Self-Cloning Yeast Strains Containing Novel FAS2 Mutations Produce a Higher Amount of Ethyl Caproate in Japanese Sake

Biosci. Biotechnol. Biochem., 68 (1), 206–214, 2004

Self-cloning Yeast Strains Containing Novel FAS2 Mutations Produce a Higher Amount of Ethyl Caproate in Japanese Sake

Kazuo ARITOMI,1 Isao HIROSAWA,2 Hisashi HOSHIDA,2 Mikio SHIIGI,1 y Yoshinori NISHIZAWA,2 Susumu KASHIWAGI,1 and Rinji AKADA2;

1Yamaguchi Prefectural Industrial Technology Institute, Asutopia, Ube 755-0151, Japan 2Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan

Received August 25, 2003; Accepted September 27, 2003

Point mutation of Gly1250Ser (1250S) of the yeast inhibitor of fatty acid synthesis,4,5) has been used to fatty acid synthase gene FAS2 confers cerulenin resist- isolate yeast mutants that produced higher amounts of ance. This mutation also results in a higher production ethyl caproate, an apple-like flavor component in sake.3) of the apple-like flavor component ethyl caproate in The cerulenin resistance and the higher production of Japanese sake. We mutated the 1250th codon by in vitro ethyl caproate were conferred by a mutation from site-directed mutagenesis to encode Ala (1250A) or Cys glycine to serine at the 1250th codon (1257th in a (1250C) and examined cerulenin resistance and ethyl different strain) of the fatty acid synthase gene FAS2.6–9) caproate production. The mutated FAS2 genes were This dominant FAS2 mutation was thought to decrease inserted into a binary plasmid vector containing a drug- the activity of the fatty acid synthase complex, which is resistance marker and a counter-selectable marker, a hexamer (66) of and subunits encoded by FAS2 GALp-GIN11M86. The plasmids were integrated into and FAS1, respectively.10) Decreased carbon chain the wild-type FAS2 locus of a sake yeast strain, and the elongation activity during fatty acid synthesis increased loss of the plasmid sequences from the integrants was the amount of C6 caproic acid, a precursor of ethyl done by growth on galactose plates, which is permissive caproate, and therefore probably resulted in the increase for loss of GALp-GIN11M86. These counter-selected of ethyl caproate.3) strains contained either the wild type or the mutated This case demonstrates that mutant selection can be FAS2 allele but not the plasmid sequences, from which effective for finding valuable sake yeast strains. How- FAS2 mutant strains were selected by allele-specific ever, random screenings such as for drug resistance, PCR. The FAS2-1250C mutant produced a higher ethanol tolerance, osmotolerance, or other stress resist- amount of ethyl caproate in sake than FAS2-1250S, ance, do not always result in the successful isolation of while FAS2-1250A produced an ethyl caproate level useful yeast mutants. Strain improvement by hybrid- intermediate between FAS2-1250S and the parental ization and fusion of two yeast strains is also a form of Kyokai no. 7 strain. Interestingly, these mutants only random selection that will not always yield valuable showed detectable cerulenin resistance. These ‘self- yeast strains. For this reason, a reasonable step-by-step cloning’ yeast strains should be acceptable to the public method to improve industrially important traits of yeast because they can improve sake quality without the strains is needed. Recombinant DNA technology is presence of extraneous DNA sequences. attractive for this purpose because one characteristic can be precisely modified without affecting other desirable Key words: Saccharomyces cerevisiae; sake yeast; properties.2) FAS2; ethyl caproate; self-cloning A major issue in the application of recombinant yeast strains for commercial use is the concern of consumers The yeast Saccharomyces cerevisiae produces flavor regarding genetically modified (GM) foods. Since the components that are important factors in the quality of development of plant genetic engineering, recombinant food and alcoholic drinks. To improve the quality of crops have been available commercially in the world but food and alcoholic drinks and the ability of fermenta- they have not received public acceptance.11,12) More- tion, yeast strains have been improved by genetic over, there are no commercially available GM foods techniques such as mutagenesis, hybridization, and directly produced by the fermentation of recombinant protoplast fusion.1,2) One such successful example is a microorganisms. The lack of public acceptance of GM cerulenin-resistant mutant in sake yeast.3) Cerulenin, an foods may be due to a fear of toxic or allergenic

y To whom correspondence should be addressed. Fax: +81-836-85-9201; E-mail: [email protected] Self-cloning Sake Yeast Strains with FAS2 Mutations 207 Table 1. Strains

