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Increased Ethyl Caproate Production by Inositol Limitation in Saccharomyces cerevisiae
Abstract
Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest amounts of ethyl caproate were produced in sake mash, and supplementation of inositol caused a decrease in ethyl caproate production. However, at a polishing ratio of over 90%, supplementation of inositol had no effect on ethyl caproate production. These results suggest that the use of rice with a polishing ratio of 70% results in increased ethyl caproate content in sake when limiting the inositol available to yeast. The reduction in ethyl caproate production following inositol addition was due to the decrease in its enzymatic substrate caproic acid, because the concentrations of middle chain fatty acids (MCFA), caproic acid, caprylic acid and capric acid in sake were lowered by inositol. A disruptant of the OPI1 gene, an inositol/choline-mediated negative regulatory gene, produced higher amounts of MCFA than the control strain both in the static culture and in sake mash when a sufficient amount of inositol was supplemented. Therefore, the enhancement of MCFA biosynthesis by inositol limitation was thought to be caused not by a posttranscriptional event, but predominantly by transcriptional enhancement of fatty acid biosynthetic genes. The overexpression of FAS1 considerably stimulated MCFA formation while that of ASC2, ACC1 and FAS2 genes was not effective. Co-overexpression of FAS1 and FAS2 resulted in a maximal stimulation of MCFA formation and substantially abolished the inhibitory effect of inositol on MCFA formation. These results suggest that the repression of FAS1 gene expression by inositol results in the decrease in MCFA formation. Therefore, it is presumed that the removal of inositol by polishing the rice used in sake brewing, increases the production of ethyl esters of MCFA, since high-level production of MCFA is achieved by the derepression of FAS1 transcription.
... The overexpression of both FAS1 and FAS2 led to a 30 % increase in lipid content from 4.3 to 5.6 % [27]. Studies have shown that the fatty acid synthesis genes, such as ACC1, FAS1, and FAS2, are transcriptionally repressed with phospholipid biosynthesis by the negative regulatory gene OPI1 [13][14][15][16]. Tasuku demonstrated that the deletion of the OPI1 gene in sake yeast can improve ethyl caproate production. ...
... In this study, OPI1 deletion exhibited different levels of decreased the repression effects for the three fatty acid synthesis genes (ACC1, FAS1, and FAS2) at the transcriptional level. As a [14]. The production of FAEEs by the recombinant strains α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 was increased in different degrees compared with α5. ...
... Thus, we work on the site-directed mutation of the ACC1 gene to decrease the phosphorylation and improve the production of ethyl esters in the future. Tasuku also found that the co-overexpression of FAS1 and FAS2 resulted in a maximal accumulation of ethyl caproate in sake yeast [14]. Therefore, we can attempt to improve the ethyl caproate accumulation through the co-overexpression of FAS1 and FAS2 in Chinese liquor yeast. ...
During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.
... Increased fatty acid synthesis was achieved by transforming Y700 (wild-type) and the Y700 pGAL-ACB1 strains with high-copy plasmids expressing either ACC1 (pG1M-ACC1, a gift from Keiji Furukawa, Kiku-Masamune Sake Brewing Co., Kobe, Japan [24]) or FAS1 (pJS229, a gift from Professor Hans-Joachim Schüller, Universität Greifswald, Greifswald, Germany [25]). The corresponding empty vectors were transformed to be used as controls. ...
... However, a signal generated from fatty acid metabolism may be involved in the regulation of inositol-mediated repression of Ino2p and Ino4p target genes. Wenz et al. [25] showed previously that FAS1, but not FAS2, increased FAS activity 2.8-fold when expressed from a highcopy plasmid. We therefore hypothesized that ectopic expression from a high-copy plasmid of FAS1 and ACC1, the rate-limiting enzyme in fatty acid biosynthesis, would increase fatty acid/acyl CoA ester availability and thereby reduce INO1 expression in Acb1p-depleted cells. ...
... 1:2.6 and 1:2.2 respectively ( Figure 6). Notably, overexpression in Acb1p-depleted cells of FAS2, which is unable to enhance FAS activity [25], did not reduce INO1 mRNA levels (results not shown). This suggests that the Acb1p-mediated removal of de novo synthesized acyl-CoA esters from the yeast FAS [16] can be overcome by increasing the availability of fatty acids and/or acyl-CoA esters though enhancement of de novo fatty acid biosynthesis or supplementation with fatty acids (Fig- ures 5 and 6). ...
In the present study, we have used DNA microarray and quantitative real-time PCR analysis to examine the transcriptional changes that occur in response to cellular depletion of the yeast acyl-CoA-binding protein, Acb1p. Depletion of Acb1p resulted in the differential expression of genes encoding proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), ion transport and uptake (e.g. ITR1 and HNM1) and stress response (e.g. HSP12, DDR2 and CTT1). In the present study, we show that transcription of the INO1 gene, which encodes inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline, and UAS(INO1) (inositol-sensitive upstream activating sequence)-driven transcription is enhanced in Acb1p-depleted cells. In addition, the reduction in inositol-mediated repression of INO1 transcription observed after depletion of Acb1p appeared to be independent of the transcriptional repressor, Opi1p. We also demonstrated that INO1 and OPI3 expression can be normalized in Acb1p-depleted cells by the addition of high concentrations of exogenous fatty acids, or by the overexpression of FAS1 or ACC1. Together, these findings revealed an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p mutant which is unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implied that gene expression is modulated either by the Acb1p-acyl-CoA ester complex directly or by its ability to donate acyl-CoA esters to utilizing systems.
... The content of ethyl caproate in B was significantly different from that in the other samples (P < 0.05). The content of ethyl caproate in the HB, HF, and HS of the MLF group was lower than that in the B, S, and F. This difference is related to MLF and may be related to the inhibition of the esterification of ethyl caproate (Furukawa, Yamada, Mizoguchi, & Hara, 2003). ...
... This process refers to the slow reaction of organic acids and alcohols to form esters. The acyltransferase pathway is the main synthesis pathway of ethyl hexanoate in Saccharomyces cerevisiae (Furukawa et al., 2003). The alcohol dehydrogenase pathway is the oxidation of hemiacetals to esters. ...
Luzhou-flavoured liquor is one of Chinese most popular distilled liquors. Hundreds of flavoured components have been detected from this liquor, with esters as its primary flavouring substance. Among these esters, ethyl hexanoate was the main component. As an essential functional microbe that produces ethyl hexanoate, yeast is an important functional microorganism that produces ethyl hexanoate. The synthesis of ethyl hexanoate in yeast mainly involves the lipase/esterase synthesis pathway, alcohol transferase pathway and alcohol dehydrogenase pathway. In this study, whole-genome sequencing of W. anomalus Y-1 isolated from a Chinese liquor fermentation starter, a fermented wheat starter containing brewing microorganisms, was carried out using the Illumina HiSeq X Ten platform. The sequence had a length of 15,127,803 bp with 34.56% GC content, encoding 7,024 CDS sequences, 69 tRNAs and 1 rRNA. Then, genome annotation was performed using three high-quality databases, namely, COG, KEGG and GO databases. The annotation results showed that the ko7019 pathway of gene 6,340 contained the Eht1p enzyme, which was considered a putative acyltransferase similar to Eeb1p and had 51.57% homology with two known medium-chain fatty acid ethyl ester synthases, namely, Eht1 and Eeb1. Ethyl hexanoate in W. anomalus was found to be synthesised through the alcohol acyltransferase pathway, while acyl-coenzyme A and alcohol were synthesised under the catalytic action of Eht1p. The results of this study are beneficial to the exploration of key genes of ester synthesis and provide reference for the improvement of liquor flavoured.
... During fermentation, longchain saturated fatty acids accumulate and start to inhibit acetyl-CoA carboxylase, which causes the release of unfinished MCFAs from the FAS complex [56,57]. Results show that the overexpression of the FAS1 and FAS2 fatty acid synthetic genes trigger more MCFA formation [58]. ...
A wine’s aroma profile is an important part of the criteria affecting wine acceptability by consumers. Its characterisation is complex because volatile molecules usually belong to different classes such as alcohols, esters, aldehydes, acids, terpenes, phenols and lactones with a wide range of polarity, concentrations and undesirable off-aromas. This review focused on mechanisms and conditions of the formation of individual aroma compounds in wine such as esters and higher alcohols by yeast during fermentation. Additionally, aroma losses during fermentation are currently the subject of many studies because they can lead to a reduction in wine quality. Principles of aroma losses, their prevention and recovery techniques are described in this review.
... MCFAs are produced by the fatty acid synthase (FAS) complex in a manner dependent on acetyl-CoA carboxylase (ACC1) (Ayrapaa and Lindstrom, 1977;Taylor and Kirsop, 1977;Wakil et al., 1983;Dufour et al., 2003;Marchesini and Poirier, 2003). FAS1 and FAS2 encode the beta and alpha subunits of the FAS complex, respectively, and both these genes are known to participate in MCFA and ethyl ester synthesis (Furukawa et al., 2003). In addition, a point mutation in FAS2 has been shown to enhance octanoic acid synthesis (Nagai et al., 2016). ...
