Improved production of isoamyl acetate by a sake yeast mutant resistant to an isoprenoid analog and its dependence on alcohol acetyltransferase activity, but not on isoamyl alcohol production

1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, strongly inhibited the growth of sake yeast at 120 microM in YPD medium, whereas at 30 microM it reduced cellular production of isoamyl acetate to 20% of the control level despite the absence of inhibitory effect on CO2 evolution. The FPy-resistant mutant A1 was characterized by the high production of flavor compounds represented by a nearly threefold increase in the level of isoamyl acetate in YPD medium in which the level of isoamyl alcohol as its precursor remained almost unchanged. The FPy resistance phenotype of strain A1 was not accompanied by cellular resistance to either the L-leucine analog or L-canavanine, which alters yeast amino acid metabolism in favor of isoamyl alcohol production. Alcohol acetyltransferase (AATase) activity was high in strain A1, which further increased in response to isoamyl alcohol accumulation in medium. Flavor compound production in sake brewing could be improved using strain A1, resulting in a 1.4-fold increase in isoamyl acetate production in spite of a limited production of isoamyl alcohol.     

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Screening of drug‐resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)‐resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched‐chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long‐term in a VPA‐containing medium. This strain, K7‐VPA^LS, is significantly more resistant to not only VPA‐induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7‐VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7‐VPA^LS‐brewed sake has milder sourness and higher saltiness or richness than K7‐brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7‐VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7‐VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7‐VPA^LS shows improved characteristics for brewing of high‐quality sake. Copyright © 2017 The Institute of Brewing & Distilling     

Isolation of a Copper‐resistant Sake Yeast Mutant with Improved Flavour Compound Production

The sake (traditional Japanese alcoholic beverage) yeast mutant A1 was previously isolated as a strain resistant to an isoprenoid analog. This strain is used for industrial sake brewing because of its increased production of isoamyl acetate. In this study, a physiological event was identified which was closely related to the elevation of alcohol acetyltransferase (AATase) activity in strain A1. This finding was applied for the isolation of another mutant with an improved capacity for flavour compound production. Strain A1 revealed an additional phenotype showing resistance to Cu²⁺, as seen from its growth and isoamyl acetate production, even in a medium with the copper ion at 6 mM. Mutant strains were successfully isolated with increased isoamyl acetate production capacity from sake yeast strain 2NF on the basis of a Cu²⁺‐resistant phenotype at a high yield. Among them, strain Cu7 was characterized by its ability to produce isoamyl acetate at the highest concentration under condition where isoamyl alcohol (its precursor) was accumulated to the lowest extent. Such a phenotype of strain Cu7 is applicable for the practical production of an alcoholic beverage of excellent quality in terms of flavour.     

Isolation and characterization of the ATF2 gene encoding alcohol acetyltransferase II in the bottom fermenting yeast Saccharomyces pastorianus

The ATF2 gene encodes alcohol acetyltransferase II, which catalyses the synthesis of isoamyl acetate from acetyl coenzyme A and isoamyl alcohol. To characterize the ATF2 gene from the bottom fermenting yeast Saccharomyces pastorianus, the S. pastorianus ATF2 gene was cloned by colony hybridization using the S. cerevisiae ATF2 gene as a probe. When an atf1 null mutant strain was transformed with a multi-copy plasmid carrying the S. pastorianus ATF2 gene, the AATase activity of this strain was increased by 2.5-fold compared to the control. The S. pastorianus ATF2 gene has 99% nucleic acid homology in the coding region and 100% amino acid homology with the S. cerevisiae ATF2 gene. Southern blot analysis of chromosomes separated by pulse-field gel electrophoresis indicated that the ATF2 gene probe hybridized to chromosome VII in S. cerevisiae and to the 1100 kb chromosome in S. pastorianus. As S. pastorianus is thought to be a hybrid of S. cerevisiae and S. bayanus, the S. bayanus-type gene, which has a relatively low level of homology with the S. cerevisiae-type gene, is also usually detected. Interestingly, an S. bayanus-type ATF2 gene could not be detected. These results suggested that the cloned ATF2 gene was derived from S. cerevisiae. Analysis using an ATF2-lacZ fusion gene in S. pastorianus showed that expression of the ATF2 gene was relatively lower than that of the ATF1 gene and that it is repressed by aeration but activated by the addition of unsaturated fatty acids. The S. pastorianus ATF1, Lg-ATF1 and ATF2 Accession Numbers in the DDBJ Nucleotide Sequence Database are D63449, D63450 and D86480, respectively.     

