesses similar mating abilities as MATa and MATα haploids generated via sporulation (Fukuda et al. 2013a). LOH is a natural genetic event that generates homozy- *Correspondence: nob-fukuda@aist.go.jp Biomedical Research Institute, National Institute of Advanced Industrial gous loci via chromosomal rearrangement of het- Science and Technology (AIST ), Higashi, Tsukuba, Ibaraki 305-8566, Japan erozygous loci (Alvaro et  al. 2006; Andersen et  al. 2008; Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fukuda et al. AMB Expr (2016) 6:45 Page 2 of 12 Daigaku et  al. 2004; Takagi et  al. 2008), whereas mitotic α-type derivative cells by removing unnecessary plasmids chromosome loss, which is also a naturally occurring and introducing new plasmids. In this work, the feasibil- event, involves the loss of single or multiple chromo- ity of this approach for sake yeasts was demonstrated by somes (Mayer and Aguilera 1990). Therefore, a LOH performing and generating modified strains through con - event at the mating-type (MAT) locus within MATa/α tinuous crossbreeding. cells produces either MATa/a or MATα/α cells, whereas yeast cells that lose one of both copies of chromosome Materials and methods III containing the MAT locus during mitotic division Strains and media become MATa or MATα cells (Fukuda et  al. 2013a). Detailed information about S. cerevisiae laboratory yeast However, because the spontaneous occurrence frequen- strain BY4742 (Brachmann et  al. 1998) and sake yeast cies of LOH and mitotic chromosome loss are less than strains Kyokai No. 6, No. 7 and No. 9 (provided by the −4 1  ×  10 , it is difficult to isolate the generated a- and Biological Resource Center, NITE, Japan), as well as the α-type cells from mixed cell populations (Hiraoka et  al. other strains used in this study, is shown in Table 1. Yeast 2000; Kumaran et al. 2013). cells were grown in YPD medium (1 % yeast extract, 2 % To isolate a- and α-type cells, we previously established peptone and 2  % glucose) or SD/MSG medium (0.17  % a growth selection system for laboratory yeast strains yeast nitrogen base without amino acids and ammo- using the auxotrophic marker URA3 (Fukuda et  al. nium sulfate [Becton–Dickinson and Company, Franklin 2013a). In this system, the expression of the marker gene Lakes, NJ, USA], 0.1  % monosodium glutamate and 2  % is induced in a mating-type-specific manner, thereby glucose). A final concentration of 2 % agar was added to permitting the efficient selection of a- and α-cells from both types of media to prepare solid media. within a mixed cell population. Using this approach, we succeeded in isolating a- and α-type derivative cells from Construction of plasmids a cell population of parental MATa/α laboratory yeasts The sequences of the oligonucleotides used in this study without any false positives, and confirmed that these cells are listed in Table  2. The plasmids used in this study were able to mate and produce new hybrid yeasts. (Table  1) were constructed as follows. Using pTriEx-2 Unlike auxotrophic laboratory yeasts, however, indus- Hygro (Novagen, Inc., Madison, WI) as a template, the trially used yeasts, including sake yeasts, are generally hygro gene was amplified with oligonucleotide pair o1 prototrophic, which prevents the use of auxotrophic and o2. P was also amplified from genomic DNA TDH3 markers for the selection of strains following mating. In derived from strain BY4742 using oligonucleotide pair addition, industrial yeast strains have remarkably low o3 and o4. The two amplified DNA fragments, P TDH3 genetic transformation efficiencies compared to labora - and hygro, were combined using the In-Fusion HD Clon- 1 2 tory strains (sake yeast, 10 ~10  cfu/μg-DNA; laboratory ing kit (Takara Bio, Inc., Shiga, Japan), and the resulting 4 5 yeast, 10 ~10   cfu/μg-DNA) (Ogata et  al. 1993). There - DNA fragment, P -hygro, was amplified with oligonu - TDH3 fore, a complete method for the transformation, isolation, cleotide pair o3 and o2, and then digested with SacI and and evaluation of a- and α-type derivative and hybrid BamHI. In addition, T was amplified from genomic PGK1 cells, is required for the efficient crossbreeding of sake DNA derived from strain BY4742 using oligonucleotide yeasts. pair o5 and o6, and the obtained fragment was digested Here, we designed and constructed two types of plas- with BamHI and XhoI. The two digested DNA fragments, mids for isolating a- and α-type sake yeasts from mixed P -hygro and T , were inserted at the SacI-XhoI TDH3 PGK1 cell populations (Fig.  1). Although drug sensitivity var- sites of pLY-3U to replace the P -URA3-T cas- STE3 CYC1 ies from strain to strain, the hygro (hygromycin B-resist- sette (Fukuda et al. 2013a), yielding a plasmid designated ance) and kanMX4 genes (G418-resistance) have been pLY-hygro. commonly used for the selection of prototrophic yeasts Using pK6 (Fukuda et  al. 2013b) as a template, the (Murakami et al. 2012). To develop a versatile method for expression cassette of the kanMX4 marker (consisting crossbreeding of sake yeasts, we used the hygro gene