A bstract
Traditional Norwegian Farmhouse ale yeasts, also known as kveik, have captured the attention of the brewing community in recent years. Kveik were recently reported as fast fermenting thermo- and ethanol tolerant yeasts with the capacity to produce a variety of interesting flavour metabolites. They are a genetically distinct group of domesticated beer yeasts of admixed origin with one parent from the “Beer 1” clade and the other unknown. While kveik are known to ferment wort efficiently at warmer temperatures, its range of fermentation temperatures and corresponding flavour metabolites produced, remain uncharacterized. In addition, the characteristics responsible for its increased thermotolerance remain largely unknown. Here we demonstrate variation in kveik strains at a wide range of fermentation temperatures and show not all kveik strains are equal in fermentation performance, flavour metabolite production and stress tolerance. Furthermore, we uncovered an increased capacity of kveik strains to accumulate intracellular trehalose, which likely contributes to its increased thermo- and ethanol tolerances. Taken together our results present a clearer picture of the future opportunities presented by Norwegian kveik yeasts and offer further insight into their applications in brewing.
... Then, acyl-CoAs are released during synthesis from the fatty acid synthesis complex, MCFAs are increased in the mash, and as a result, the MCFA ethyl ester is increased (Äyräpää & Lindström, 1977). Moreover, it is reported that the expression of EEB1 encoding acyl-CoA:ethanol O-acyltransferase (Takahashi et al., 2017) and FAS1 and FAS2 encoding fatty acid synthase (Furukawa et al., 2003) contribute to the enhancement of MCFA ethyl esters. We did not determine the reason for the increase in MCFA content in the mash made Black bars show the content of reducing sugar produced by enzyme digestion, which was estimated from the difference between the reducing sugar content after enzyme digestion and reducing sugar content in koji. ...
We investigated the contribution of the complex expressions of two α-amylases, namely acid-stable α-amylase (AS-amylase) and acid-labile α-amylase (AL-amylase), from Aspergillus kawachii to the microstructure of koji and the brewing. AL-amylase was found to be the primary contributor to the decomposition of starch in the early stage of koji making. From the middle stage, both α-amylases decomposed the starch in a coordinated manner, and at the final stage, acid-stable α-amylase and glucoamylase decomposed the starch granules. Characterization of koji prepared by the single disruptions of AS-amylase or AL-amylase genes, double disruption of both α-amylase genes, and triple disruption of two α-amylase and glucoamylase genes in A. kawachii cells indicated that both α-amylases can work in a synergistic manner to decompose starches during koji making. AL-amylase was found, for the first time, to play an important role in starch decomposition in koji. The speed of alcohol fermentation and ester contents of the fermented mash were higher the mash prepared the control strain, followed by single, double, and triple disruptants. These results indicate that the microstructure of koji plays a role in promoting alcohol fermentation and flavor development.
... During the sake fermentation trials, S. arboricola produced significantly less ethyl hexanoate than either the hybrid or S. cerevisiae wine strain M22. Increased inositol concentrations have been shown to decrease production of ethyl hexanoate during fermentation [76]. This relationship was seen in PC1 of the hybrids and suggests S. arboricola's inositol biosynthesis pathway is more active, in turn producing low ethyl hexanoate levels seen in this study. ...
The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. Hierarchical clustering heatmapping with principal component analysis for metabolic profiling was used in finding low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic profile of parental strains, while metabolic analysis suggested S. arboricola had a lower amino acid net uptake than S. cerevisiae. Additionally, this research found an increase in ethanolic fermentation from pyruvate and increased sulfur metabolism. Together, these results suggest S. arboricola is poised for in-depth metabolomic exploration in sake fermentation.
... During our sake fermentation trials, S. arboricola produced significantly less ethyl hexanoate than either the hybrid or S. cerevisiae wine strain M22. Increased inositol concentrations have been shown to decrease production of ethyl hexanoate during fermentation [75]. This relationship was seen in PC1 of the hybrids and suggests S. arboricola's inositol biosynthesis pathway is more active, in turn producing low ethyl hexanoate levels seen in this study. ...
The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. We used hierarchical clustering heatmapping with principal component analysis for metabolic profiling and found that the low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic profile of parental strains, while metabolic analysis suggested S. arboricola had a lower amino acid net uptake than S. cerevisiae. Additionally, we found an increase in ethanolic fermentation from pyruvate and increased sulfur metabolism. Together, our results suggest S. arboricola is poised for in-depth metabolomic exploration in sake fermentation.
... Medium-chain fatty acids formation entails the activity of acetyl-CoA carboxylase and the fatty acid synthase (encoded by ACC1, FAS1, and FAS2). ACC1 and FAS1, which are involved in the synthesis of medium-chain fatty acyl-CoA (MCFA-CoA), and their up-regulations, are usually accompanied with the high production of MCFAs [32]. FAT3 encodes protein required for fatty acids uptake and ELO1 involved in the elongation of fatty acids. ...
To understand the individual enological function of different unsaturated fatty acids (UFAs), the separated effects of three different UFAs, linoleic acid (LA), oleic acid (OA), and α-linolenic acid (ALA), on yeast fermentation and aroma compounds were investigated in the alcoholic fermentation of Cabernet Sauvignon wine. The results showed that, besides concentration, UFAs types could also influence fermentation process and volatiles in final wine. Low concentrations of UFAs (12 and 60 mg/L), especially LA and OA, significantly promoted fermentation activity and most volatiles when compared to the control, however, the effect became the inhibition with increasing concentrations of UFAs (120 and 240 mg/L). It was interesting to find that OA addition (12 and 60 mg/L) could generate more acetate esters (especially isoamyl acetate) in wine, while 12 mg/L LA facilitated more fatty acids formation (octanoic acid and decanoic acid). In comparison, 120 and 240 mg/L ALA produced more amount of C6 alcohols (1-hexanol) and higher alcohols (isobutyl alcohol and 2,3-butanediol). UFAs additions were unfavorable for ethyl esters formation, except for an increment of ethyl hexanoate in 12 mg/L OA wine. As a result, different aromatic profiles of wines were generated by variations of UFAs types and levels, as shown by PCA. The transcriptional data revealed that the expressions of aroma-related genes, such as BAT1, BAT2, PDC1, PDC5, PDC6, ACC1, FAS1, ATF1, EEB1, and EHT1 were correlated with aroma compounds productions in different treatments. Our data suggested that the three UFAs have different enological functions and they could generate different aromatic profiles. Thus, besides concentrations, it is essential to consider the types of UFAs when applying the strategy to adjust UFAs contents to modulate the aromatic quality of wines.
... ACC1 and FAS1 involve in the synthesis of medium-chain fatty acyl-CoA (MCFA-CoA), and their up-regulations are usually accompanied with high production of MCFA (Furukawa, Yamada, Mizoguchi, & Hara, 2003). The expressions of ACC1 and FAS1 presented similar profiles during fermentation, and reached a maximum level in the late stationary phase (Fig. 4). ...
... In strain selection, specifi c attention to the levels of expression of other genes (e.g. fatty acid synthesis genes) appears to be more relevant than the levels of expression of the EEB1 and EHT1 genes for the fi nal ethyl ester levels [35]. ...
... Noticeably, ethyl hexanoate and isoamyl acetate formation was enhanced via SSF, whereas the synthesis of these compounds was restricted via SmF of OP hydrolysate using free yeast cells. It seems that SSF overcomes limitations associated with the reduction of hexanoic acid production by liberated inositol from phytic acid hydrolysis in acid medium by repressing FASI (-subunit of fatty acid synthetase gene) expression [43] and the loss of amino acids as precursors due to some degree of degradation during hydrolysis. Noticeably, by the SSF, maximum yield and productivity of ethyl hexanoate (Table 3) surpassed the respective values in SmF of OP hydrolysate using immobilized cells by 15 and 23 folds, respectively [7]. ...
Consumer demand for natural products and the requirement for eco-friendly processes promote development of innovative processes for flavour synthesis via biotechnology. In this direction, cultivation of selected industrial yeast strain under solid state fermentation of orange peel (OP) was studied. For this purpose, autoclave sterile OP for the elimination of D-limonene and natural microflora was evaluated with regard to yeast viability, nutrient consumption and cell ability to produce flavour active compounds. Non-sterile OP was also used to follow pros and cons of the sterilization process. Yeast cells showed better growth performance under sterilized process conditions, than under non-sterilized ones. In the first case, the enhanced de novo synthesis of “fruity” esters was demonstrated (48.7, 25.2, 9.3, 6.3 and 4.5 mg/kg of fermented OP for isoamyl acetate, ethyl dodecanoate, decanoate, octanoate and phenyl ethyl acetate, respectively, after 72 h). Yeast cells exhibited accelerated synthesis of ethyl hexanoate (154.2 mg/kg OP at 48 h). Biotransformation of naturally occurring aroma compounds by yeast may be considered in this process. The proposed process, resulting in high yields of industrially important volatile aroma esters (total of ~250 mg/kg OP), could be applied to a sustainable biorefinery for the valorization of OP waste.