Strain Genotype and characteristics Source Kyokai no. 7 Sake yeast BSJa RAK649 FAS2wt/FAS2-1250S derived from Kyokai no. 7 9 RAK1988 Kyokai no. 7 integrant [pGG119FAS2A] This study RAK1750 Kyokai no. 7 integrant [pGG119FAS2C] This study RAK2071 FAS2wt/FAS2-1250A, Self-cloning derived from RAK1988 This study RAK2075 FAS2wt/FAS2-1250A, Self-cloning derived from RAK1988 This study RAK2035 FAS2wt/FAS2-1250C, Self-cloning derived from RAK1750 This study RAK2039 FAS2wt/FAS2-1250C, Self-cloning derived from RAK1750 This study YPH250 MATa ura3-52 his3-200 leu2-1 trp1-1 ade2-10 lys2-80 17

a Brewery Society of Japan. products expressed by foreign genes or bacterial Materials and Methods sequences introduced into GM organisms.13) Introduc- tion of a valuable gene derived from the same organism Strains and media. The strains used are shown in and elimination of other undesirable DNA sequences Table 1. All strains were isogenic to Japanese sake yeast used for gene manipulations would eliminate this issue. Kyokai no. 7 strain from the Brewery Society of Japan, To help solve this problem, we used a strategy to except for the laboratory strain YPH250.17) The DH5 make yeast gene mutations conferring valuable traits strain of Escherichia coli was used for plasmid rationally rather than randomly, and eliminate all constructions. The yeast strains were grown in YPD unnecessary foreign DNA sequences from recombinant medium (2% glucose, 2% polypepton and 1% yeast yeast strains after the introduction of the yeast mutant extract along with 2% agar if necessary). Synthetic genes and its vectors. For the latter, we have developed a dextrose medium (SD) contained 0.17% Yeast Nitrogen novel counter-selection marker, GALp-GIN11M86,to Base without amino acids and ammonium sulfate, 0.5% remove unwanted sequences from yeast.9) The marker ammonium sulfate and 2% glucose. YPGal medium was consists of a galactose-inducible overexpression pro- the same as YPD medium but contained 2% galactose moter and the GIN11 growth-inhibitory sequence.14) instead of glucose. Cerulenin, cycloheximide (Sigma Cells that lost the marker grew well on galactose, but Chemical Co., St. Louis, MO, USA), aureobasidin A cells that retained the marker did not, because of the (Takara Shuzo Co., Ltd.) and nystatin (Wako Pure growth inhibitory effect of GIN11 overexpression. This Chemical) were used for preparing drug plates and marker is dominant, and can therefore be used in most added after autoclaving. YM-5 (5% glucose, 0.3% yeast yeast strains, including industrial yeast strains, without extract, 0.3% malt extract and 0.3% polypepton) was the need for mutations that are required in traditional used for the analysis of ethyl caproate production. counter-selections.9,14,15) This construction process, which is called ‘self-cloning’, should be the most DNA manipulation. The primers used are shown in efficient way to use recombinant DNA technology in Table 2. Mutant FAS2 genes were constructed by the the food market.16) method of Barettino et al.18) For the first PCR reaction, We took advantage of prior information on the pRSFAS2-69) was used as a template and the primers cerulenin-resistant FAS2 mutation to make useful with mutations and a counter primer (FAS2-C5178) mutations. The 1250th codon is mutated in the cerule- were mixed in a 25 l reaction mixture containing KOD nin-resistant FAS2 mutation from glycine to serine.6) DNA polymerase and reactions were done according to Given that this codon appears to be necessary for both the activity of the fatty acid synthase complex and for ethyl caproate production, we speculated that conversion Table 2. Primers to other amino acids with structures similar to serine (i.e. alanine, cysteine and threonine) might affect the activity Primer Sequence of fatty acid synthase complex. Using a gene replace- FAS2-1250A 5-GGTTCTGCTATGGGTGGTGTTT-3 ment protocol, we constructed mutant FAS2 strains to FAS2-1250C 5-GGTTCTTGTATGGGTGGTGTTT-3 contain only the FAS2 mutations but not the other FAS2-1250T 5-GGTTCTACTATGGGTGGTGTTT-3 extraneous DNA sequences derived from the vector. The FAS2-C5178 5-CTTATCCTTAGATACACG-3 FAS2-2001 5-CTTCAACGGTGTCACCTT-3 self-cloning yeast strains containing the FAS2 mutations FAS2-WGG 5-GGTAACTGTTCTGGTTCTGG-3 produced higher amounts of ethyl caproate in sake. The FAS2-MGC 5-GGTAACTGTTCTGGTTCTGC-3 difference between those FAS2 mutants and the wild FAS2-WAG 5-TGGTAACTGTTCTGGTTCAG-3 type yeast strain is only a single base. These GM yeast FAS2-MAT 5-TGGTAACTGTTCTGGTTCAT-3 strains will be applicable to commercial sake produc- Mutated bases are in bold. The 30-terminal nucleotides complementary to the tion. mutations are underlined. Italics of A are additional nucleotide mismatches. 