as a of P , ORF and terminator) was amplified with oligo - TEF1 transformation marker and the kanMX4 gene as a marker nucleotide pair o7 and o8, and inserted in place of the for isolation of a- or α-type derivative cells. In this P -EGFP-T cassette at the SacII-XhoI sites of PGK1 ADH1 approach, the a- and α-type derivative cells must have pHY-PGA (Fukuda et  al. 2013a), yielding a plasmid des- different marker genes. Because yeast transformation ignated pHY-kan. is performed using plasmids in our system, the marker Using pLY-hygro as a template, a DNA fragment con- genes are lost in the absence of selection pressure. There - taining the P -hygro-T cassette was amplified TDH3 PGK1 fore, through plasmid exchange, different marker genes with oligonucleotide pair o3 and o9. Subsequently, a for hybridization can be introduced into the target a- and DNA fragment containing the P -a1-T cassette PGK1 ADH1 Fukuda et al. AMB Expr (2016) 6:45 Page 3 of 12 ac CEN6/ CEN6/ ARSH4 ARSH4 r r Amp Amp PTDH3 PTDH3 pLhyS-2K-Pa1 pHhyS-3K-2α TCYC1 TADH1 hygro hygro kanMX4 kanMX4 TPGK1 TPGK1 TADH1 TADH1 PSTE2 PSTE3 a1 α2 PPGK1 PSTE2 b a-type of cell d a-type of cell a1 a1 α2 G418 resistance kanMX4 kanMX4 α-type of cell α-type of cell α2 a1 α2 G418 resistance kanMX4 kanMX4 a/α-type of cell a/ -type of cell a1 a1 α2 α2 kanMX4 kanMX4 Fig. 1 Schematic outline of the strategy used for isolation of a- and α-type yeast cells. a Plasmid map of pLhyS-2 K-Pa1, which was used for the isolation of a-type yeast cells. b Isolation of a-type yeast cells using G418 selection. Formation of the a1-α2 complex in α-type and a/α-type cells represses expression of the kanMX4 marker gene. Only a-type cells are able to survive in culture medium containing G418 by expressing the kanMX4 marker gene. c Plasmid map of pHhyS-3 K-2α, which was used for the isolation of α-type yeast cells. d When introduced into a-type and a/α-type cells, the α2 protein represses expression of the kanMX4 marker gene. Only α-type cells are able to survive in culture medium containing G418 by expressing the kanMX4 marker gene was amplified with oligonucleotide pair o10 and o11 from sites of pLS-2 K (Fukuda et al. 2013a), yielding a plasmid pHPY-a1 (Fukuda et  al. 2013b). Using the In-Fusion kit, designated pLhyS-2 K-Pa1. the two amplified DNA fragments, P -hygro-T Similarly, a DNA fragment containing the P -α2- TDH3 PGK1 STE2 and P -a1-T , were inserted into the SacI-SacII T cassette was amplified with oligonucleotide pair PGK1 ADH1 ADH1 Fukuda et al. AMB Expr (2016) 6:45 Page 4 of 12 Table 1 Yeast strains and plasmids used in this study Name description Description Reference source Yeast strains BY4742 MATα his3∆1 ura3∆0 leu2∆0 lys2∆0 Brachmann et al. (1998) MCF4741 MATa his3∆1 ura3∆0 leu2∆0 met15∆0 Fig. 1::Fig. 1-EGFP-loxP-kanMX4-loxP Fukuda et al. (2013b) HR42-11T MATα his3∆1 ura3∆0 leu2∆0 lys2∆0 hmra ::HMRa -loxP-kanMX4-loxP Fukuda et al. (2013b) inc cut K6 Sake yeast; Kyokai No. 6; MATa/α NBRC2346 K7 Sake yeast; Kyokai No. 7; MATa/α NBRC2347 K9 Sake yeast; Kyokai No. 9; MATa/α NBRC2377 K6A a-type of strain derived from Kyokai No. 6 Present study K6AL α-type of strain derived from Kyokai No. 6 Present study K7A a-type of strain derived from Kyokai No. 7 Present study K7AL α-type of strain derived from Kyokai No. 7 Present study K9A a-type of strain derived from Kyokai No. 9 Present study K9AL α-type of strain derived from Kyokai No. 9 Present study K67 Hybrid strain generated by zygosis of K6A and K7AL Present study K69 Hybrid strain generated by zygosis of K6A and K9AL Present study K76 Hybrid strain generated by zygosis of K7A and K6AL Present study K79 Hybrid strain generated by zygosis of K7A and K9AL Present study K96 Hybrid strain generated by zygosis of K9A and K6AL Present study K97 Hybrid strain generated by zygosis of K9A and K7AL Present study K76A a-type of strain derived from K76 Present study K76AL α-type of strain derived from K76 Present study K76x9 Hybrid strain generated by zygosis of K76A and K9AL Present study Plasmids pLY-hygro 2μ ori, LEU2 marker and P -hygro Present study TDH3 pHY-kan 2μ ori, HIS3 marker and P -kanMX4 Present study TEF1 pAUR112 CEN4/ARS1 ori, URA3, AUR1-C Takara Bio, Inc., Shiga, Japan pLhyS-2 K-Pa1 CEN6/ARSH4 ori, LEU2, P -hygro, P -kanMX4 and P -a1 Present study TDH3 STE2 PGK1 pHhyS-3 K-2α CEN6/ARSH4 ori, HIS3, P -hygro, P -kanMX4 and P -α2 Present study TDH3 STE3 STE2 Resources were provided by Biological Resource Center (NBRC), NITE, Japan Table 2 Sequences of  oligonucleotides used to  construct o12 and o13 from pL3G-2α (Fukuda et al. 2013b). Using plasmids the In-Fusion kit, the two DNA fragments P -hygro- TDH3 T and P -α2-T were inserted into the SacI- PGK1 STE2 ADH1 Number Sequence SacII sites of pHS-3  K (Fukuda et  al. 2013a), yielding a 1 5′-CAAAgcggccgcATGGATAGATCCGGAAAGCC-3′ plasmid designated pHhyS-3 K-2α. 2 5′-AATTTATTTCggatccCTATTCCTTTGCCCTCGGAC-3′ Each constructed plasmid was introduced into yeast 5′-TATAGGGCGAATTGgagctcGAATAAAAAACACGCTTTTT-3′ cells using the lithium acetate method (Gietz et al. 1992). 