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 ( 6 6) 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-Á200 leu2-Á1 trp1-Á1 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.