208 K. ARITOMI et al. the manufacturer’s instructions (Toyobo, Osaka, Japan). were picked and used for further analyses as counter- The first PCR reaction was started at 94C for 1 min and selected strains. followed by 25 cycles of 94C for 20 s, 50C for 2 s, and 74C for 30 s. A 1.4-kb PCR product was isolated by Southern blot analysis and allele-specific PCR. agarose gel electrophoresis and used as a megaprimer Chromosomal DNA was isolated by a protoplast method for the second PCR. We used pAURFAS2-69) as a using Zymolyase 100T (Seikagaku Kogyo, Tokyo, template for the second PCR reaction because it lacks an Japan), followed by phenol/chloroform extraction and annealing site for FAS2-C5178. In addition, the second ethanol precipitation.21) Chromosomal DNA was digest- PCR was done using the primers FAS2-2001 and FAS2- ed with NruI and separated by agarose gel electro- C5178. The second PCR cycle was similar to the first phoresis. Next, the DNA was transferred to a nylon PCR except that extension time was 74C for 1 min membrane. The FAS2 probe was a 1.5-kb fragment instead of 30 s. The 3.0-kb PCR product from this isolated from AflII-NheI digested pRSFAS2-6, and the second PCR was digested with AflII and BstEII, and the bacterial sequence probe was BamHI-digested pBlue- resulting 2.4-kb fragment was isolated by agarose gel script KSþ. Probe preparation and hybridization was electrophoresis and inserted into AflII-BstEII sites of done as described9) using the DIG system (Roche pRSFAS2-6. The resulting plasmids contained URA3, Diagnostics, GmbH). ARS/CEN and mutant FAS2, which we named p1250A To detect mutant alleles, allele-specific PCR was done for alanine, p1250T for threonine, and p1250C for using KOD Dash DNA polymerase (Toyobo) and cysteine mutations. The mutations were confirmed by specific primers and a counter primer (0.1 g/l) sequencing using a Dye Terminator Cycle Sequencing (Table 2) according to Akada et al.22) The PCR mixture kit and ABI PRISM 310 Genetic Analyzer (Perkin was initially incubated at 94C for 60 s, and then Elmer Applied Biosystems). The p1250A, p1250T, and subjected to 25 or 30 cycles of the following program in p1250C plasmids were digested with XhoI and NotI and a PCR machine (Progene: Techne Ltd., Cambridge, the 7.0-kb fragments containing mutated FAS2 genes UK): 94C for 20 s ! 56Cor57C for 2 s ! 74C for were inserted into SalI-NotI sites of pGG11915) to form 60 s.22) pGG119FAS2A, pGG119FAS2T, and pGG119FAS2C, respectively. The plasmids used in this study are listed in Fermentation test. For a pilot experiment, cells were Table 3. inoculated in 10 ml of YM-5 medium and incubated at 15C for 7 d. The concentration of ethyl caproate in the Yeast manipulations. Yeast transformation was done culture supernatant was measured as described.9) For by a simple lithium acetate/PEG method.19) Trans- laboratory scale sake experiments, 500 g of rice was formants of Kyokai no. 7 strain were selected on SD used and sake fermentation was done at 10C. Fermen- plates containing 1.0 g/ml cerulenin. Colonies ap- tation profiles were monitored by a loss of the weight by peared after incubation at 28 C for 3–5 d. The colonies CO2 production. General components of the sake were were picked and grown on SD plates containing 2.0 g/ analyzed by the standard method established by the ml cerulenin. The cells grown on the 2.0 g/ml National Tax Administration Agency of Japan.23) cerulenin plates were then streaked on SD plates containing 0.5 g/ml cycloheximide. Cells growing on Results the cycloheximide plates were used for further analysis.20) Construction of FAS2 mutant genes For counter-selection, transformants were grown for We speculated that, like the cerulenin-resistant 1250S 1 d in liquid YPGal medium. Cells (103–104Þ were mutation, changes in the 1250th amino acid of Fas2 spread on a YPGal plate as described previously.9) might dominantly affect the activity of fatty acid Colonies growing on the YPGal plates after 36–48 h synthase. Therefore, we mutated the 1250th codon from