4 5′-CATgcggccgcTTTGTTTGTTTATGTGTGTT-3′ 5 5′-GCTTATGTAAggatccGAAATAAATTGAATTGAAT-3′ Investigation of cell growth characteristics 6 5′-CGGGCCCCCCctcgagAGCTTTAACGAACGCAGAA-3′ Each yeast strain was grown at 30  °C in 500  μL YPD 7 5′-AATTGGAGCTCCAccgcggATCTGTTTAGCTTGCCTCGT-3′ medium supplemented with or without the antibiotics 5′-CGGGCCCCCCctcgagCTCGTTTTCGACACTGGAT-3′ hygromycin B (HYG), geneticin (G418) or aureobasidin 9 5′-CTCGAGGGGGGGCCCGgagctcAGCTTTAACGAACGCA- A (AUR; Takara Bio, Inc., Shiga, Japan) at the indicated GAA-3′ concentrations. The initial optical density at 600  nm 5′-GGTGATATTGGATaccgcggAGATGCCGATTTGGGC-3′ (OD ) values of the cultures were 0.03, and changes in 11 5′-CGGGCCCCCCctcgag-3′ the OD were monitored using a UV/visible spectro- 12 5′-TTTTCAACAAAATccgcgg-3′ photometer (Ultrospec 3100 pro; GE Healthcare Japan 13 5′-CGGGCCCCCCctcgagGAGCGACCTCATGCTATA-3′ Corp., Tokyo, Japan). Fukuda et al. AMB Expr (2016) 6:45 Page 5 of 12 Isolation of yeast cells with target mating‑type mating responses in both cell types. In contrast, a/α-type Parental a/α-type cells were grown in 500  μL YPD cells express both the a1 and α2 genes from MAT loci, medium containing 500 μg/mL HYG at 30 °C for 2 days, resulting in the formation of the a1-α2 complex, which and were passaged daily with 10,000-fold dilution. Yeast represses the expression of hsg (Fukuda et  al. 2013b). cells were then harvested and washed and resuspended Here, using machinery for mating-type-dependent gene in distilled water. Cell suspensions were spread on expression (Fukuda et  al. 2013a), the kanMX4 marker YPD + G418 (500 μg/mL for the isolation of a-type cells, gene was expressed in a- and α-type derivative cells. and 1.0  mg/mL for α-type cells) plates to isolate a- and The plasmid pLhyS-2  K-Pa1 (Fig.  1a) was constructed α-type yeast cells. for the selection of a-type yeast cells and contains the hygro gene as a transformation marker (hygromycin Mating assay B-resistance), the a1 gene for preventing undesirable Evaluation of mating ability was performed by cultivating mating between derivatives from the same parent by yeast cells with a mating partner in 1  ml YPD medium formation of the a1-α2 complex (Fukuda et al. 2013a, b), at 30  °C for 1.5  h. The initial OD of each strain was and the kanMX4 marker gene (repressed by α2 alone) 0.1. After cultivation, yeast cells were harvested, washed, (Fig. 1b). For the selection of α-type yeast cells, the plas- and resuspended in distilled water to give cell suspen- mid pHhyS-3  K-2α (Fig.  1c), which contains the hygro sions with OD values of 1, 0.1, and 0.01. A total of gene as a transformation marker, the α2 gene for the pre- 10  μl of each cell suspensions was spotted on SD/MSG venting undesirable mating by formation of the a1-α2 solid medium (without amino acids) containing 500  μg/ complex, and the kanMX4 marker gene (repressed by the mL G418 for the growth selection of zygotes. After incu- a1-α2 complex), was constructed (Fig.  1d). More details bation of the plates at 30  °C for 2  days, image data was of the gene expression regulation system used in these recorded for colonies that formed on the solid medium. constructs are described in previous reports (Fukuda et al. 2013a, b). Ploidy analysis using FACS The basic experimental design for the generation of Each yeast strain was grown overnight in 500  μL YPD a- and α-type sake yeast cells is schematically illustrated medium at 30 °C. The cells then were harvested, washed in Fig.  2a. Briefly, the plasmid pLhyS-2 K-Pa1 was intro - with 500 μL distilled water, and resuspended in 500 μL of duced into parental a/α-type sake yeast strains to isolate 70  % ethanol. After a 1-h incubation at room tempera- a-type of derivatives, whereas the plasmid pHhyS-3 K-2α ture, the ethanol-treated cells were harvested, washed was used to transform other a/α-type strains to iso- with 500  μL phosphate buffered saline (PBS), and resus - late α-type derivatives. In both cases, positive transfor- pended in 90  μL PBS containing 0.5  mg/mL RNase A. mants were selected on solid medium containing HYG After 1 h of incubation at 37 °C, 10 μL of 1 mg/mL pro- (YPD + HYG plates). After several passages of the trans- pidium iodide (PI) solution was added to the cell suspen- formant cultures, each cell mixture was individually sion, which was further incubated at 37  °C for 30  min spread on solid medium containing G418 (YPD +  G418 to stain DNA. The stained cells were harvested, washed plates) for the selection of a- or α-type cells expressing with 100 μL PBS, and resuspended in 1 mL sheath solu- the kanMX4 gene. tion (Becton, Dickinson and Co., Franklin Lakes, NJ, Hybrid cells cannot be generated via the mating of USA). After a 5-s sonication to reduce cell flocculation, derivative a- and α-type cells, because both cell types PI-fluorescence of yeast cells was detected using a BD express the same marker genes (hygro and kanMX4). To FACS Canto II flow cytometer (Becton, Dickinson and allow differentiation of the a - and α-type cells, plasmid Co., Franklin Lakes, NJ, USA) equipped with a 488-nm exchanges