Sake: Production and flavor

Abstract

Sake, its history in Japan, the unique brewing process, and microbes concerned with the characteristics of flavor are described. The main flavor components derived mainly from the fermentation process are higher alcohols, isoamyl acetate, ethyl caproate, and phenethyl acetate. These are the compounds which give an accent to the flavor of sake. The esters are formed mainly by yeast during mash fermentation. Isoamyl acetate is produced by the reaction of acetyl CoA with isoamyl alcohol catalyzed by alcohol acetyl transferase. The enzyme, bound to the yeast cell membrane, is unstable to heat and unsaturated fatty acids. The ester formation is regulated by the amount of isoamyl alcohol produced. Acyl CoA alcohol acyl transferease catalyzes the formation of ethyl caproate from caproyl CoA and ethanol. In this reaction, the amount of caproyl CoA supplied is important. The mechanism of higher alcohol formation, including the biosynthetic pathway of amino acids and its feedback regulation, is discussed. Finally, breeding and the practical use of sake yeast with high productivity of higher concentrations of higher alcohols and esters are described.     

Effects of overexpression of the alcohol acetyltransferase‐encoding gene ATF1 and disruption of the esterase‐encoding gene IAH1 on the flavour profiles of Chinese yellow rice wine

Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis. On the other hand, isoamyl acetate is degraded into a higher alcohol under the catalysis of IAH1‐encoded esterase. In this study, Chinese Saccharomyces cerevisiae was used as the parent strain to construct an ATF1 overexpression and IAH1 disruption mutant. The results show that after 5 days of pre‐fermentation, the concentrations of ethyl acetate, isoamyl acetate and isobutyl acetate in the yellow rice wines fermented with EY1 (pUC‐PIAK) increased to 468.94 mg L^?1 (which is approximately 22‐fold higher than that of the parent cell RY1), 99.86 and 7.69 mg L^?1 respectively. Meanwhile, isoamyl alcohol production was reduced to 56.37 mg L^?1 (which is approximately 50% of that produced by the parent strain RY1). Therefore, ATF1 overexpression and IAH1 disruption can significantly increase acetate esters contents and reduce isoamyl alcohol content in Chinese yellow rice wine, thereby paving the way for breeding an excellent yeast strain for high‐quality Chinese yellow rice wine production.     

LIPID COMPOSITION OF AEROBICALLY AND ANAEROBICALLY PROPAGATED BREWER'S BOTTOM YEAST

Aerobically grown pitching yeast is very rich in unsaturated fatty acids and sterol esters compared to traditional, anaerobic yeast. The principal fatty acids in aerobic yeast cells are unsaturated palmitoleic and oleic acids, whereas in anaerobic cells saturated palmitic acid predominates. The difference in fatty acid distribution between aerobic and anaerobic cells is most marked in the sterol esters. The fatty acids of phospho-lipids are more stable, although remarkable differences are observed. The sterols of aerobic cells are almost entirely in esterified form and zymosterol is the principal sterol. During the first hours of fermentation a rapid synthesis of palmitoleic acid is observed when anaerobic yeast is used for pitching and the wort is aerated. The synthesis of oleic acid requires more oxygen and time than is available under normal brewing conditions. When aerobic pitching yeast is used no more unsaturated fatty acids are synthesised and the lipid stores of pitching yeast are distributed among the daughter cells. The decrease in acetate ester production by aerobic pitching yeast is concluded to be due to a decrease in acetyl CoA synthesis, which may be caused by the high proportion of unsaturated fatty acids in membrane lipids.     

