Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

Reduced production of ethyl carbamate for wine fermentation by deleting <Emphasis Type="Italic">CAR1</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.     

Breeding of a non-urea producing sake yeast with killer character using a kar1-1 mutant as a killer donor

Arginase-deficient (car1/car1) sake yeasts can brew sake without urea, a main precursor of ethyl carbamate, which is a suspected carcinogen in various fermented beverages. For the use of car1/car1 yeasts in sake production, contamination by wild-type (CAR1/CAR1) yeasts is a major problem. To protect sake mash against such contamination, killer character was introduced into the car1/car1 sake yeast HL163 by rare mating and protoplast fusion, using a kar1–1 haploid harboring killer dsRNA plasmids as a killer donor. All killer yeasts obtained showed no arginase activity and the same DNA content per cell as strain HL163, and produced sake with ordinary quality and very low levels of urea. We also demonstrated that one of these killer yeasts could effectively eliminate contaminant cells of a CAR1/CAR1 yeast from sake mash. Journal of Industrial Microbiology & Biotechnology (2000) 24, 203–209. Received 09 August 1999/ Accepted in revised form 07 December 1999     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO₂ production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.     

Non-foaming Mutants of Sake Yeasts

Two selection methods of non-foaming mutants of sake yeasts (a kind of cell wall mutants lacking the ability to form froth head in sake mash) are described. The mutants, being different in both the affinity to gas bubble and in the agglutinability from the parent, were concentrated, by removing the wild type cells with froth in froth flotation method and by removing them by agglutination caused by lactobacillus cells in cell agglutination method. Spontaneous non-foaming mutants of Kyokai No. 7 strain were isolated from the concentrates after 9 successive trials of each selection procedure at the rates of 50% by the former and 81% by the latter. The UV-induced mutants were also isolated from the concentrates after the 7 successions at the rates of 80% and 100%, respectively, by the former and by the latter. There were two types among the non-foaming mutants with respect to the agglutinability; the one was non- or almost non-agglutinable type (type 1) and the other was weakly-agglutinable one (type 2). The spontaneous mutants isolated by the froth flotation method were all of type 2, while 54% of those isolated by the cell agglutination method was of type 1 and the rest was type 2. On the other hand, only type 1 was found with the UV-induced mutants. It is inferred from the concentration rate determined by the froth flotation method in a model experiment that the mutation would occur spontaneously at a rate of 10^?8 and be stimulated about 100 fold by the UV-irradiation in Kyokai No. 7 strain. The usefulness of the non-foaming mutants are discussed from a practical point of view for sake production.     

History,lineage and phenotypic differentiation of sake yeast

ABSTRACT

Sake yeast was first isolated as a single yeast strain, Saccharomyces sake, during the Meiji era. Yeast strains suitable for sake fermentation were subsequently isolated from sake brewers and distributed as Kyokai yeast strains. Sake yeast strains that produce characteristic flavors have been bred in response to various market demands and individual preferences. Interestingly, both genetic and morphological studies have indicated that sake yeast used during the Meiji era differs from new sake yeasts derived from Kyokai Strain No. 7 lineage. Here, we discuss the history of sake yeast breeding, from the discovery of sake yeast to the present day, to highlight the achievements of great Japanese scientists and engineers.     

Sake Yeast Suppresses Acute Alcohol-Induced Liver Injury in Mice

Brewer’s and baker’s yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methonine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.     

Histone deacetylases in sake yeast affect fermentation characteristics

ABSTRACT

Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3Δ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3Δ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1 may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation.

Abbreviations: HDAC: Histone deacetylase; HAT: histone acetyltransferase; K701: sake yeast Kyokai No. 701; PCR: polymerase chain reaction; HPLC: high performance liquid chromatography; E/A: Ester/Alcohol; BCAA: branched chain-amino acid; Atf: alcohol acetyltransferase.     

Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.     

LAMMER kinase contributes to genome stability in Ustilago maydis

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1^Q488*) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1^Q488* rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1^Q488* mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1^Q488* mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.     