were performed to introduce unique selection blue laser, and the collected data were an analyzed using maker gene (Fig.  2a). The plasmids pLhyS-2  K-Pa1 and FlowJo software (Tree star, Ashland, Oregon, USA). The pHhyS-3  K-2α were removed by passaging cells in the fluorescence signal was collected through a 585/21  nm absence of selection pressure, and the plasmids pLY-hygro band-pass filter. (HYG-resistance) and pHY-kan (G418-resistance) were then introduced into derivative a-type and α-type cells, Results respectively. Hybrid a/α-type cells were selected on solid General isolation strategy for a‑ and α‑type yeast cells YPD medium containing HYG and G418 (YPD  +  HYG, An outline of the strategy used for the isolation of a- G418 plates) and were then isolated on YPD plates after and α-type sake yeast cells is shown in Fig.  1. Typically, the removal of both plasmids through passage cultures a-type cells express the a1 gene, whereas α-type hap- performed with YPD medium without antibiotics. loids express the α2 gene from their respective MAT loci. Although repetitive hybridizations using the newly Haploid-specific genes (hsg ) are expressed and induce generated a/α-type sake yeast strains as the parent Fukuda et al. AMB Expr (2016) 6:45 Page 6 of 12 backcrossing a/α-type of cell a-type of cell (parent) (derivative) without + G418 a/α-type of cell selection (hybrid) pLhyS- pLY- pressure hygro 2K-Pa1 Multi- hybridization mating without a/α-type of cell α-type of cell selection (parent) (derivative) pressure without + G418 selection pHY- pHhyS- pressure 3K-2 kan backcrossing backcrossing a/α-type of cell a-type of cell (parent) (derivative) + G418 a/α-type of cell a-type of cell (hybrid) (derivative) pLhyS- 2K-Pa1 Multi- hybridization Mating without a/α-type of cell α-type of cell + G418 AUR (parent) (derivative) without + G418 selection pAUR112 pHhyS- pressure 3K-2 a/α-type of cell a-type of cell (parent) (derivative) without + G418 a/ -type of cell α α-type of cell selection (hybrid) (derivative) pAUR112 pLhyS- pressure 2K-Pa1 Multi- hybridization Mating without a/α-type of cell α-type of cell + G418 AUR (parent) (derivative) + G418 backcrossing pHhyS- 3K-2α Fig. 2 Schematic outline of the experimental design for the crossbreeding of sake yeast strains. a Outline of the basic method used for the cross- breeding of sake yeast strains. Plasmids pLhyS-2 K-Pa1 and pHhyS-3 K-2α were used for isolation of a- and α-type derivative cells, respectively, and plasmids pLY-hygro and pHY-kan were used for the hybridization of derivatives. Unnecessary plasmids were removed from yeast cells by cultivat- ing cells in the absence of selection pressure. b Improved method for continuous crossbreeding without the requirement for removal of plasmid pLhyS-2 K-Pa1. c A second improved method for continuous crossbreeding without the requirement for removal of plasmid pHhyS-3 K-2α. Plasmid pAUR112 was used for the hybridization of obtained derivatives in (b) and (c) Fukuda et al. AMB Expr (2016) 6:45 Page 7 of 12 (continuous crossbreeding) can be performed using the or pHhyS-3  K-2α were selected on YPD plates con- scheme described in Fig. 2a, it is necessary to re-transform taining 300  μg/ml HYG, and after repeated passag- the strains with pLhyS-2 K-Pa1 or pHhyS-3 K-2α. To avoid ing, cell suspensions of each transformant were spread the labor required for plasmid exchange for continuous on YPD  +  G418 (>500 μg/mL) plates. The plasmids crossbreeding, a third selection marker gene, AUR1-C, was pLhyS-2  K-Pa1 and pHhyS-3  K-2α were removed from used to generate a/α-type hybrid strains (Fig. 2b, c). the isolated a- or α-type yeast cells, as shown in Fig.  2a, Figure  2b shows the procedure used to acquire a-type yielding strains K6A, K7A and K9A (a-type), and K6AL, cells derived from the generated hybrids. After isolation K7AL and K9AL (α-type) (Table  1). The removal of the of the derivatives from the parent a/α-strains, α-type cells plasmids containing the hygro and kanMX4 genes was were cultivated without selection pressure to remove the confirmed by PCR (Additional file  1: Table S3 and Fig. plasmid pHhyS-3  K-2α, and plasmid pAUR112 (Takara S1). Bio, Inc., Shiga, Japan), which contains the AUR1-C To verify the mating type and examine the mating marker gene, was then introduced into the α-type deriva- abilities of the derivative strains, a mating assay was per- tive cells. The mating of a- and α-type derivative cells was formed with the auxotrophic and G418-resistant labora- performed in mixed cultures in YPD medium, and a/α- tory haploid strains HR42-11T (α-type) and MCF4741 type hybrid cells were the isolated on solid YPD medium (a-type) (Fukuda et  al. 2013b) (Table  1). Although all of containing HYG and AUR (YPD  +  HYG, AUR plates). the derivative strains are prototrophs, they do not have After the removal of pAUR112 from cells by repeated resistance to G418, and therefore only zygotes can grow passage in medium containing only HYG (YPD  +  HYG on SD/MSG plates containing G418, but lacking any medium), a-type derivative cells expressing