Table 3. Plasmids

Plasmid Genotype or characteristics References pRS316 URA3 CEN/ARS 17 pGG119 PGKp-YAP1 GALp-GIN11M86 15 pRSFAS6 URA3 CEN/ARS FAS2-1250S 9 pAURFAS2-6 AUR1-C FAS2-1250S 9 p1250A-60 FAS2-1250A URA3 CEN/ARS This study p1250C-9 FAS2-1250C URA3 CEN/ARS This study p1250T-50 FAS2-1250T URA3 CEN/ARS This study pGG119FAS2A FAS2-1250A PGKp-YAP1 GALp-GIN11M86 This study pGG119FAS2C FAS2-1250C PGKp-YAP1 GALp-GIN11M86 This study pGG119FAS2T FAS2-1250T PGKp-YAP1 GALp-GIN11M86 This study Self-cloning Sake Yeast Strains with FAS2 Mutations 209 glycine (GGT) to alanine (GCT), threonine (ACT), or cysteine (TGT) by in vitro site-directed mutagenesis.18) The mutant FAS2 genes were inserted into pRSFAS2-6, resulting in p1250A-60, p1250C-9 and p1250T-50. The plasmids were used to transform YPH250 by the URA3 selection, and ethyl caproate production was measured after incubation in YM-5 medium. The pilot experiment showed that transformants produced slightly higher amounts of ethyl caproate (0.03–0.07 ppm) than the vector control (0–0.02 ppm). Because the production of ethyl caproate from the YM-5 culture of the YPH250 laboratory strain was usually poor in our hands, we introduced the mutant FAS2 genes into the Kyokai no. 7 sake yeast strain.

Self-cloning of FAS2 mutant genes To replace wild type FAS2 with the mutant FAS2 in Fig. 1. Structure of pGG119FAS2 Plasmids. the Kyokai no. 7 sake yeast strain, we inserted the pGG119 contained the PGKp-YAP1 drug-resistant transformation 15) marker and the GALp-GIN11M86 counter-selection marker. In vitro- mutant FAS2 genes into pGG119, resulting in generated FAS2-1250 mutant genes were inserted into pGG119 to pGG119FAS2A, pGG119FAS2C, and pGG119FAS2T generate pGG119FAS2A (alanine), pGG119FAS2C (cysteine) and (Fig. 1). The pGG119 contained a dual selective pGG119FAS2T (threonine). transformation marker, PGKp-YAP1, which enables eﬃcient transformation selection on plates with the The plasmids were linearized with a single-cut NheI site antibiotics cerulenin or cycloheximide,20) and a counter- located within the FAS2 open reading frame and used to selection marker to remove plasmid sequences from transform Kyokai no. 7 strain by selection on SD transformants.15) containing cerulenin. The cerulenin-resistant colonies The self-cloning strategy is represented in Fig. 2A. were picked and placed on SD plates containing 0.5

Fig. 2. Self-cloning of FAS2 Mutant Genes. A. Schematic representation of self-cloning strategy. pGG119FAS2 plasmid containing FAS2 mutation was integrated into FAS2 locus of Kyokai no. 7. The resulting strains contained vector sequences between the direct repeats of mutant and wild-type FAS2 alleles. Homologous recombination between the repeats resulted in loss of the plasmid sequences. Counter-selected strains contain either the wild-type FAS2 or the mutant FAS2 allele. B. Southern blot analysis. Chromosomal DNAs were digested with NruI and hybridized with the FAS2 probe (left) and the pBluescript KSþ probe (right). RAK2071 and RAK2075 were selected from RAK1988, and RAK2035 and RAK2039 were from RAK1750. 210 K. ARITOMI et al.