... Inability of free cells to synthesize ethyl hexanoate is probably correlated with the inhibitory effect of liberated inositol from phytic acid (inositol hexakisphosphate) and its hydrolysates by the action of yeast phytases (Kaur et al. 2007). The inhibitory mechanism is through the reduction of hexanoic acid production by repressing FASI (β-subunit of fatty acid synthetase gene) expression (Furukawa et al. 2003). The protective role of immobilization using Ca alginate beads against inositol can be explained by the binding affinity of the inositol polyphosphates for Ca 2+ present in the medium or after solubilization from the gel. ...
The rising trend of bioflavour synthesis by microorganisms is hindered by the high manufacturing costs, partially attributed to the cost of the starting material. To overcome this limitation, in the present study, dilute-acid hydrolysate of orange peel was employed as a low-cost, rich in fermentable sugars substrate for the production of flavour-active compounds by Saccharomyces cerevisiae. With this purpose, the use of immobilized cell technology to protect cells against the various inhibitory compounds present in the hydrolysate was evaluated with regard to yeast viability, carbon and nitrogen consumption and cell ability to produce flavour active compounds. For cell immobilization the encapsulation in Ca alginate beads was used. The results were compared with those obtained using free-cell system. Based on the data obtained immobilized cells showed better growth performance and increased ability for de novo synthesis of volatile esters of "fruity" aroma (phenylethyl acetate, ethyl hexanoate, octanoate, decanoate and dodecanoate) than those of free cells. The potential for in situ production of new formulations containing flavour-active compounds derive from yeast cells and also from essential oil of orange peel (limonene, α-terpineol) was demonstrated by the fact that bioflavour mixture was found to accumulate within the beads. Furthermore, the ability of the immobilized yeast to perform efficiently repeated batch fermentations of orange peel hydrolysate for bioflavour production was successfully maintained after six consecutive cycles of a total period of 240 h.
... The two classes of esters are formed through discrete sets of precursor compounds through the action of specific enzymes. Overexpression of ATF1 causes an increase in acetate esters, whereas overexpression of FAS1 or FAS2 increases the concentration of ethyl esters (Furukawa et al., 2003;Varela et al., 2012). Groups 1 and 4 have the highest acetate ester to ethyl ester ratio, providing insight into important phenotypic and potential genetic differences between the clusters of strains. ...
Wine has been made for thousands of years. In modern times, as the importance of yeast as an ingredient in winemaking became better appreciated, companies worldwide have collected and marketed specific yeast strains for enhancing positive and minimizing negative attributes in wine. It is generally believed that each yeast strain contributes uniquely to fermentation performance and wine style because of its genetic background; however, the impact of metabolic diversity among wine yeasts on aroma compound production has not been extensively studied. We characterized the metabolic footprints of 69 different commercial wine yeast strains in triplicate fermentations of identical Chardonnay juice, by measuring 29 primary and secondary metabolites; we additionally measured seven attributes of fermentation performance of these strains. We identified up to 1000-fold differences between strains for some of the metabolites, and observed large differences in fermentation performance, suggesting significant metabolic diversity. These differences represent potential selective markers for the strains that may be important to the wine industry. Analysis of these metabolic traits further builds on the known genomic diversity of these strains and provides new insights into their genetic and metabolic relatedness. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
... Specifically, feedback inhibition of acetyl-CoA carboxylase has been suggested to cause the premature release of unfinished MCFAs from the FAS complex (Dufour, Malcorps, & Silcock, 2003). It also has been shown that overexpression of the genes encoding the FAS complex, FAS1 and FAS2, enhances MFCA formation (Furukawa, Yamada, Mizoguchi, & Hara, 2003). Thus, lipid addition would result in an inhibition of the FAS complex causing the release of MFCAs increasing their concentration and subsequently increasing the formation of ethyl esters. ...
... Increased phospholipid metabolism in the middle and late phases of fermentation at 13 1C could also be inferred from the upregulation of DEP1, which encodes the positive regulator of the phospholipid synthesis pathway, and downregulation of OPI1, which encodes a negative regulator of phospholipid biosynthesis. The repression of OPI1 was further consistent with increased levels of medium-chain fatty acids (Table 2), and agreed with a previous report that indicated a correlation between OPI1 repression and increased content of medium-chain fatty acids (Furukawa et al., 2003). However, the higher expression of this class of genes at 13 1C compared to 25 1C was not accompanied by an increase in cardiolipin, which is an endproduct of this metabolic pathway (Table 4), but by an increase in phosphatidic acid, the obligate intermediate in the synthesis of several phospholipids, including sphingolipid (Daum et al., 1998). ...
... Increased phospholipid metabolism in the middle and late phases of fermentation at 13 1C could also be inferred from the upregulation of DEP1, which encodes the positive regulator of the phospholipid synthesis pathway, and downregulation of OPI1, which encodes a negative regulator of phospholipid biosynthesis. The repression of OPI1 was further consistent with increased levels of medium-chain fatty acids (Table 2), and agreed with a previous report that indicated a correlation between OPI1 repression and increased content of medium-chain fatty acids (Furukawa et al., 2003). However, the higher expression of this class of genes at 13 1C compared to 25 1C was not accompanied by an increase in cardiolipin, which is an endproduct of this metabolic pathway (Table 4), but by an increase in phosphatidic acid, the obligate intermediate in the synthesis of several phospholipids, including sphingolipid (Daum et al., 1998). ...
Wine produced at low temperature is often considered to have improved sensory qualities. To investigate the effects of temperature on winemaking, the expression patterns during the industrial fermentation process carried out at 13 degrees C and 25 degrees C were compared, and correlated with physiological and biochemical data, including viability, fermentation byproducts and lipid content of the cells. From a total of 535 ORFs that were significantly differentially expressed between the 13 degrees C and 25 degrees C fermentations, two significant transcription programmes were identified. A cold-stress response was expressed at the initial stage of the fermentation, and this was followed by a transcription pattern of upregulated genes concerned with the cell cycle, growth control and maintenance in the middle and late stages of the process at 13 degrees C with respect to 25 degrees C. These expression patterns were correlated with higher cell viability at low temperature. The other relevant transcriptomic difference was that several genes implicated in cytosolic fatty acid synthesis were downregulated, while those involved in mitochondrial short-chain fatty acid synthesis were upregulated in the fermentation process conducted at 13 degrees C with respect to that at 25 degrees C. These transcriptional changes were qualitatively correlated with improved resistance to ethanol and increased production of short-chain (C(4)-C(8)) fatty acids and their corresponding esters at 13 degrees C as compared to 25 degrees C. While this increase of ethyl esters may account in part for the improved sensory quality of wine fermented at 13 degrees C, it is still unclear how the esterification of the short-chain fatty acids takes place. On the basis of its strong upregulation at 13 degrees C, we propose a possible role of IAH1 encoding an esterase/ester synthase in this process.
... Hence, by altering these parameters, beneficial adaptation of the flavor profile may also be possible. In strain selection, specific attention to the levels of expression of other genes (e.g., fatty acid synthesis genes [14] ) appears to be more relevant than the levels of expression of the EEB1 and EHT1 genes for final ethyl ester levels. ...
Volatile esters are responsible for the fruity character of fermented beverages and thus constitute a vital group of aromatic compounds in beer and wine. Many fermentation parameters are known to affect volatile ester production. In order to obtain insight into the production of ethyl esters during fermentation, we investigated the influence of several fermentation variables. A higher level of unsaturated fatty acids in the fermentation medium resulted in a general decrease in ethyl ester production. On the other hand, a higher fermentation temperature resulted in greater ethyl octanoate and decanoate production, while a higher carbon or nitrogen content of the fermentation medium resulted in only moderate changes in ethyl ester production. Analysis of the expression of the ethyl ester biosynthesis genes EEB1 and EHT1 after addition of medium-chain fatty acid precursors suggested that the expression level is not the limiting factor for ethyl ester production, as opposed to acetate ester production. Together with the previous demonstration that provision of medium-chain fatty acids, which are the substrates for ethyl ester formation, to the fermentation medium causes a strong increase in the formation of the corresponding ethyl esters, this result further supports the hypothesis that precursor availability has an important role in ethyl ester production. We concluded that, at least in our fermentation conditions and with our yeast strain, the fatty acid precursor level rather than the activity of the biosynthetic enzymes is the major limiting factor for ethyl ester production. The expression level and activity of the fatty acid biosynthetic enzymes therefore appear to be prime targets for flavor modification by alteration of process parameters or through strain selection.
