Improvement of isoamyl acetate productivity in sake yeast by isolating mutants resistant to econazole

Sake yeast strains were improved so as to produce larger amounts of isoamyl acetate than the parental strain by isolating econazole-resistant mutants. Econazole, an imidazole antimycotic, directly interacts with unsaturated fatty acids in the yeast cell membrane, where it also inhibits the synthesis of ergosterol and decreases the ratio of unsaturated to saturated fatty acids. In contrast, alcohol acetyltransferase (AATase), which catalyzes the synthesis of isoamyl acetate, is inhibited by unsaturated fatty acids. Fifty econazole-resistant mutants were isolated from a sake yeast, Kyokai no. 701, several of which produced approximately 1.4 to 2.4 times more isoamyl acetate and an almost equal amount of isoamyl alcohol compared with the parental strain. The AATase activities of the mutants in koji extract were 1.2 to 1.4 times higher, and the unsaturated to saturated fatty acid ratios were lower, than in the parental strain.     

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.     

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.     

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     

YDL080C和LEU2基因敲除对工业黄酒酵母异戊醇生成量的影响

通过敲除生成途径中关键酶的编码基因,对异戊醇在工业黄酒酵母N85中的生成进行了研究。采用融合PCR技术,分别构建类丙酮酸脱羧酶基因（YDL080C）缺失组件\     

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1- and ATF2-encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1-encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1-encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester-synthesizing and ester-degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2-phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester-producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles.     

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of deltapdr5 and deltasnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity - 1) x 1443] of the deltapdr5 and deltasnq2 strains rose faster than that of HL69. On the other hand, a deltayor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the deltayor1 slowed down compared with that of CTZ21. A deltayor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the deltapdr5 and deltasnq2 strains of CTZ21. In contrast, the deltayor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.     

利用酒精废糟液无蒸煮发酵生产酒精的研究

通过对几种酶制剂的液化、糖化能力以及几种防腐剂的抑菌效果进行了试验选择性比较 ,确定了在原料无蒸煮废液全回用的状态下 ,酶制剂C的适宜浓度是 0 1g酶蛋白 / 10 0g原材料 ,防腐剂B的适宜浓度是 10 0× 10 -6。在以玉米粉为原料的无蒸煮废液全回用发酵试验中 ,发酵 9天后的乙醇浓度达到 9 0 %v/v ,且酒精收得率可达到 46 5mL/kg以上的极好水平。     

一株耐高浓度乙醇酒精酵母的筛选及发酵特性的研究

为获得乙醇耐受性较高的酵母菌,通过富集培养,从白酒窖池的酒糟中分离筛选出一株耐18%vol乙醇浓度的菌株A2。其最适生长温度为32℃,最适pH值为4.5。通过正交试验得出A2的最优发酵条件为葡萄糖浓度20%(w/v),温度34℃,pH值为4.5。发酵72h后,发酵液酒精浓度达到9.5%vol。     

Effect of short‐term anaerobic conditions on the production of volatiles,activity of alcohol acetyltransferase and other quality traits of ripened bananas

Yellow, ripened (yellow with green tips) bananas were treated with nitrogen gas for 12, 24 and 48 h at 20 °C to study the effects of anaerobic conditions on the fruit quality, including the production of volatile compounds and activity of alcohol acetyltransferase (AAT) during the post‐harvest period. Significantly higher concentrations of acetaldehyde and ethanol were found in the treated fruit than in the untreated bananas after removal from the conditions. The 48‐h treatment showed higher levels of ethanol and acetaldehyde after storage. Immediately after releasing the fruit from the anaerobic atmosphere, the amounts of isobutyl acetate and isoamyl acetate decreased. The production of ethyl acetate increased markedly in each treatment unit 1 day after removing the fruit. Regardless of recovery from the production of isobutyl acetate and isoamyl acetate 1 day after treatment, the banana‐like aroma was denatured because of the production of high levels of ethyl acetate and ethanol. The increase in AAT was slightly lower in nitrogen‐treated fruit than untreated fruit. However, the activity in treated fruit was sufficient for ester production in bananas. The endogenous alcohol levels were increased during nitrogen gas treatment in the fruit due to the inhibition of ester production. Colour development of the bananas was greatly inhibited by the treatments, while the sugar content did not show any differences between the treated and untreated fruit. The results suggest that, even if bananas are removed from the short‐term anaerobic conditions, a loss in quality, except sweetness, easily occurs afterwards, especially the characteristic aroma of the fruit during the post‐harvest period. Copyright © 2006 Society of Chemical Industry     