Fig. 3. Allele-specific PCR to Detect FAS2 Mutant Alleles. PCR was done with specific primers designed to amplify either the wild-type or mutant FAS2 by changing its 30-end nucleotide. The counter primer was FAS2-C5178. K7: Kyokai no. 7 strain. A. Amplification with FAS2-MAT (T) primer for the detection of 1250C allele and FAS2- WAG (G) for wild type allele. B. Amplification with FAS2-MAC (C) primer for the detection of 1250A allele and FAS2-WGG (G0) for wild type allele.

g/ml cycloheximide, and colonies resistant to both 1250C allele did not produce a fragment from the cerulenin and cycloheximide but not to amphotericin B chromosomal DNA of wild type Kyokai no. 7 but (15 g/ml in YPD) and nystatin (25 g/ml in YPD) produced a fragment from the pGG119FAS2C integrant were selected.20) Transformants of pGG119FAS2T (RAK1750) and the two self-cloning strains, RAK2035 could not be obtained, thus we could not perform and 2039, indicating that RAK2035 and 2039 contained further studies on the threonine mutant of FAS2. FAS2-1250C allele (Fig. 3A). FAS2-WAG primer for Chromosomal integration of the plasmids was confirmed wild type allele detection produced a fragment from by Southern blot analysis. The NruI digest of chromo- Kyokai no. 7 as well as the integrant and self-cloning somal DNA isolated from integrants is expected to yield strains. Amplification with FAS2-MAC primer produced a 22-kb band from the integrated chromosome and a 7.5- a fragment from RAK2071 and RAK2075 but not from kb band from the wild type chromosome because the other strains, indicating that RAK2071 and 2075 Kyokai no. 7 is diploid, while the wild-type strain contained FAS2-1250A allele (Fig. 3B). yields only a 7.5-kb band (Fig. 2A). The result showed that the integrant yielded two bands, one integrated band Drug-resistance phenotype of FAS2 mutants (arrow in Fig. 2B), which was also detected with a The self-cloning FAS2 mutants and plasmid trans- bacterial plasmid probe (Fig. 2B, right), and one wild- formants were examined for their growth on YPD, SD, type 7.5-kb band. SD+cerulenin, and SD+cycloheximide plates (Fig. 4). The integrants contained plasmid sequences between pGG119FAS2A and pGG119FAS2C transformants were direct repeats of the mutant and wild type FAS2 genes. resistant to 1.0 g/ml cerulenin and 0.5 g/ml cyclo- The loss of plasmid sequences through homologous heximide due to the presence of the PGKp-YAP1 drug- recombination between the FAS2 genes was selected by resistance marker.20) However, the self-cloning strains using the GALp-GIN11M86 marker.9,14,15) The integrants were not resistant to a 3-d incubation with 1.0 g/ml, were grown overnight in liquid YPGal medium and the although they showed weak resistance to a 7-d incuba- cultures were diluted and spread on YPGal plates. tion with 0.2 g/ml cerulenin. The cerulenin-resistant Colonies that appeared on the plates were picked, and FAS2-1250S mutant was resistant to concentrations the loss of plasmid sequences was confirmed by South- higher than 2.0 g/ml cerulenin.6) Therefore, compared ern blot analysis (Fig. 2B). Counter-selected strains to FAS2-1250S, the FAS2-1250A and FAS2-1250C showed only a 7.5-kb band and lost the 22-kb band mutants were weakly resistant to cerulenin. because of the reversion to either the wild-type or mutant allele. More than 90% of the colonies showed Ethyl caproate production of FAS2 mutants complete loss of plasmid sequences. The properties of sake produced by the FAS2 mutants To select FAS2 mutants, we designed primers were investigated by laboratory-scale fermentation using containing mutated nucleotides at the 30 ends (Table 2) 500 g of rice. Ethanol production by the mutant strains for an allele-specific PCR.22) Specific amplification of was as same as with the wild-type Kyokai no. 7 and mutant alleles but not of the wild type allele can be RAK649 (FAS2-1250S/FAS2) strains, but the FAS2- achieved by adjusting the annealing temperature. Am- 1250C strain produced more caproic acid and ethyl plification with FAS2-MAT primer for the detection of caproate than the FAS2-1250S cerulenin-resistant mu- Self-cloning Sake Yeast Strains with FAS2 Mutations 211