Isoamyl acetate and ethyl caproate are the primary aroma compounds responsible for the fruity fragrance characteristic of Ginjo sake. Simultaneous high-level production of both compounds is crucial to achieving a balanced aroma and complex flavor. Isoamyl acetate is predominantly produced by hda1∆/hda1∆ and LEU4(G516S)/LEU4(G516S), while ethyl caproate is produced in high quantities by FAS2(G1250S)/FAS2(G1250S). In this study, to maximize the production of both aroma compounds, genome editing was employed to generate sake yeast strains combining these mutations. After small-scale fermentation tests were conducted to evaluate the production of aroma compounds, we found that the isoamyl acetate-enhancing effect of hda1∆/hda1∆ was almost completely masked by FAS2(G1250S)/FAS2(G1250S). In contrast, the effects of LEU4(G516S)/LEU4(G516S) were not entirely masked by FAS2(G1250S)/FAS2(G1250S), resulting in 2.4- and 5.4-fold greater production of isoamyl acetate and ethyl caproate, respectively. This study highlights the utility of genome editing in the combinatorial breeding of sake yeast.
In UniProtKB, each protein is linked to numerous publications covering topics such as sequence, function, and structure, which are annotated manually or through automated methods. Given the vast number of proteins and literature, manual annotation is timeconsuming and labour-intensive. Although UniProtKB offers automated annotations, their quality often falls short. Therefore, developing an accurate automated classifier to identify the topics of publications associated with each protein is imperative for advancing biomedical knowledge discovery. Classifying publications in UniProtKB involves protein-publication pairs characterized by multi-label, label co-occurrence, and class imbalance, which increases complexity. This paper proposes a novel method called PubLabeler, which simultaneously considers protein description and scientific literature texts as input. PubLabeler employs the PubMedBERT model to encode input texts and integrates label co-occurrence information into the model parameters. Additionally, it uses focal loss to update parameters, allowing the model to focus more on classes with a few instances. Using newly annotated literature from Swiss-Prot in 2023 as a test set, PubLabeler achieved superior results in both micro and macro metrics, showing a 28.5% improvement in macro-F1 compared to UniProtKB's automated annotation method, UPCLASS. Furthermore, we validated PubLabeler's effectiveness in TrEMBL annotation, showcasing its comprehensive prediction results compared to TrEMBL's automated annotations. These findings highlight PubLabeler's reliability and potential to advance protein-related information extraction and knowledge discovery.
Carbonic maceration (CM) consists in placing intact grape bunches into a sealed tank to have a natural or artificially-created carbon dioxide atmosphere. No articles have been published on the comparison between CM and nitrogen maceration (NM). Therefore, the aim has been the use of alternative maceration technique NM in alternative to CM, to create the conditions of anoxia on the Gamay variety obtaining an aromatic wine as well. The results showed a higher concentration of polyphenols and anthocyanins in the macerated wines, especially in NM wine. Concerning VOCs (volatile organic compounds), the CM wine showed the highest content in esters while the total alcohol content was slightly higher in the NM wines. The CM wine had the highest content in volatile medium chain fatty acids (MCFA). E-nose measurements revealed a clear separation among the samples, and the E-nose data, in PCA computation, were generally overlapped by the PCA of VOCs analysis. The NM can be an useful technique to have new style aromatic wine in an environment and economic sustainable way.
EHT1 and EEB1 are the key Saccharomyces cerevisiae genes involved in the synthesis of ethyl esters during wine fermentation. We constructed single (Δeht1, Δeeb1) and double (Δeht1Δeeb1) heterogenous mutant strains of the industrial diploid wine yeast EC1118 by disrupting one allele of EHT1 and/or EEB1. In addition, the aromatic profile of wine produced during fermentation of simulated grape juice by these mutant strains was also analyzed. The expression levels of EHT1 and/or EEB1 in the relevant mutants were less than 50% of the wild-type strain when grown in YPD medium and simulated grape juice medium. Compared to the wild-type strain, all mutants produced lower amounts of ethyl esters in the fermented grape juice and also resulted in distinct ethyl ester profiles. ATF2, a gene involved in acetate ester synthesis, was expressed at higher levels in the EEB1 downregulation mutants compared to the wild-type and Δeht1 strains during fermentation, which was consistent with the content of acetate esters. In addition, the production of higher alcohols was also markedly affected by the decrease in EEB1 levels. Compared to EHT1, EEB1 downregulation had a greater impact on the production of acetate esters and higher alcohols, suggesting that controlling EEB1 expression could be an effective means to regulate the content of these aromatic metabolites in wine. Taken together, the synthesis of ethyl esters can be decreased by deleting one allele of EHT1 and EEB1 in the diploid EC1118 strain, which may modify the ester profile of wine more subtly compared to the complete deletion of target genes.
Wine has very rich and complex aromas but is paradoxically obtained from grape juice that has very little odor itself. This aromatic transformation is mainly due to the action of yeasts during the alcoholic fermentation. First, yeasts produce secondary metabolites that give to wines its basic “winy” characteristic. Second, some enzymatic activities may transform nonodorous compounds of certain grape varieties into varietal aromas, giving the specific characteristics of “cépage.” Depending on the yeast strain, the concentration of aromatic molecules is determined, modifying the organoleptic perception of wine. The enzymatic activities of yeast metabolism and their genetic control have been widely studied, allowing genetic selection approaches as well as molecular engineering for selecting more specialized commercial starters.
L’expression aromatique fruitée des vins rouges a été le sujet de nombreuses études qui démontrent qu’au moins une composante de cette expression est le reflet d’interactions perceptives impliquant des esters. La synthèse de ces esters peut être affectée par la levure réalisant la fermentation alcoolique mais aussi par d’autres paramètres, plus technologiques, comme le niveau de maturité de la vendange. De plus, peu d’études ont été réalisé afin de valider le rôle physiologique tenu par les esters pour la levure.Dans le but de mettre en évidence plus précisément le rôle de ces facteurs dans la synthèse des esters, des souches de levures délétés des majeures estérases ont été construites, et testées en fermentation en milieu œnologique, dans des moûts de merlot et de tempranillo récoltés à deux maturités différentes. Ainsi, la concentration en chaque esters linéaires et substitués a pu être déterminée dans chaque modalité de vinification.L’étude des différents mutants de délétion, a permis, pour la première fois, de valider le rôle des principales estérases dans la synthèse des esters linéaires chez la levure mais aussi, la mise en évidence de deux gènes, non étudiés jusqu’alors en condition œnologique, sur la synthèse des esters substitués. L’analyse de l’expression génétique de la délétion des estérases chez la levure a permis aussi de valider que ces gènes permettent une véritable stabilité physiologique de la levure en conditions stressante.L’impact du degré de maturité de la vendange a été étudié à la fois chez les vins fermentés avec une levure standard commerciale mais aussi fermentés avec une levure délétée des 4 principales estérases. Une maturité avancée, des raisins de merlot seulement, entraine une baisse de 50% de la teneur en esters linéaire avec la levure standard, ce qui n’est plus observé avec la levure mutante. Cette diminution de la concentration en esters linéaire dans ces vins de merlot de maturité avancée, est bien corrélée à une diminution de leur perception fruitée. De plus, des reconstitutions aromatiques faites dans ces matrices, ont permis de valider l’implication totale des esters dans la perception de l’arôme fruité des vins rouges réalisés avec des matrices de maturité normale. En revanche en ce qui concerne les vins de merlot de maturité avancée, il existerait d’autres composés en plus des esters qui pourraient expliquer les différences sensorielles observées dans ces vins.Enfin, une approche de transcriptomique est mise en œuvre pour tâcher d’éclairer les facteurs à l’origine des modifications de production d’esters en fonction du niveau de maturité des raisins de merlot. Il en est sorti que l’effet maturité n’existe pas seul mais est bien combiné avec l’avancée de la fermentation alcoolique. L’effet global de la matrice qui peut expliquer les différences observées entre les vins de merlot.
Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry scenario with zinc, likely involving the Zrt family of zinc transporters and involving arrestin mediated endocytosis. Reactive oxygen species were generated by copper nanoparticles that induced lipid peroxidation, altering the phosphatidylcholine and phosphatidylethanolamine membrane composition, resulting in a disfigured cell surface. Following this study, I designed experiments aimed at furthering this dissertation's focus on metabolism by describing the metabolism of a novel species, Saccharomyces arboricola, thus filling a gap in knowledge in the industrial fermentation field. Low levels of endogenous amino/organic acids separated S. arboricola from the S. cerevisiae industrial strains and their interspecific hybrids showed a mosaic metabolic profile of parental strains. Overall, my dissertation research identified mechanisms of cellulosic copper nanotoxicity that included transport, metal homeostasis, reactive oxygen species, and the cellular membrane composition. Perturbations of Saccharomyces yeast by exogenous exposure to nanocopper or by interspecific hybridization had effects on the cellular metabolism.