the kanMX4 amino acids (Fig.  3a). In the mating assay, the deriva- gene were selected on YPD  +  G418 plates. The isolated tive strains K6A, K7A and K9A mated with HR42-11T a-type cells can be directly utilized for hybridization, (α-type), whereas the parental strains (Kyokai No. 6, No. such as backcrossing and multi-hybridization, without 7 and No. 9) did not mate (Fig. 3b). Similarly, the deriva- removing the plasmid pLhyS-2 K-Pa1. tive strains K6AL, K7AL and K9AL mated with MCF4741 Figure  2c illustrates the procedure used to acquire (a-type), whereas the parental strains again did not mate α-type cells from the a/α-type hybrids. After isolation (Fig.  3c). These results confirmed that the growth selec - of the derivatives from the parent a/α-type of strains, tion system designed here promoted the generation and a-type cells were cultivated without selection pressure allowed for the isolation of a- and α-type yeast derivative to remove the plasmid pLhyS-2  K-Pa1, and plasmid cells. pAUR112 was then introduced into the a-type deriva- Outcrossing of Kyokai No. 6, No. 7 and No. 9 was next tive cells. The a- and α-type derivative cells were mated conducted according to the scheme illustrated in Fig. 2a. in YPD medium, and a/α-type hybrid cells were then After transformation of a- and α-type derivative cells isolated on YPD  +  HYG, AUR plates. After removal with the plasmids pLY-hygro and pHY-kan, cells were of pAUR112 by repeated passage in YPD  +  HYG selected on YPD + HYG and YPD + G418 plates, respec- medium, a-type cells expressing the kanMX4 gene were tively. Zygotes (hybrid cells) were isolated from mixed selected on YPD  +  G418 plates. As described for the cultures of the derivative cells using YPD +  HYG, G418 a-type cells generated using this procedure, the plas- plates, and all plasmids were then removed from each mid pHhyS-3  K-2α does not need to be removed from hybrid cell, yielding hybrid strains K67, K69, K76, K79, the isolated α-type cells prior to performing subsequent K96 and K97 (Table 1). hybridizations. Improvement of the continuous crossbreeding procedure Outcrossing of Kyokai No. 6, No. 7 and No. 9 Although the feasibility of the basic hybrid selec- The basic method for generating strains for continuous tion method (Fig.  2a) was demonstrated in the above- crossbreeding (Fig. 2a) was utilized for the outcrossing of described outcrossing experiment, each stage of the the sake yeast strains Kyokai No. 6, No. 7 and No. 9. The continuous crossbreeding procedure requires the minimum inhibitory concentration (MIC) of HYG and exchange of plasmids. As plasmid exchange is a time- G418 was first determined for these three strains (Addi - and labor-consuming process, we attempted to improve tional file  1: Table S1 and S2). The MIC values of HYG the continuous crossbreeding method (Fig.  2b, c) using for strains Kyokai No. 6, No. 7 and No. 9 were 300, 200 a third marker gene (AUR1-C; AUR-resistance). Using and 300  μg/ml, respectively, and the MIC of G418 was this approach, a- and α-type derivative cells were isolated 200 μg/ml for all three strains. from hybrid strain K76. To isolate a- and α-type cells of Kyokai No. 6, No. In this modified selection system, the isolation of a- 7 and No. 9, cells transformed with pLhyS-2  K-Pa1 and α-type derivative cells based on transformation with Fukuda et al. AMB Expr (2016) 6:45 Page 8 of 12 pAUR112 on YPD  +  AUR plates. K6AL/pAUR112 cells Parents (a/α) Partner (a or α) Derivatives Partner (a or α) were cultured together with K7A/pLhyS-2  K-Pa1 cells, prototroph G418-resistance prototroph G418-resistance and hybrid K76/pLhyS-2 K-Pa1/pAUR112 cells were then isolated on YPD  +  HYG, AUR plates and further culti- vated in YPD  +  HYG medium (without AUR). The cells were transferred to YPD  +  G418 plates for the selec- Zygote tion of a-type derivative cells (K76A/pLhyS-2  K-Pa1). prototroph To obtain α-type derivative cells from hybrid strain K76 G418-resistance according to the scheme illustrated in Fig.  2c, plasmid No colonies on SD/MSG+G418 plates Colony formation on SD/MSG+G418 pAUR112 was introduced into strain K7A after removing (No zygotes are generated) plates (when zygosis occurs) plasmid pLhyS-2  K-Pa1, yielding K7A/pAUR112 trans- OD formants, which were selected on YPD  +  AUR plates. Hybrid K76/pHhyS-3  K-2α/pAUR112 cells were then isolated on YPD  +  HYG, AUR plates from mixed cul- tures of K7A/pAUR112 and K6AL/pHhyS-3  K-2α cells, and were further cultivated in YPD  +  HYG medium (without AUR). Finally, a-type derivative cells, K76AL/ Kyokai No.6 K6A Kyokai No.7 K7A Kyokai No.9 K9A pHhyS-3 K-2α, were isolated on YPD + G418 plates. × × × × × × HR42-11T HR42-11T HR42-11T HR42-11T For determination of mating type, all plasmids were HR42-11T HR42-11T removed via cultivation in YPD medium to yield strains OD600 K76A and K76AL (Table  1). In a mating assay, strain K76A successfully mated with HR42-11T (α-type) and K76AL mated with MCF4741 (a-type) (Fig.  4). These results suggest that a- and α-type derivative cells can be generated from hybrid strains (polyploids) as well as parental diploid strains. Notably, the generated K76A/ Kyokai No.7 K7AL Kyokai No.9 K9AL Kyokai No.6 K6AL pLhyS-2  K-Pa1and K76AL/pHhyS-3  K-2α derivative × × × × × × MCF4741 MCF4741 MCF4741 MCF4741 MCF4741 MCF4741 cells can be directly utilized for hybridization during Fig. 3 Evaluation of the mating abilities of derivative strains. a Sche- continuous crossbreeding without removing plasmids matic outline of the mating assay. Using haploid strains HR42-11T pLhyS-2 K-Pa1and pHhyS-3 K-2α, respectively. (α-cell) and MC4741 (a-cell) as the mating partners, zygotes were To verify the feasibility of performing continu- selected on solid medium containing G418, but lacking amino acids ous crossbreeding using the a- and α-type derivative (SD/MSG + G418 plates). The Parent strains were Kyokai No. 6, No. 7 and No. 9 (a/α-type cells), and the Derivative strains were K6A, K7A and K9A (isolated as a-type cells) and K6AL, K7AL and K9AL (isolated OD as α-type cells), respectively. b Images showing colony formation in the mating assay for strains K6A, K7A and K9A. c Images show- ing colony formation in the mating assay for strains K6AL, K7AL and K9AL. The OD values of 10-μL cell suspensions spotted on the solid media were set at 1.0, 0.1 and 0.01 the plasmids pLhyS-2  K-Pa1 and pHhyS-3  K-2α was performed as described in Fig.  2a. The obtained deriva - tive cells, K7A/pLhyS-2 K-Pa1 and K6AL/pHhyS-3 K-2α, were then subjected to plasmid exchange using one of K76 K76AL K76 K76A two methods. Because the MIC values of AUR against × × × × strains K7A and K6AL were 100 and 300  ng/ml, respec- MCF4741 MCF4741 HR42-11T HR42-11T tively (Additional file  1: Table S4), yeast transformation Fig. 4 Evaluation of mating abilities of the derivative strains K76A with plasmid pAUR112 was performed using YPD plates and K76AL. Images showing colony formation on SD/MSG + G418 containing 300 ng/ml AUR. plates in the mating assay are shown. The Parent strains were K76 To obtain a-type derivative cells from hybrid strain K76 (a/α-type cells), and the Derivative strains were K76A (isolated as a-type cells) and K76AL (isolated as α-type cells), respectively. The according to the scheme illustrated in Fig.  2b, plasmid OD values of 10-μL cell suspensions spotted on the solid media pAUR112 was introduced into strain K6AL after remov- were as in Fig. 3b ing plasmid pHhyS-3 K-2α, yielding transformants K6AL/ Fukuda et al. AMB Expr (2016) 6:45 Page 9 of 12 cells, multi-hybridization was performed with K76A/ hybrid strain K76 was much larger than that of strains pLhyS-2  K-Pa1 and K9AL/pHhyS-3  K-2α cells (isolated K7A and K6AL, suggesting that the DNA content within as described above) according to the scheme illustrated in strain K76 cells had increased. In contrast, the cell size of Fig. 2b. The plasmid pAUR112 was introduced into strain multi-hybrid strain K76x9 was clearly smaller than that K9AL after removing the plasmid pHhyS-3  K-2α, yield- of strains K76 and K76A, suggesting that a major loss ing K9AL/pAUR112 cells after selection on YPD +  AUR of DNA occurred after the hybridization of K76A and plates. Mixed cultures of K76A/pLhyS-2  K-Pa1 and K9AL. K9AL/pAUR112 were plated on YPD  +  HYG, AUR To more accurately estimate the DNA content of cells, medium to select for hybrid K76x9/pLhyS-2  K-Pa1/ FACS analysis was performed after propidium iodide pAUR112 cells, and all plasmids were then removed from (PI)-staining of each yeast strain (Fig.  5). In DNA con- the hybrid cells via cultivation in YPD medium without tent histograms, two PI-fluorescence signal peaks, cor - antibiotics to yield multi-hybrid strain K76x9 (Table 1). responding to the G0/G1 and G2/M phases, are typically observed (Qiu et  al. 2013). The DNA content of diploid Estimation of cellular DNA content strains Kyokai No. 6, No. 7 and No. 9 was measured, and To further characterize the derivative and hybrid cells, reference values of 2 N (two sets of all chromosomes) and microscopic observation of strains K7A, K6AL, K9AL, 4  N (four sets of all chromosomes) were defined as the K76, K76A and K76x9 was performed using BY4742 average of each peak value. The 3 N value was calculated and Kyokai No. 7 as control haploid and diploid strains, as the average of the 2 and 4 N values, and 1, 1.5, 2.5, 4.5, respectively (Additional file  1: Fig. S2). The diameter of 5, 6, 8 and 9 N values were then calculated from the 3 N a yeast cell increases with increasing amounts of nuclear value (Table  3). Notably, although haploid strain BY4742 DNA (Amodeo and Skotheim 2016). The cell size of is a laboratory yeast strain, the two peak values obtained Fig. 5 DNA content histogram of PI-stained yeast cells using FACS. PI-fluorescence data was collected from yeast cells within a gate drawn in the FSC-SSC dot plots (Additional file 1: Fig. S3) according to the data collected from control diploid strains. For relative comparison of DNA content, ref- erence values of 2 N (two sets of all chromosomes) and 4 N (four sets of all chromosomes) were defined as the average of each peak value of Kyokai