The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis

For beer wort fermentation the addition of unsaturated fatty acids has sometimes been suggested as an alternative to wort oxygenation. This can however negatively affect the synthesis of acetate esters and consequently beer flavour. This work investigates the effect of supplementing a cropped yeast with an unsaturated fatty acid on the fermentation performance of the pitching yeast. Cropped yeast is in a different physiological state to yeast pitched in unfermented wort. Using a synthetic medium for the fermentations, it was found that the incubation of cropped yeast with linoleic acid resulted in two important changes in the yeasts composition: (1) the ratio of unsaturated fatty acids to total fatty acids increased from 0.53 to 0.66 and (2) the ratio of trehalose to glycogen increased from 0.17 to 0.49. The performance of this yeast in subsequent fermentations was compared to unsupplemented yeast under three conditions: medium pre‐aeration, de‐aerated medium and de‐aerated medium with newly added unsaturated fatty acid. It was found that the supplemented pitching yeast showed growth, attenuation and ethanol formation profiles similar to those obtained with unsupplemented yeast in pre‐aerated medium, which simulated the normal brewing practice. Compared to fermentations with unsaturated fatty acids added to the medium, the supplemented cropped yeast did not induce a reduction in acetate ester synthesis. Results indicated that the supplementation of cropped yeast with unsaturated fatty acids could be an interesting alternative to wort oxygenation to restore the optimal membrane fluidity of the yeast.     

Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains

Esters and higher alcohols produced by yeast during the fermentation of wort have the greatest impact on the smell and taste of beer. Alcohol acetyltransferase, which is mainly encoded by the ATF1 gene, is one of the most important enzymes for acetate ester synthesis. Cytosolic branched-chain amino acid aminotransferase, on the other hand, which is encoded by the BAT2 gene, plays an important role in the production of branched-chain alcohols. The objective of this study is to construct engineered brewer’s yeast strains that produce more acetate esters and less higher alcohols. Industrial brewer’s yeast strain S5 was used as the parental strain to construct ATF1 overexpression and BAT2 deletion mutants. The engineered strains S5-2 and S5-4, which feature partial BAT2 allelic genes replaced by the constructed ATF1 overexpression cassette, were obtained. The ester production of the engineered strains was observed to increase significantly compared with that of the parental cells. The concentrations of ethyl acetate produced by the engineered strains S5-2 and S5-4 increased to 78.88 and 117.40 mg L^?1, respectively, or about 7.7-fold and 11.5-fold higher than that produced by parental S5 cells. The isoamyl acetate produced by S5-2 and S5-4 also increased to 5.14 and 9.25 mg L^?1, respectively; by contrast, no isoamyl acetate was detected in the fermentation sample of the parental strain S5. Moreover, S5-2 and S5-4, respectively, produced about 65 and 51 % of higher alcohols produced by the parental strain. The increase in acetate ester content and decrease in higher alcohol concentration shown by the engineered brewer’s yeast strains at the end of fermentation process indicate that the new strains are useful in future developments in the wheat beer industry.     

Using promoter replacement and selection for loss of heterozygosity to generate an industrially applicable sake yeast strain that homozygously overproduces isoamyl acetate

By application of the high-efficiency loss of heterozygosity (HELOH) method for disrupting genes in diploid sake yeast (Kotaka et al., Appl. Microbiol. Biotechnol., 82, 387–395 (2009)), we constructed, from a heterozygous integrant, a homozygous diploid that overexpresses the alcohol acetyltransferase gene ATF2 from the SED1 promoter, without the need for sporulation and mating. Under the conditions of sake brewing, the homozygous integrant produced 1.4 times more isoamyl acetate than the parental, heterozygous strain. Furthermore, the homozygous integrant was more genetically stable than the heterozygous recombinant. Thus, the HELOH method can produce homozygous, recombinant sake yeast that is ready to be grown on an industrial scale using the well-established procedures of sake brewing. The HELOH method, therefore, facilitates genetic modification of this rarely sporulating diploid yeast strain while maintaining those characteristics required for industrial applications.     

Isolation of sake yeast mutants producing a high level of ethyl caproate and/or isoamyl acetate

In the case of sake, ethyl caproate and isoamyl acetate are considered to be closely associated with flavor. Various mutant yeast strains producing a higher level of flavor compounds (ethyl caproate and/or isoamyl acetate) than the parent strain were isolated by ethyl methane sulfonate treatment followed by global selection. Two of the mutants obtained also showed a high malate productivity. These mutants would be promising for practical sake fermentation.     