The production of esters is largely influenced by functional gene expressions and the nutrition status of grape must. In this study, the effects of unsaturated fatty acids (UFAs) mixture (including linoleic acid, oleic acid and linolenic acid) on volatile esters of wine fermented with T73::ATF1 (overexpressing ATF1-encoded alcohol acetyltransferases) and T73::EEB (overexpressing EEB1-encoded acyl-CoA: ethanol O-acyltransferases) were investigated in alcoholic fermentation, respectively. The inoculation of T73::ATF1 into UFAs added juice significantly increased acetate esters production (ethyl acetate, isoamyl acetate and phenylethyl acetate) compared to in the control juice. The promotion on ethyl esters biosynthesis was also found in T73::EEB1 when supplemented with low level of UFAs, but the function became inhibition with the further increment of UFAs concentration. The levels of higher alcohols, medium fatty acids and the major genes expression (ADH1, BAT1, PDC1, ACC1, FAS1 and IAH1) were changed and varied among the both strains. These results indicated that UFAs can modulate the effect of overexpressing ATF1 and EEB1 on esters biosynthesis by influencing the substrates availability. The combination of increasing ATF1 and EEB1 expressions and modulating UFAs content is a potential means to increase the esters production and improve the aromatic quality of wines.
Esters are important flavor compounds in alcoholic beverages. Although they are present in trace levels, esters play a key role in flavor formation, especially fruity flavors, in beverages. Low ester contents result in bland beer and unpleasant flavor. In this study, three recombinant strains, ethanol O-acyltransferase-encoding EEB1 overexpression strain (31194::EEB1), 2-enoyl thioester reductase-encoding ETR1 overexpression strain (31194::ETR1), and EEB1/ETR1 co-overexpression strain (31194::EEB1::ETR1), were constructed. Ethyl hexanoate productions by 31194::EEB1 and 31194::EEB1::ETR1 were all 106% higher than that by the parental strain. Further, ethyl octanoate production by 31194::EEB1 and 31194::EEB1::ETR1 were enhanced by 47% and 41%, respectively, compared to that by the parental strain 31194. However, no difference was observed between 31194::ETR1 and the parental strain in terms of ethyl hexanoate and ethyl octanoate production. This indicates that while the EEB1 overexpression in Saccharomyces pastorianus enhanced ethyl hexanoate and ethyl octanoate production, ETR1 expression level did not affect the extracellular concentration of these esters.
Sake yeast produces a fruity flavor known as ginjo-ko-which is mainly attributable to ethyl caproate and isoamyl acetate-during fermentation in sake brewing. The production of these flavor components is inhibited by unsaturated fatty acids derived from the outer layer of rice as raw material. We isolated three mutants (hec2, hec3, and hec6) with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio from a cerulenin-resistant mutant derived from the hia1 strain, which shows enhanced isoamyl acetate productivity. The hec2 mutant had the homozygous FAS2 mutation Gly1250Ser, which is known to confer high ethyl caproate productivity. When the homozygous FAS2 mutation Gly1250Ser was introduced into strain hia1, ethyl caproate productivity was increased but neither this nor intracellular caproic acid content approached the levels observed in the hec2 mutant, indicating that a novel mutation was responsible for the high ethyl caproate productivity. We also found that the expression of EEB1 encoding acyl-coenzyme A: ethanol O-acyltransferase (AEATase) and enzymatic activity were increased in the hec2 mutant. These results suggest that the upregulation of EEB1 expression and AEATase activity may also have contributed to the enhancement of ethyl caproate synthesis from ethanol and caproyl-CoA. Our findings are useful for the brewing of sake with improved flavor due to high levels of isoamyl acetate and ethyl caproate.
Wine has very rich and complex aromas but is paradoxically obtained from grape juice that has very little odour itself. This aromatic transformation is mainly due to the action of yeasts during the alcoholic fermentation. First, yeasts produce secondary metabolites that give to wines its basic 'winy' characteristic. Second, some enzymatic activities may transform non-odorous compounds of certain grape varieties into varietal aromas, giving the specific characteristics of '. cépage'. Depending on the yeast strain, the concentration of aromatic molecules is determined, modifying the organoleptic perception of wine. The enzymatic activities of yeast metabolism and their genetic control have been widely studied, allowing genetic selection approaches as well as molecular engineering for selecting more specialized commercial starters.
The objective of this study was to investigate the effect of adding selected amino acids (proline, leucine, cysteine, valine, glutamine and isoleucine) to synthetic wort on the generation of aroma compounds with respect to the transcription levels of specific genes involved in their biosynthetic pathway under brewery fermentation. The results showed that the changes in the selected amino acid levels had no eminent impact on the general course of fermentation. Addition of leucine increased the production of isoamyl alcohol and isoamyl acetate and 2-methylbutyl acetate. Adding valine and isoleucine increased the production of isobutanol and 2-methyl butanol, respectively. Overall, total higher alcohol production increased by amino acid supplementation; this effect could be associated with the upregulation of pyruvate decarboxylases and phenylpyruvate decarboxylase. Amino acid supplementation resulted in a reduction in the final total concentration of esters, especially ethyl acetate. This reduction might have been caused by the downregulation of the genes participating in ester biosynthesis. Our results demonstrate that during lager yeast fermentation, the production of aroma-active compounds can be significantly affected by changing the supply of even one amino acid.
Understanding the ways by which yeasts respond to changes in their physicochemical environment is very important in the food and beverage industries. For example, it is important for the maintenance of yeast viability and vitality in the production and utilisation of yeasts for food and fermentation processes, and it is additionally important for the control of yeasts that act as spoilage agents of foods and beverages. In the former situation, yeasts are confronted with several environmental stresses including insults caused by changes in temperature, pH, osmotic pressure, ethanol concentration and nutrient availability that individually or collectively can deleteriously affect yeast physiology. These changes may result in lowered yeast growth yield and impaired fermentation performance. In the case of food spoilage yeasts, such organisms have adapted to survive stress caused by low temperature and oxygen levels, anhydrobiosis and high salt/sugar concentrations and their effective elimination is often based on measures to counteract the inherent stress tolerance of these yeasts. Chapter 11 covers food spoilage yeasts in more detail. The present chapter describes both physiological and molecular aspects of stress on yeast cells and will focus on yeasts’ responses to changes in their environment which are pertinent in situations where survival of the yeast is both desirable (e.g. industrial fermentations) and undesirable (e.g. foods and beverages spoilage). The stresses of particular relevance for the food industry are thermostress, pH shock, osmostress, nutrient starvation, ethanol toxicity, oxidative stress, prolonged anaerobiosis, and exposure to chemical preservatives. This chapter will not review biologically related stress factors in yeasts such as cellular ageing, genotypic changes and competition from other organisms, the last of these having been dealt with in Chap. 4. Chapter 5
Because many questions arise regarding the use of immobilization technology to consistently produce a high quality beer, this
work focuses on the effects of using an immobilization matrix in the fermentation process. The aim of this study was to explore
the feasibility and potential uses of immobilization on sensorial characteristics such as color, flavor, and headspace compounds
of stout beer, when using batch fermentation. Batch production of beer was conducted as a standard ale process for stout beer
production. For the immobilized yeasts fermentation, cells were microencapsulated in alginate, by using the Thiele modulus
procedure for microcapsule design. Glucose concentration, cell multiplication, cell viability, specific gravity, pH, Brix,
and ethanol were monitored throughout the fermentation process. Both, sensorial analysis (statistic triangle tests) and instrumental
methods (gas chromatography to measure headspace compounds and visible spectrophotometer to quantify the color) were used
to evaluate characteristics of the beer that was produced from immobilized and free yeast fermentations. Free and immobilized
yeasts fermentation showed no significant difference (p > 0.05) for all variables of interest. The profile of headspace compounds was different, perhaps because of changes in yeast’s
behavior and the presence of secondary metabolites. However, immobilization did not have a significant impact on the beer
flavor, as detected by the sensorial triangle test.
KeywordsAlginate–Beer–Fermentation–Microencapsulation–Stout–Yeast
We show that the concentration of total free fatty acids (FFAs) in sake produced by yeast with high productivity of ethyl caproate could be approximated by the concentration of 2 FFAs, caproic and caprylic acids. Measurement of the total FFAs concentration by an enzymatic method proved useful for both estimating the ethyl caproate concentration in sake and also for yeast breeding.