Fig. 4. Drug-resistance Phenotype of FAS2 Mutants. Kyokai no. 7 (K7), plasmid integrants, and self-cloning FAS2 mutants were streaked on plates of YPD, SD, and SD containing 0.5 g/ml cycloheximide, 1.0 g/ml cerulenin, and 0.2 g/ml cerulenin. Cells were incubated at 28C for the indicated times.

Fig. 5. Laboratory-scale Sake Fermentation Using the FAS2 Mutants. Sake yeast strains containing FAS2-1250 mutations were fermented with boiled rice. The net weight loss due to CO2 production (left) and production of ethyl caproate (right) were measured. Symbols are , Kyokai no. 7; , FAS2-1250S (RAK649); , pGG119FAS2A integrant (RAK1988); , self-cloning FAS2-1250A (RAK2071); , pGG119FAS2C integrant (RAK1750); , self-cloning FAS2-1250C (RAK2035). tant (Fig. 5). The FAS2-1250A strain produced a level of Discussion caproic acid and ethyl caproate that was between the levels produced by the FAS2-1250S and Kyokai no. 7 Many GM yeast strains have been constructed to strain. Therefore, the ethyl caproate/caproic acid pro- improve foods and alcoholic beverages by introducing duction (ppm) by the strains, from low to high was: or disrupting speciﬁc genes.1,2,16,24–27) Manipulations of FAS2þ-1250G (1.0/6.5) < FAS2-1250A (3.4/28.4) < the ARO4, ATF1, LEU4, and FAA1 genes in sake or FAS2-1250S (4.9/36.1) < FAS2-1250C (6.1/42.2) wine yeast strains can alter the production of aromatic (Table 4). In contrast, integrants of the FAS2-1250A compounds.28–31) However, none of these strains are and FAS2-1250C plasmids showed slightly weak fer- currently commercially available. All of these GM yeast mentation ability and produced a lower amount of ethyl strains possess bacterial DNA sequences due to the use caproate (2.6 and 2.2 ppm, respectively) than the of plasmid vectors. This non-self DNA may be problem- corresponding self-cloning strains (3.4 and 6.1 ppm, atic for commercial applications.16) To solve this respectively). problem, we have constructed a recombinant yeast strain that possesses only the FAS2-1250S mutation but does not include extraneous DNA sequences.9) The FAS2-1250S mutant could also be obtained by mutant 212 K. ARITOMI et al. Table 4. Sake Proﬁle

FAS2 strains Kyokai no. 7 RAK649 RAK1988 RAK1750 RAK2075 RAK2035 Parameters Wild type Self-cloning Integrant Integrant Self-cloning Self-cloning 1250G 1250S 1250A 1250C 1250A 1250C Incubation (days) 27 25 28 34 27 27 Sake meter þ9.3 þ8.9 þ10.8 þ9.2 þ9.2 þ11.4 Acidity (ml) 1.9 1.6 1.6 1.4 1.7 1.5 Amino acid (ml) 1.0 1.0 0.9 1.3 1.0 1.0 Acetic acid (g/l) 0.24 0.11 0.15 0.11 0.11 0.09 Succinic acid (g/l) 0.07 0.08 0.08 0.05 0.08 0.09 Ethanol (%) 15.5 15.3 15.3 15.2 15.2 15.5 N Caproic acid (ppm) 6.5 36.1 25.7 27.5 28.4 42.2 Ethyl caproate (ppm) 1.0 4.9 2.6 2.2 3.4 6.1