Breeding of a high tyrosol‐producing sake yeast by isolation of an ethanol‐resistant mutant from a trp3 mutant

A novel breeding strategy for a high tyrosol‐producing sake yeast was developed by isolating an ethanol‐resistant mutant from a tryptophan auxotrophic mutant of a sake brewery yeast. Since tyrosol has antioxidant, cardioprotective and taste‐sharpening effects, increasing the tyrosol level of alcohol beverages could be beneficial in alcohol production. Since the transporters of aromatic amino acids are degraded by several stresses and mutants defective in the synthesis of aromatic amino acids are sensitive to ethanol, it was hypothesized that the degradation of these transporters should be inhibited in ethanol resistant mutants isolated from the auxotrophic mutants of aromatic amino acids, and that the uptake of aromatic amino acids would be increased in the mutants. Consistent with this hypothesis, sake was brewed with the ethanol‐resistant mutant of a tryptophan auxotrophic mutant and the sake was found to contain a lesser content of tyrosine and a higher content of tyrosol relative to the sake brewed with the parental strains. The taste of the sake brewed with the mutant strain could be discriminated from the sake brewed with the parental strains, probably because of the altered concentrations of tyrosol and certain amino acids and organic acids. The results suggest that combining the isolation of an ethanol‐resistant mutant and an auxotrophic mutant is an effective method to breed a brewing strain with a modified metabolism of these substances. Copyright © 2012 The Institute of Brewing & Distilling     

Nitrogen addition influences formation of aroma compounds,volatile acidity and ethanol in nitrogen deficient media fermented by Saccharomyces cerevisiae wine strains

The effects of nitrogen addition into nitrogen deficient/depleted media on the release of aroma compounds post-fermentation were investigated in three commercial yeast strains of Saccharomyces cerevisiae which highlight the yeast strain effect as well as nitrogen effects. By comparing the two timings of nitrogen addition, prior to fermentation or later at stationary phase (72 h), it was shown that nitrogen addition at stationary phase significantly decreases ethanol and acetic acid formation and significantly increases the following compounds: 2-phenylethanol, ethyl isobutyrate, 2-phenylethyl acetate, ethyl 2-methylbutyrate and ethyl propionate in the three strains, and also isovaleric acid, isoamyl alcohol and ethyl isovalerate in both PYCC4072 and UCD522. The strain EC1118 produced significantly less medium chain fatty acids, hexanoic, octanoic and decanoic acids and their respective esters after nitrogen addition. Therefore, timing of nitrogen addition to a ferment media can vary the concentration of certain aroma compound and might provide a means for varying wine composition.     

CHEMICAL CHARACTERIZATION AND STEREOSPECIFIC ANALYSIS OF LIPIDS SYNTHESIZED BY CERTAIN YEAST STRAINS

Fatty acid analysis of lipids from yeast strains, Apiotrichum curvatum ATCC 10567, Cryptococcus albidus ATCC 56297, Lipomyces starkeyi ATCC 12659, and Rhodosporidium toruloides ATCC 10788, grown on whey permeate revealed palmitic, stearic, oleic and linoleic acids as the predominant fatty acids in the triacylglycerol fractions. The phospholipid fractions were dominated by oleic and linoleic acids. The stereospecific distribution of fatty acids in the triacylglycerol fractions of lipids from two yeast strains, Apiotrichum curvatum ATCC 10567 and Lipomyces starkeyi ATCC 12659, was determined and revealed the presence of almost entirely unsaturated fatty acids at the sn-2 position (91.9% and 82.8%, respectively). The dominance of unsaturated fatty acids (in the range of 61.5 to 72.3%) at the sn-1 position was also observed. Oleic acid was the predominant fatty acid at positions sn-1 and sn-2 for both yeast strains. Position sn-3 had the greatest concentrations of saturated fatty acids, with palmitic acid as the predominant fatty acid.     