The need to understand and control ester synthesis is driven by the fact that esters play a key role in the sensorial quality of fermented alcoholic beverages like beer, wine and sake. As esters are synthesized in yeast via several complex metabolic pathways, there is a need to gain a clear understanding of ester metabolism and its regulation. The individual genes involved, their functions and regulatory mechanisms have to be identified. In alcoholic beverages, there are two important groups of esters: the acetate esters and the medium-chain fatty acid (MCFA) ethyl esters. For acetate ester synthesis, the genes involved have already been cloned and characterized. Also the biochemical pathways and the regulation of acetate ester synthesis are well defined. With respect to the molecular basis of MCFA ethyl ester synthesis, however, significant progress has only recently been made. Next to the characterization of the biochemical pathways and regulation of ester synthesis, a new and more important question arises: what is the advantage for yeast to produce these esters? Several hypotheses have been proposed in the past, but none was satisfactorily. This paper reviews the current hypotheses of ester synthesis in yeast in relation to the complex regulation of the alcohol acetyl transferases and the different factors that allow ester formation to be controlled during fermentation.
A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.
The effect of cellular inositol content on the ethanol tolerance of sake yeast was investigated. In a static culture of strain K901 in a synthetic medium, when cells were grown in the presence of inositol in limited amount (L-cells), the inositol content of cells decreased by one-third that of cells grown in the presence of inositol in sufficient amount (H-cells). L-cells exhibited a higher death rate constant than H-cells in the presence of 12-20% ethanol, while no difference in specific ethanol production rate in the presence of 0-18% ethanol between the two cell types was observed. L-cells leaked more intracellular components, such as nucleotides, phosphate and potassium, in the presence of ethanol than H-cells. L-cells exhibited a lower intracellular pH value than H-cells, which represented the lowering of cell vitality by the decrease in H(+) extrusion activity. Furthermore, the plasma membrane H(+)-ATPase activity of L-cells was approximately one-half of that of H-cells. Therefore, it was considered that the decrease in viability in the presence of ethanol due to inositol limitation results from the lowering of H(+)-ATPase activity, which maintains the permeability barrier of the yeast membrane, ensuring the homeostasis of ions in the cytoplasm of yeast cells. It is assumed that the lowering of H(+)-ATPase activity due to inositol limitation is caused by the change in lipid environment of the enzyme, which is affected by inositol-containing glycerophospholipids such as phosphatidylinositol (PI), because in the PI-saturated mixed micellar assay system, the difference in H(+)-ATPase activity between L- and H-cells disappeared. In the early stage of sake mash, inositol limitation lowers the ethanol tolerance due to the decrease in H(+)-ATPase activity as in static culture. In the final stage of sake mash, the disruption of the ino1 gene responsible for inositol synthesis, resulted in a decrease in cell density. Furthermore, the ino1 disruptant, which was not capable of increasing the cellular inositol level in the final stage, exhibited a significantly higher methylene blue-staining ratio than the parental strain. It was suggested that the yeast cellular inositol level is one of the important factors which contribute to the high ethanol tolerance implied by the increased cell viability in the presence of ethanol.
Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest isoamyl acetate/isoamyl alcohol (E/A) ratio in sake was obtained, and inositol addition caused a decrease in E/A ratio. In several strains tested, inositol addition to the mash decreased isoamyl acetate content and E/A ratio in sake Inositol addition significantly decreased alcohol acetyltransferase (AATase) activity which is responsible for the synthesis of acetate esters from alcohols and acetyl coenzyme A. The results of Northern blot analysis and disruption of the OPII gene, an inositol/choline-mediated negative regulatory gene, showed that the decrease in AATase activity following inositol addition is not due to a transcriptional event. Inositol addition increased phosphatidylinositol (PI) content 3-fold in sake mash yeast cells, while it had no effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl-serine (PS) contents. When cell-free extracts prepared from sake mash yeast cells were treated with chloroform or phospholipase C to remove PI, no difference in AATase activity in sake mash between with (Ino+) and without (Ino-) inositol addition was observed. PI prepared from sake mash yeast cells inhibited AATase activity more strongly than PC and PE. Furthermore, when PI, PC, PE and PS at a ratio (1.0:1.28:0.70:0.09) corresponding to the phospholipid composition of Ino+ sake mash yeast cells were added to a reaction mixture, the AATase activity decreased to 26-55% that of yeast cells from the Ino- mash with a phospholipid composition of 0.34:1.28:0.7:0.09. Approximately all of the PI was recovered in the ammonium sulfate precipitate of the cell-free extract, while only half of the PC and PE was recovered. The acidic phospholipid, phosphatidylglycerol, as well as PI inhibited AATase activity more strongly than PC, despite its having the same fatty acid composition as PC. These results suggest that the strong inhibition of AATase activity by PI is due to its high adsorptive capacity for the AATase protein. Therefore, rice polishing can remove inositol from rice leading to an increase in AATase activity, and resulting in a high E/A ratio in sake.
The abnormal accumulation of lipids due to myo-inositol deficiency in Saccharomyces carlsbergensis, and the mechanism involved was investigated. The deficient cells contained much more neutral lipids with a greater ratio of unsaturated fatty acids compared to the supplemented cells, whereas there was no significant change in their phospholipid contents. The biosynthesis of fatty acids and sterols from acetate, and of triacylglycerols and sterol esters from palmitate was markedly augmented in the deficient cells. Acetyl-CoA carboxylase activity of the deficient supernatant was 2- to 5-fold higher than that of the supplemented. However, the activity from both sources was not significantly different after Sephadex G-25 gel filtration of the supernatant, suggesting the presence of low molecular effector(s) in the deficient supernatant. There was a great increase in acid-soluble glycogen, trehalose, and fructose-1,6-P2, as well as a drastic decrease in citrate in the deficient cells. Their intracellular levels were calculated so that their effects on acetyl-CoA carboxylase was examined over the range of physiological concentration. Citrate strongly inhibited the enzyme activity of the supernatant, but it had no effect on the preparation after gel filtration. On the other hand, fructose-1,6-P2 stimulated the enzyme activity both before and after gel filtration. The acetyl-CoA carboxylase activity in the gel filtrate was measured as a function of citrate concentration at several fixed concentrations of fructose-1,6-P2. Citrate counteracted the activation by fructose-1,6-P2 in a dose-dependent manner. Citrate lacked the inhibitory effect in the absence of fructose-1,6-P2. It was concluded from these results that neutral lipid accumulation in the deficient cells reflected an increase in the synthesis of fatty acids, at least partly based on an enhancement of acetyl-CoA carboxylase activity, and that the operation of a reciprocal regulation of the enzyme by fructose-1,6-P2 and citrate caused a marked elevation of the enzyme activity in the deficient cells with a high fructose-1,6-P2 level and a low citrate level.
A systematic search for upstream controlling elements necessary for efficient expression of the yeast fatty acid synthase genes FAS1 and FAS2 revealed identical activation sites, UASFAS, in front of both FAS genes. The individual element confers, in a heterologous yeast test system, an approximately 40-fold stimulation of basal gene expression. The UASFAS motifs identified have the consensus sequence TYTTCACATGY and function in either orientation. The same sequence motif is found in the upstream regions of all so far characterized yeast genes encoding enzymes of phospholipid biosynthesis. In gel retardation assays, a protein factor, Fbf1 (FAS binding factor), was identified which interacted with UASFAS. The UASFAS motif proved to be an inositol/choline responsive element (ICRE) conferring strict repression by exogenous inositol and choline on a heterologous reporter gene. Its core sequence perfectly matches the CANNTG motif typical of basic helix-loop-helix DNA-binding proteins. In contrast to the individual UASFAS element, the intact yeast FAS promoters are not significantly influenced by inositol and choline, and thus allow nearly constitutive fatty acid synthase production. Available evidence suggests that additional cis- and trans-acting elements, other than UASFAS and Fbf1, are involved in this constitutive FAS gene expression.
We have isolated a 1.2-kilobase pair cDNA fragment in a screening for yeast genes regulated at the level of transcription by soluble lipid precursors, inositol and choline. Sequence analysis and comparison of the deduced amino acid sequence to protein databases unveiled 68% similarity of a 374-amino acid peptide fragment to published C termini of chicken and rat acetyl-CoA carboxylase and almost 100% identity to the product of the FAS3 gene from yeast. Several lines of evidence confirm that the cloned gene represents the yeast structural gene ACC1 encoding acetyl-CoA carboxylase. Overexpression of the ACC1 gene from a high copy number plasmid resulted in overexpression of a 250-kDa biotin-enzyme and enzymatic activity of acetyl-CoA carboxylase. Disruption of one ACC1 allele in a diploid wild-type strain resulted in 50% reduction of ACC1-specific mRNA and acetyl-CoA carboxylase specific activity and a marked decrease of biotin associated with a 250-kDa protein, compared to wild-type. After sporulation of diploid disruptants, spores containing the disrupted acc1 allele failed to enter vegetative growth, despite fatty acid supplementation, suggesting that acetyl-CoA carboxylase activity is essential for a process other than de novo fatty acid synthesis and that only a single functional copy of the ACC1 gene exists. ACC1 transcription was repressed 3-fold by lipid precursors, inositol and choline, and was also controlled by regulatory factors Ino2p, Ino4p, and Opi1p, providing evidence that the key step of fatty acid synthesis is regulated in conjunction with phospholipid synthesis at the level of gene expression. The 5'-untranslated region of the ACC1 gene contains a sequence reminiscent of an inositol/choline-responsive element identified in genes encoding phospholipid biosynthetic enzymes.