Variability of the lipolytic activity and volatile molecules production by a strain of Yarrowia lipolytica in pork fat and its dependence on environmental conditions

The effects of selected variables, i.e. temperature, water activity and yeast inoculation level, on the lipolytic pattern and volatile production by Yarrowia lipolytica Y16A (chosen on the basis of a previous screening) were assessed. The variables were varied according to a central composite design and the models obtained enabled evaluation and weighting of the effects of the independent variables on the free fatty acids (FFAs) and volatile profiles in pork fat based medium. The polynomial models showed the levels temperature, water activity of the pork fat based system and yeast strain inoculation were able to maximize the release of specific FFAs or molecules of sensory importance.     

复合诱变对酵母产脂的影响及检测方法的研究

采用紫外、微波以及紫外和微波复合诱变,对假丝酵母进行诱变处理。结果显示,不同的处理方法效果不同。与紫外、微波诱变相比,复合诱变在提高油脂产量、产油率方面效果较好,通过复合诱变获得1株油脂高产菌株B2。在最佳条件下,菌株的生物量、油脂产量最大提高率分别为38.99%和35.62%。同时对诱变中所用的检测方法进行了研究,找出了一种适于高产油脂菌株筛选的快速、准确、简便的检测方法。     

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of Δpdr5 and Δsnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity-1) × 1443] of the Δpdr5 and Δsnq2 strains rose faster than that of HL69. On the other hand, a Δyor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the Δyor1 slowed down compared with that of CTZ21. A Δyor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the Δpdr5 and Δsnq2 strains of CTZ21. In contrast, the Δyor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.     

Effect of extraction conditions on the yield and purity of apple pomace pectin precipitated but not washed by alcohol

ABSTRACT:  A study of the influence of extraction conditions (pH: 1.5 to 2; temperature: 80 to 90 °C; extraction time: 1 to 3 h), on the yield and purity of apple pomace pectin without elimination of impurities by alcohol washing was carried out. The alcohol precipitate yields varied from 2.9% to 8.9% depending on the pH. At pH 1.5, these yields were higher than those obtained at pH 2 contrary to the galacturonic acid purity (%w/w). Compounds other than pectins were solubilized from the cell walls of apple pomace at pH 1.5, and they were precipitated with alcohol. The apple pectins obtained from the different extraction procedures were highly methylated (54.5% to 79.5%), especially when the conditions (temperature, pH) were drastic. Similar conclusions can be drawn for the neutral sugar content that decreased at pH 1.5 (arabinose, xylose, and galactose) or at the highest temperatures and extraction times (arabinose and galactose). The phenomenon of demethylation and pectin degradation of neutral sugars chains can be observed at acid pH, and for long extraction times. The presence of high quantities of mannose or fructose, glucose, and xylose in the alcohol precipitate showed that pectin precipitation with ethanol was not specific.     

Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.     

The maximum take-up velocity as a spinnability criterion and its dependence on the composition of coagulation bath,illustrated by an example of sodium alginate

We present the results of a study of sodium alginate solutions spinnability in a coagulation bath containing CaCl₂ and HCl. The spinnability is estimated by means of the measurement of the maximum take-up velocity (MTUV) of the fiber at the time of breaking. We have demonstrated that MTUV represents a function of parameters associated with the peculiar features of coagulation. A simplified relation is suggested which allows to consider MTUV as spinnability criterion.     

Processing yeast to reduce its nucleic acid content. Induction of intracellular RNase action by a simple heat-shock procedure,and an efficient chemical method based on extraction of RNA by salt solutions at low pH

Autolytic breakdown of the RNA of Saccharomyces cerevisiae can be induced by subjecting a suspension of yeast in NaCl solution at pH 6 to heat shock, produced by bringing the suspension to 50°C and then adding enough water at 100°C to increase the temperature momentarily to 68°C, followed after 1 min by the addition of cold water to reduce it to 50°C. During continued incubation of the suspension (60 g yeast solids and 0.5 mol NaCl/litre) at 50°C, the rate of degradation of yeast RNA is as rapid as when autolysis is triggered by treatment of the yeast with organic solvents, or by drying and rehydration. The procedure is compared with an efficient chemical method for reduction of RNA content, by which SCP can be produced with a protein/nucleic acid ratio of over 100. This is based on heating a suspension of yeast in 0.5 M-NaCl at pH 1.4 to 70°C for a period of 2 h. This technique can be applied to yeast from which low-molecular weight constituents have first been removed by extraction with water at 70-100°C, with a considerable reduction in nuisance due to odours.