EFFECTS OF LINOLEIC ACID SUPPLEMENTS ON THE SYNTHESIS BY YEAST OF LIPIDS AND ACETATE ESTERS

The concentrations of acetate esters in beer were reduced by up to 85% by addition of linoleic acid to the fermentation or by pitching with yeast previously enriched with this unsaturated fatty acid. Linoleic acid was rapidly incorporated into yeast lipids and was effective in reducing the rate of ethyl acetate formation within 2 h. Addition of linoleic acid altered the pattern of synthesis of fatty acids by yeast, causing a shift from medium toward long chain acids. Secondly, the amount of squalene in yeast was reduced by up to 70% whereas that of lanosterol was increased threefold. Since total yeast lipid synthesis was reduced by up to 40%, we conclude that less acetylCoA is synthesized in the presence of linoleic acid. Further, high concentrations of linoleic acid decreased the proportion of acetylCoA consumed by the synthesis of acetate esters. Therefore linoleic acid may directly decrease acetate ester synthesis in addition to its effect via reduction of acetylCoA availability.     

SPME-GC-MS分析不同商业葡萄酒酵母香气物质合成的差异性

采用顶空固相微萃取(HS-SPME)和气质联用法(GC-MS)分析了4株常用商业葡萄酒酵母的挥发性香气成分。对酵母代谢的19种香气成分进行定性定量分析,菌株CY3079具有较高的高级醇生成能力,其中异戊醇和2-苯乙醇的合成均高于其它酵母菌株。D254酵母具有较高的酯类和挥发酸类合成能力,其中乙酸乙酯和乙酸对酵母D254总酯和总挥发酸的含量贡献较大。EC1118高级醇生成量最低,但是具有较高的乙酸-2-苯乙酯生成能力。RC212酒样具有较低的酯类和挥发酸。感官品评试验表明,D254酒样的果香比较明显;CY3079酒样的刺激性大于其它菌株;EC1118酒样花香比较突出。     

REDUCTION OF HIGHER ALCOHOLS BY FERMENTATION WITH A LEUCINE-AUXOTROPHIC MUTANT OF WINE YEAST

Several auxotrophic mutants requiring branched chain amino acids (valine, leucine, or isoleucine) were isolated in a strain of Montrachet wine yeast. They were tested for their ability to produce lowered amounts of higher alcohols (‘fusel oil’: isobutyl, active amyl, and isoamyl alcohols) in grape juice fermentations. One strain which required leucine was especially good in this respect. This mutation is recessive and is the result of a deficiency for the enzyme α-isopropylamate dehydratase. In trial fermentations with this mutant, the resulting wines contained up to 20% less total fusel oil and 50% less isoamyl alcohol compared to the parent Montrachet strain. An experienced taste panel did not discern any gross degradation of taste quality in wine made with the mutant strain compared to that made with the parent strain. The mutant strain could be of commercial importance in preparation of distilling material for alcoholic beverages since the reduced fusel oil content would not require any special distillation procedures which are normally used to avoid the unpleasant flavour associated with concentrated higher alcohols. Reduction of the isoamyl alcohol content is particularly significant since this fusel oil component is usually present in the highest amount.     

Production of alcohol‐free beer with elevated amounts of flavouring compounds using lager yeast mutants

Arrested or limited fermentation process is a widespread method used for production of alcohol‐free beer (AFB). However, it usually leads to worty off‐flavour and a lack of pleasant fruity flavour/aroma. The aim of this study was to isolate spontaneous mutants of Saccharomyces pastorianus strain 2 resistant to 5,5,5‐trifluoro‐dl ‐leucine, since the resistance is related to overproduction of flavour active isoamyl alcohol (IAAL) and isoamyl acetate (IAAC). The mutants were the subject of selection during series of screening tests aimed at selecting the best producer of target compounds (IAAL and IAAC). Subsequently, the overproduction stability of the selected mutant strain was verified in four consecutive fermentations of AFBs. A higher pitching rate and increased fermentation temperature did not result in an overall improvement in the production of the target compounds. Sensory analysis proved the flavouring effect of the spontaneous lager yeast mutant on the AFB produced by arrested fermentation. Copyright © 2013 The Institute of Brewing & Distilling