The reduction of acetate ester synthesis by aeration and the addition of unsaturated fatty acids to the medium has been reported to be the result of the reduction in alcohol acetyltransferase (AATase) activity induced by inhibition of this enzyme. However, regulation of the AATase gene ATF1 has not been reported. In this study, ATF1 gene expression was studied by Northern analysis, and the results showed that the ATF1 gene was repressed both by aeration and by unsaturated fatty acids. The results also showed that the reduction of AATase activity is closely related to the degree of repression of ATF1 mRNA, which suggested that the gene repression is the primary means of reducing AATase activity in vivo. Using the Escherichia coli lacZ gene as a reporter gene, it was shown that a 150-bp fragment of the 5' flanking sequence played a major role in the repression by aeration and unsaturated fatty acid addition.
Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (AURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (Δ URA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only ura3 mutants can grow. Direct URA3 gene disruption with pURA36 (Δ URA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.
A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (Δ TRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.
Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.
In sake brewing, the supply of inositol to the sake mash is markedly limited due to the polishing of the rice used. Additionally, free inositol in polished rice is further decreased during the steeping process. In the present study, free inositol in the supernatant of sake mash decreased until very little could be detected because of its uptake by yeast in the early period of the fermentation. Contrastingly, bound inositol was not utilized by yeast directly, and was not converted into a free form by either phytase or the enzyme extracted from koji. Under such an inositol-limited condition, the number of yeast cells was lower and their size was larger than when inositol was fully supplied. The inositol-limited condition also resulted in a low inositol content in yeast cells in the early period of the sake fermentation. The inositol content of yeast cells in sake mash in which an ordinary yeast strain (INO1) was used increased gradually, and exceeded the level of yeast fully supplied with inositol in the final period of the fermentation. The total amount of inositol in the sake mash also increased during the fermentation. On the other hand, the inositol content of the cells in sake mash made with an inositol-requiring strain (ino1) increased little during the fermentation. These results showed that the increase in the inositol content of yeast in the final period of sake fermentation was mainly due not to uptake from the raw materials but to biosynthesis by the yeast itself. This finding was supported by the induction of inositol-1-phosphate synthase activity under inositol-limited condition.
The influence of acid phosphatase and phytase on the population of yeast and the parallel fermentation in small scale sake brewing was investigated. It was revealed that the moromi period was shortened and sake-cake yield was lowered in the sake brewing with the addition of commercial acid phosphatase preparation, because the dissolution of steamed rice, the production of glucose and the fermentation in the moromi mash were enhanced by the addition of the enzyme. On the other hand, in the sake brewing with the addition of commercial phytase preparation, it was shown that the supply of glucose was the overall rate-limiting step because of the depressed dissolution of steamed rice in the moromi mash.
The influence of phytic acid and related compounds on the parallel fermentation in small scale sake brewing was investigated. It was revealed that the digestion of steamed rice was enhanced inithe sake brewing with the addition of phytic acid because of the release of adsorption of α-amylase onto rice protein, resulting in the increase of α-amylase activity in the liquid phase of moromi mash.
On the other hand, it was found that α-amylase was inhibited by the degradation products of phytic acid which were cleaved by phytase and others in sake-koji.
The activity of alcohol acetyltransferase, bound to the cell membrane and responsible for the formation of acetate esters, was affected by the fatty acid composition of the cell membrane. When saturated fatty acids, which only slightly inhibit alcohol acetyltransferase activity, were incorporated into the cell membrane, the enzyme activity and ester formation were only slightly affected. On. the other hand, when unsaturated fatty acids, which strongly inhibit the enzyme activity, accumulated in the cell membrane, ester formation was suppressed with inhibition of the enzyme activity. The mechanism of formation of acetate esters by brewers' yeast was explained by the alcohol acetyltransferase activity under the influence of the fatty acid composition of the cell membrane.
Alcohol acetyltransferase responsible for the formation of acetate esters during beer fermentation was found to be localized at the cell membrane of brewers' yeast. This cell membrane-bound enzyme was purified 120-fold by solubilization with Triton X-100, gel filtration on a Sepharose 6B column and chromatography on a DEAE-Sephadex A-50 column. The enzyme was most active at 30°C at pH 7-8. It was least active against C3 alcohol among C1-C6 alcohols, and slightly more active against straight-chain alcohols than against branched-chain alcohols with the same carbon number. The enzyme was strongly inhibited by unsaturated fatty acids, heavy metal ions and sulfhydryl reagents.
Uracil auxotrophic mutants were constructed from the sake yeasts K-701 and K-901 by successive URA3 gene disruption. First, as sake yeast is diploid, one URA3 gene was disrupted with pURA38 (ΔURA3 SMR1) and the heterozygous disruptant was isolated on an SM (sulfometuron methyl) plate. The other URA3 gene was disrupted with pURA36 (ΔURA3) and homozygous URA3 disruptants were isolated on FOA (5-fluoro-orotic acid) plate on which only URA3 mutants can grow. Direct URA3 gene disruption with pURA36 (ΔURA3) was also done and the uracil auxotrophic mutant was isolated. Four types of URA3 disruptants were isolated, two of which had no bacterial DNA.
A tryptophan auxotrophic mutant was constructed from one of the URA3 disruptant using pTRP14 (ΔTRP1 URA3) by gene disruption. This TRP1 disruptant was also lacking bacterial DNA.
Laboratory scale sake brewing using the auxotrophic mutants showed that these strains are very useful as recipient strains for molecular breeding of sake yeasts.
The vitamin requirements of various brewery yeast strains have been tested at 25° and at 12° C. All the flocculent type strains of brewery yeasts partially require inositol for their growth at 25° C., whereas at this temperature non-floccuient type strains do not show any requirement for inositol. At the lower temperature (12° C.), however, almost all the brewery yeast strains require inositol absolutely. Some brewery yeasts, which can grow adaptively in a pantothenate-deficient medium at 25° C, require pantothenate absolutely at 12° C. Some other vitamins-such as thiamine, pyridoxin, and nicotinic acid—are also partially required at the lower temperature by most of the brewery yeast strains tested. The cause of such an increased requirement for vitamins at lower temperatures is discussed.
Although the yeast strain K901 could grow in a low-phosphate culture supplemented with phytate, it could not grow when the amount of phosphate was high, suggesting that yeast repressible acid phosphatase (AP) liberates inositol from phytate. PHO5-3 (a PHO5-PHO3 fusion gene) and PHO11 from K901, and PHO5 from X2180-1A, were constitutively expressed in UT-1. The expression of PHO11 markedly influenced the effect of phytate on growth. Additionally, crude AP originating from PHO11 liberated inositol from phytate more than from the other PHO genes. These results revealed that the expression of PHO11 enables yeast to hydrolyze phytate and subsequently to utilize it for growth. Yeast could grow sufficiently even when a commercial enzyme was used instead of koji in sake mash, whereas it grew to a lesser extent when phosphate was added exogeneously. These findings suggest that in sake mash, yeast AP also liberates inositol from phytate in steamed rice, Phytate in steamed rice was not solubilized in the liquid phase and remained in the residue even if starch was degraded. However the degradation of protein led to the solubilization of phytate. Phytate solubilization through the degradation of protein by koji or a commercial enzyme made it possible for yeast AP to liberate inositol from phytate in sake mash. Although the phytate-hydrolyzing activity of yeast AP in the supernatant of sake mash is about 1/3 of that of koji in the initial stage, yeast AP contributes to yeast growth as much as koji-enzyme because cell-bound yeast AP activity is greater than the extracellar activity and is effective in liberating inositol from solubilized phytate.
In yeast biosynthesis of long-chain fatty acids depends essentially on 4 distinct enzyme systems, i.e. acetyl-CoA carboxylase (ACC), fatty acid synthetase (FAS), desatcrase and, in some instances, a malonyl-CoA-independent chain-elongation system. FAS and ACC are soluble proteins which have been purified to homogeneity from the yeast cytoplasm. The desaturation and elongation systems, on the other hand, are particulate enzymes associated with the microsomal fraction of the cell. For a long time, both ACC and FAS catalyzing multistep reaction sequences were considered to be classical multi enzyme complexes composed of several non-identical and functionally different subunits. Recently, however, this view proved to be wrong when it was demonstrated that in yeast both enzymes were multifunctional proteins combining several different catalytic activities within a single polypeptide chain. Though first indications for this structure came from protein-chemical studies, final proof was obtained from genetic investigations by determining the number of gene loci involved in ACC and FAS biosynthesis. For these studies, yeast proved to be an especially well-suited organism: extensive work, especially in Lynen's laboratory, on these two yeast enzymes had characterized them biochemically in extraordinary detail. Furthermore, genetic manipulation of yeast is easy and, thus Saccharomyes cerevisiae has become the best genetically characterized eukaryote so far.