Sake yeast suppresses acute alcohol-induced liver injury in mice

Brewer's and baker's yeasts appear to have components that protect from liver injury. Whether sake yeast, Saccharomyces cerevisiae Kyokai no. 9, also has a hepatoprotective effect has not been examined. Here we show that sake yeast suppresses acute alcoholic liver injury in mice. Male C57BL/6 mice that had been fed a diet containing 1% sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed sake yeast, ethanol-induced increases in triglyceride (TG) and glutamate pyruvate transaminase (GPT) were significantly attenuated and hepatic steatosis was improved. In addition, sake yeast-fed mice showed a smaller decrease in hepatic S-adenosylmethionine (SAM) level and a smaller increase in plasma homocysteine (Hcy) level after ethanol treatment than the control mice, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by sake yeast. These results indicate that sake yeast protects against alcoholic liver injury through maintenance of methionine metabolism in the liver.     

The role of novel genes rrp1+ and rrp2+ in the repair of DNA damage in Schizosaccharomyces pombe

We identified two predicted proteins in Schizosaccharomyces pombe, Rrp1 (SPAC17A2.12) and Rrp2 (SPBC23E6.02) that share 34% and 36% similarity to Saccharomyces cerevisiae Ris1p, respectively. Ris1p is a DNA-dependent ATP-ase involved in gene silencing and DNA repair. Rrp1 and Rrp2 also share similarity with S. cerevisiae Rad5 and S. pombe Rad8, containing SNF2-N, RING finger and Helicase-C domains. To investigate the function of the Rrp proteins, we studied the DNA damage sensitivities and genetic interactions of null mutants with known DNA repair mutants. Single Δrrp1 and Δrrp2 mutants were not sensitive to CPT, 4NQO, CDPP, MMS, HU, UV or IR. The double mutants Δrrp1 Δrhp51 and Δrrp2 Δrhp51 plus the triple Δrrp1 Δrrp2 Δrhp51 mutant did not display significant additional sensitivity. However, the double mutants Δrrp1 Δrhp57 and Δrrp2 Δrhp57 were significantly more sensitive to MMS, CPT, HU and IR than the Δrhp57 single mutant. The checkpoint response in these strains was functional. In S. pombe, Rhp55/57 acts in parallel with a second mediator complex, Swi5/Sfr1, to facilitate Rhp51-dependent DNA repair. Δrrp1 Δsfr1 and Δrrp2 Δsfr1 double mutants did not show significant additional sensitivity, suggesting a function for Rrp proteins in the Swi5/Sfr1 pathway of DSB repair. Consistent with this, Δrrp1 Δrhp57 and Δrrp2 Δrhp57 mutants, but not Δrrp1 Δsfr1 or Δrrp2 Δsfr1 double mutants, exhibited slow growth and aberrations in cell and nuclear morphology that are typical of Δrhp51.     

Inherent G418-resistance in hybridization of industrial yeasts

Eight strains of sake yeast exhibited inherent-resistance to 100 μg/ml of Geneticin (G418). Fourteen wine yeasts and 1 shochu yeast (Saccharomyces cerevisiae) and 1 miso yeast (Zygosaccharomyces rouxii) were inherent G418-sensitive. The petites converted from inherent G418-resistants by treatment with ethidium bromide retained G418-resistance (ϱ⁻ G418R), and thus were hybridized by electrofusion with the wine yeast W3 (ϱ⁺ G418S, wild type). A lag phase of 12–18 h was required prior to administration of the drug in glycerol medium when selecting G418-resistant hybridization products. Colonies were formed in the regeneration medium at a frequency of about 1 × 10⁻⁵ per used protoplasts. No growth of any parental strain (10⁶/_~10⁷ protoplasts) separately subjected to electrofusion and regeneration was observed. The hybridization products were G418-resistant “grande” strains (ϱ⁻ G418R) in which the genetic traits of parental strains had been complemented. Uninucleate cells (DAPI staining) of the hybridization products showed CHEF electrophoretic karyotypes similar to that of wine yeast, but possessed a single chromosome (approx. 320 kb) presumably from sake yeast.     

Frequent occurrence of diploid a or α sporogenous mater cells and construction of ploidy series in the sake yeast kyokai no. 9

Diploid a or α sporogenous mater cells were frequently obtained in the sake yeast Kyokai no. 9 by heat treatment followed by random spore plating. Polyploid cells were constructed between the opposite mater cells by the mass mating technique, followed by the isolation of the zygotes using a micromanipulator.     

The Awa1 gene is required for the foam-forming phenotype and cell surface hydrophobicity of sake yeast

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast.     

Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.