No.6, No.7 and No.9. The 1 and 8 N values were calculated from the 2 and 4 N values Fukuda et al. AMB Expr (2016) 6:45 Page 10 of 12 for this strain were well accorded with the calculated 1 N isolation of a- and α-type derivative cells from a/α-type and averaged 2 N value. parental cells. The constructed plasmids required at least The DNA content of sake yeast strains generated in the two types of marker genes: one for the selection of yeast present study was measured and evaluated based on com- transformants and one for the selection of a- or α-type parison to the reference values (from 1 to 9  N) (Table  4; derivative cells. To this end, we employed two commonly Fig. 5). The results of FACS analysis (Additional file  1: Fig. used marker genes for industrial yeast strains, namely S3) and microscopic observation (Additional file  1: Fig. the hygro and kanMX4 genes, for the transformation and S4) indicate that generated strains have cell flocculation isolation of a- and α-type derivative cells, respectively ability, unlike the control strains. To exclude signals origi- (Murakami et al. 2012). nating from the flocculated cells, a data collection gate As a- and α-type yeast cells can be generated by spon- was drawn according to FSC-SSC dot plots from the con- taneous chromosomal mutations, such as LOH in the trol strains (Additional file  1: Fig. S3). As shown in Table 4 region containing the MAT locus or the mitotic loss and Fig.  5, strains K7A and K7AL had similar DNA con- of chromosome III (Alvaro et  al. 2006; Andersen et  al. tents to those of the parental strain (Kyokai No. 7). In 2008; Daigaku et  al. 2004; Mayer and Aguilera 1990; contrast, strains K6A, K9A and K9AL appeared to have Takagi et al. 2008), we examined the nuclear DNA of the partially lost chromosomal DNA due to unequal distribu- generated a- or α-type derivative strains. The DNA con - tion of DNA during mitotic cell division, whereas strain tent of cells after the removal of all plasmid DNA was K6AL acquired additional chromosomal DNA. evaluated qualitatively by the microscopic analysis of In the outcrossing experiments, the DNA content of cell size and quantitatively by the FACS measurement hybrid strains K67, K69, K76, K79, K96 and K97 was found of DNA content based on PI fluorescence signal inten - to be X ± 0.5 N, where X is the total DNA content of the sity. In cases of derivative strains K6A, K9A, K9AL and two parental strains. However, strain K76A appeared to K76A, the DNA content was clearly smaller compared to have lost an amount of DNA that was nearly equivalent to those of the parental strains, suggesting that the loss of one set of chromosomes, whereas strain K76AL acquired chromosomal DNA occurred. In contrast, other deriva- additional DNA that was almost equivalent to a half set tive strains had DNA contents that were nearly equiva- of chromosomes. It was also determined that the multi- lent to those of the parental strains. Notably, however, hybrid strain K76x9 lost a substantial amount of chromo- it is not possible to determine the rearrangement event somal DNA during the mating process and maintained a that occurred in the obtained derivative strains based on near-diploid DNA content. These results are consistent these results alone, because the MAT locus only occu- with the microscopic observations of cellular morphology pies 2 % of all chromosomes (Kumaran et al. 2013). u Th s, described above (Additional file 1 : Fig. S2). although these methods do not require complicated or time-consuming procedures, estimation of chromo- Discussion some III number within cells by real-time PCR (Fukuda The aim of this study was to establish a versatile method et  al. 2013a), array comparative genomic hybridization for the crossbreeding of sake yeasts, which have low spor- (Abunimer et  al. 2016), or whole-genome sequencing ulation rates. We constructed two new plasmids for the would be needed to investigate the underlying events in more detail. Although it was demonstrated that the basic method developed here (Fig.  2a) was applicable for the isola- Table 3 Reference values used in the FACS analysis tion of yeast cells with mating ability from common sake Peak Value of PI‑fluorescence yeast strains, due to the requirement for the repeated exchange of plasmids, this approach still required multi- 1 N 189.18 ple steps to perform the final hybridization and isolated 1.5 N 283.77 a- or α-type derivatives. To reduce the need for plasmid 2 N 379.28 ± 18.59 exchange steps, we introduced a third marker gene, which 2.5 N 472.95 was compatible with the hygro and kanMX4 marker 3 N 567.54 genes, into the selection system. Using the commercial 4 N 755.79 ± 35.58 plasmid pAUR112, which contains the AUR1-C marker 4.5 N 851.30 gene, we successfully isolated hybrid cells without the 5 N 945.89 need for the time-consuming plasmid exchange process. 