selection in the presence of cerulenin.3) Because the incubated for 7 d on a plate containing 0.2 g/ml selected mutant yeast strains produce increased aroma, cerulenin. This implies that cerulenin recognizes and they are now commonly used in the Japanese sake binds weakly the structure around the 1250th amino acid market.6,22) Our recombinant FAS2-1250S yeast strain, of fatty acid synthase even if glycine is replaced with which is genetically identical to the mutationally alanine or cysteine, although this is not the case if selected strain, has been approved by the Japanese glycine is replaced with serine. Due to the weakness of Ministry of Health, Labor, and Welfare as a self-cloning the cerulenin resistance in these mutants, traditional yeast, and can be used freely in Japan.16) In this study, cerulenin-resistance screening might not isolate the we have constructed novel FAS2 mutant alleles, in 1250Ala and 1250Cys mutants. which the 1250th codon of FAS2 was mutated from In contrast to the cerulenin-resistant phenotype, both glycine to alanine or cysteine, and self-cloning strains 1250Ala and 1250Cys mutants elicited increased pro- containing these mutations were constructed in a sake duction of caproic acid and ethyl caproate in sake. This yeast by gene replacement.9,32) suggests that the structural change caused by a mutation The self-cloning is not only helpful for commercial of 1250Ala or 1250Cys leads to a dominant phenotype application but may be valuable for fermentation. similar to the Ser mutation with respect to the ethyl Fermentation ability of the plasmid integrants is slightly caproate production. Interestingly, the amounts of ethyl weaker than the wild type and self-cloning strains caproate corresponded to the sizes of side chain (Fig. 5). In addition, the production of ethyl caproate and structures of 1250th amino acid; the order of ethyl caproic acid was also lower than the self-cloning strains. caproate production, from low to high, was Gly (–H) < These phenotypes may be caused by the overproduction Ala (–CH3) < Ser (–CH2OH) < Cys (–CH2SH). It of YAP1, a transcription factor, used as a transformation might be possible that increasing of the size of side marker. Otherwise, it is possible to think that a gene chain structure resulted in greater inhibition of fatty acid dosage effect may affect the ethyl caproate production. synthase enzyme complex. Unfortunately, for unknown A diploid strain inserted with the FAS2 mutant plasmid reasons, we could not obtain integrants of a plasmid resulted in having one mutant and two wild type FAS2 containing FAS2-1250T, in which the glycine was alleles within a cell. The self-cloning mutants contained replaced with a threonine, an even larger amino acid. one mutant and one wild type gene. The dominant effect A possible explanation for this is that this mutation of the mutant FAS2 gene may be eliminated by an causes a greater level of inhibition of fatty acid increased dosage of the wild type FAS2. A combination synthesis, which leads to a growth defect. Further of the inhibitory effect of fatty acid synthesis by the studies for enzyme structure-activity relationship will be FAS2 mutations and the unfavorable effect of YAP1 required to examine this hypothesis. overexpression could be the cause of weakness of The strategy that we used to make useful mutant fermentation ability in plasmid integrants. Consequent- genes may be applicable to other enzymes. One ly, the results suggest that self-cloning is not only important finding in this study is that different amino helpful for commercial application but also valuable to acid mutations in Fas2 produced levels of ethyl caproate avoid unexpected negative effects caused by the plasmid in sake that did not correspond with the level of introduction. cerulenin resistance. This indicates that mutant screen- The cerulenin-resistant FAS2-1250S strains were ing using drug resistance can provide only limited resistant to >2.0 g/ml cerulenin,3,6) but the alanine strains. In contrast, in vitro site-directed mutagenesis can and cysteine FAS2 mutants constructed were less produce an unlimited number of mutants. In addition, resistant than the FAS2-1250S mutant. Resistance was the mutants constructed in this study can be used detected only when the 1250A and 1250C mutants were commercially because they lack extraneous DNA Self-cloning Sake Yeast Strains with FAS2 Mutations 213 sequences derived from other species. In fact, according T. J., McGloughlin, M. N., and Akhlaghi, A. R., Food to Japanese government guidelines, self-cloning micro- biotechnology: benefits and concerns. J. Nutr., 132, organisms do not need to be treated as a recombinant 1384–1390 (2002). microorganism in Japan.33) The first approved self- 13) Jonas, D. A., Elmadfa, I., Engel, K.-H., Heller, K. J., cloning GM microorganism is the FAS2-1250S sake Kozianowski, G., Ko¨nig, A., Mu¨ller, D., Narbonne, J. 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