6 N 1135.07 In a back-crossing comprising n cycles of hybridization, 8 N 1513.43 only a single plasmid exchange step would be required in 9 N 1702.61 the scheme illustrated in Fig.  2b and c, whereas (2n +  1) ±means standard deviations from three diploid strains Fukuda et al. AMB Expr (2016) 6:45 Page 11 of 12 Table 4 Evaluation of DNA content and ploidy assessment In general, crossbreeding induces genomic shuffling by FACS analysis based on mechanisms of sexual reproduction (Snoek et al. 2015), and the resulting derivative strains may therefore Strain Peak 1 Peak 2 Ploidy acquire phenotypes different from those of the parent Control strains. In the present approach, LOH and the mitotic BY4742 188.34 345.07 1 N loss of chromosomal DNA occur randomly, and the gen- Kyokai No.6 366.04 730.97 2 N erated hybrid yeast strains may undergo additional chro- Kyokai No.7 366.22 730.30 2 N mosomal loss after mating to reach stable genomic states. Kyokai No.9 405.57 806.11 2 N Such changes in ploidy are termed meiosis-like adaptation Tested (Storchova 2014) and can result in the formation of deriv- K6A 207.37 413.17 1 N ative and hybrid cells with diverse genotypes and pheno- K7A 379.61 806.97 2 N types, even from the same parental polyploid strain. The K9A 280.76 616.19 1.5 N genomic instability of polyploids is beneficial for cross - K6AL 433.52 865.21 2.5 N breeding because it has contributed to the adaptation and K7AL 331.98 728.50 2 N evolution of yeasts in nature (Snoek et al. 2015). K9AL 280.73 520.07 1.5 N In conclusion, we have established a complete proce- K67 717.23 1475.10 4 N dure for the crossbreeding of industrially used sake yeasts K69 547.61 1125.63 3 N possessing sporulation defects. The outcrossing of sake K76 718.22 1527.81 4 N yeasts was achieved in all examined combinations, and K79 739.99 1528.83 4 N the feasibility of continuous crossbreeding was demon- K96 717.00 1532.82 4 N strated by generating a multi-hybrid strain. The method K97 766.30 1637.93 4 N developed here may allow the numerous and valuable K76A 529.92 1022.16 3 N yeast resources, including sake yeasts, to be efficiently K76AL 821.08 1640.31 4.5 N used for generation of new strains with desirable proper- K76x9 367.34 756.46 2 N ties for industrial applications. Additional file plasmid exchange steps would be needed in the scheme Additional file 1. Supporting Information for Materials and Methods. described in Fig. 2a. To demonstrate the feasibility of the continuous cross- breeding approach developed in the present work, we Authors’ contributions NF designed the study; NF and MK conducted experiments; NF, JI, AK and SH generated a multi-hybrid strain, K76x9, from strains K76A analyzed data; and NF and SH co-wrote the manuscript. All authors read and and K9AL, with the expectation that the obtained strain approved the final manuscript. would inherit genetic characteristics from strains Kyokai Author details No. 6, No. 7 and No. 9. Microscopic observation and FACS Biomedical Research Institute, National Institute of Advanced Industrial analysis revealed that both strains K76A and K9AL under- Science and Technology (AIST ), Higashi, Tsukuba, Ibaraki 305-8566, Japan. went partial loss of chromosomal DNA, whereas the multi- Department of Chemical Science and Engineering, Graduate School of Engi- neering, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan. Organization hybrid strain K76x9 possessed a near-diploid DNA content, of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, having lost a major part of its DNA during the mating pro- Kobe, Japan. cess. There are two possible factors that lead to decreased Acknowledgements genomic stability in the generated strains. First, ane- The sake yeast strains Kyokai No. 6, No. 7, and No. 9 were provided by the uploidy (a chromosomal content differing from multiple Biological Resource Center (NBRC), NITE, Japan. This work was supported in sets of haploid chromosomes) induces genomic instability part by JSPS KAKENHI Grant Number 25820406 and 16K14497. (Skoneczna et  al. 2015). Strain K76x9 was generated from Competing interests aneuploid strain K9AL and suspected aneuploid strain N. Fukuda and S. Honda declare that they are inventors on a pending patent K76A, which appeared to have lost DNA), and hence, the using aspects of this work. M. Kaishima, J. Ishii and A. Kondo declare that they have no competing interests. zygotes must also be aneuploid, which might induce chro- mosomal loss. The other possible factor leading to genomic Compliance with ethical standards instability is an increase in the ploidy of yeast cells. Accord- This article does not contain any studies with human participants or animals performed by any of the authors. ing to a previous report (Kumaran et  al. 2013), chromo- somal stability is reduced as the ploidy of a cell increases. Received: 15 June 2016 Accepted: 25 June 2016 These two factors presumably had synergistics effects on the genomic instability of the multi-hybrid strain K76x9. Fukuda et al. AMB Expr (2016) 6:45 Page 12 of 12 References Kishimoto M. Fermentation characteristics of hybrids between the cryophilic Abunimer AN, Salazar J, Noursi DP, Abu-Asab MS. 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Published: Jul 8, 2016

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