In sake brewing, yeast cells grown in the traditional seed mash (kimoto) are enriched in palmitic acid, and those in the popular seed mash (sokujo-moto) are enriched in linoleic acid. Lipid composition of yeast in the main mash seeded with respective seed mash was determined. The amount of intracellular phospholipids (PLs) of yeast showed little change throughout the fermentation period, while intracellular triglyceride (TG) content decreased dramatically during the growth phase in the main mash. In the main mash seeded with sokujo-moto, the linoleic acid content of intracellular phosphatidyl ethanolamine (PE) was reduced from 30.6% of the original seed mash yeast to 6–9%, whereas the content of intracellular phosphatidyl choline (PC) was merely reduced from the original 48.4% to 26–30% during fermentation. Moreover, even after 4–5 generations of growth in the main mash, the percent of linoleic acid in PC did not notably decrease. The intracellular TG of seed mash yeast is therefore supposed to be a major source of linoleic acid in the early stage of growth in main mash. On the other hand, linoleic acid content in PLs of yeast were detectable only in low quantities (5–10%) during the fermentation of main mash seeded with kimoto as well as that seeded with kimoto (5.5%). Since there was no increase in linoleic acid content, the main mash during rapid growth phase seemed to be deficient in linoleic acid and, therefore, the digestion of raw materials, which contains large amounts of linoleic acid, failed to actively proceed. The ratio of of the cells was 0.67 in kimoto, and 1.62 in sokujo-moto. The same sort of difference in the ratio was also seen between the corresponding main mash. The presence of linoleic acid increased the ratio at the stationary phase of growth, and was decreased in the presense of 16% ethanol. Thus, kimoto could be differentiated from sokujo-moto on the basis of fatty acid composition in PC and by the ratio of the yeast in the main mash.
1.1. Saccharomyces carlsbergensis, ATCC 9080, was grown on three different carbon sources: glucose (1 or 10%), ethanol and lactate, in the presence and in the absence of added inositol.2.2. Lipids were extracted from whole cells and analyzed. In deficient cells, triglycerides were greatly enhanced with glucose as the carbon source, independant of the concentration of the nutrient. Triglyceride accumulation was less pronounced in ethanol-grown cells.3.3. The total phospholipid content of deficient cells grown on glucose was identical to that of normal cells. In ethanol-grown cells, the total phospholipids were decreased as compared to glucose-grown cells. The relative proportion of phospholipid classes was unaltered by inositol deficiency, with the exception of a lower phosphatidyl inositol content. The fatty acid composition of different lipid classes was almost identical in supplemented and deficient cells, although deficient cells contained slightly more medium-chain and saturated fatty acids.4.4. With cells grown on lactate without addition of inositol, lipid content, respiration and growth characteristics were identical to supplemented cells.5.5. Lipid accumulation occurred in deficient cells in the presence of glucose during the growth phase.
1.1. The incorporation of labeled precursors ([14C6]glucose, [i-14C]acetate, [i-14C]-labeled fatty acids, H332PO4) into lipids of cells grown in the presence or absence of inositol has been investigated. The following enzymic activities were determined in subcellular preparations from cells grown under these two conditions: fatty acid synthetase, acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase, acyl-CoA:dihydroxyacetone phosphate O-acyltransferase, CTP:phosphatidase cytidyltransferase.2.2. In the early growth phase, supplemented cells incorporate more activity from [14C6]glucose into triglycerides than deficient cells. The triglyceride content of supplemented cells decreases with time of growth, whereas, it increases in deficient cells. [14C]Acetate is incorporated into fatty acids more rapidly by deficient cells with a high proportion being incorporated into triglycerides.3.3. The labeling pattern of different lipid classes, after uptake of exogenous [i-14C]-labeled fatty acids, appeared to be very similar for both supplemented and deficient cells. Fatty acids from endogenous triglycerides are utilized for phospholipid synthesis by supplemented cells.4.4. The turnover of phospholipids is similar in supplemented and deficient cells, if related to the duplication time of the cultures, with the exception of the phosphatidyl inositol fraction which decreases at a much faster rate in deficient cells.5.5. Acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase activity is equally distributed between the mitochondria and the mitochondria-free supernatant fraction. Acyl-CoA:dihydroxyacetone phosphate O-acyltransferase exhibits a higher specific activity in the mitochondrial fraction. No difference was observed between supplemented and deficient cells with regard to the activity of these two enzymes.
In Saccharomyces cerevisiae, FAS1, FAS2, and FAS3 are the genes involved in saturated fatty acid biosynthesis. The enzymatic activities of both fatty acid synthase (FAS) and acetyl-CoA carboxylase are reduced 2- to 3-fold when yeast cells are grown in the presence of exogenous fatty acids. The mRNA levels of the FAS genes are correspondingly lower under repressive conditions. Expression of the FAS-lacZ reporter gene is also regulated by fatty acids. When a FAS2 multicopy plasmid is present in the cells, expression of both FAS1 and FAS3 increases. Thus, the FAS genes are coordinately regulated. Deletion analyses of the regulatory regions of FAS1 and FAS2 revealed common regulatory sequences. These include the GGCCAAAAAC and AGCCAAGCA sequences that have a common GCCAA core sequence and the UASINO (upstream activation sequence). Derepression of the FAS genes in the absence of exogenous inositol is not observed when UASINO is mutated, indicating that this cis element is a positive regulator of these genes. The GCCAA elements and UASINO act synergistically for optimal expression of the FAS genes.
A set of four yeast shuttle vectors that incorporate sequences from the Saccharomyces cerevisiae 2 mu endogenous plasmid has been constructed. These yeast episomal plasmid (YEp)-type vectors (pRS420 series) differ only in their yeast selectable markers, HIS3, TRP1, LEU2 or URA3. The pRS420 plasmids are based on the backbone of a multifunctional phagemid, pBluescript II SK+, and share its useful properties for growth in Escherichia coli and manipulation in vitro. The pRS420 plasmids have a copy number of about 20 per cell, equivalent to that of YEp24. During non-selective yeast growth, pRS420 plasmids are lost through mitotic segregation at rates similar to other YEp vectors and yeast centromeric plasmid (YCp) vectors, in the range of 1.5-5% of progeny per doubling. The pRS420 series provides high-copy-number counterparts to the current pRS vectors [Sikorski and Hieter, Genetics 122 (1989) 19-27].
The Saccharomyces cerevisiae genes FAS1 and FAS2 encoding the beta and alpha subunit of yeast fatty acid synthetase (FAS), respectively, were individually deleted by one-step gene disruption. Northern blot analysis of RNA from the resulting fas null allele mutants indicated that deletion of FAS2 did not influence the transcription of FAS1, while FAS2 transcription was significantly reduced in the delta fas1 strain. These data suggest an activating role of subunit beta on FAS2 gene expression or, alternatively, a repression of FAS2 by an excess of its own gene product. Compared to the intact alpha 6 beta 6 complex, the individual FAS subunits synthesized in the delta fas1 or delta fas2 strains exhibit a considerably increased sensitivity towards the proteinases present in the yeast cell homogenate. Using yeast mutants specifically defective in the vacuolar proteinases yscA (PRA1/ PEP4 gene product) and/or yscB (PRB1 gene product), it was shown that in vitro, subunit alpha is efficiently degraded by proteinase yscA while for degradation of subunit beta, the combined action of proteinases yscA and yscB is necessary. In vivo, besides the vacuolar proteinases, an additional proteolytic activity specifically affecting free FAS subunit alpha becomes increasingly apparent in cells entering the stationary growth phase. In contrast, under similar conditions uncomplexed FAS subunit beta is stable in strains lacking the vacuolar proteinases yscA and yscB. The reduced FAS subunit levels, at the stationary phase, were independent of the corresponding FAS transcript concentrations. Thus, differential degradation pathways are obviously removing an excess of either FAS subunit, at least under starvation conditions. A combination of both regulation of FAS gene expression and proteolysis of free FAS polypeptides may therefore explain the equimolar amounts of both FAS subunits observed in yeast wild-type cells.
This chapter describes vector systems for constitutive and inducible gene expression in Saccharomyces cerevisiae. Three new plasmids (pG-1, pG-2, and pG-3) are constructed that direct high-level constitutive gene expression in yeast. These vectors, derived from plasmids originally developed for expression of the rat glucocorticoid receptor cDNA in yeast, contain the very efficient yeast glyceraldehyde-3-phosphate dehydrogenase promoter. Each plasmid contains the yeast TRPI gene and 2-μm origin of replication and the ampicillin resistance gene and prokaryotic origin of replication from pUC18. In addition, a hormone-inducible expression vector is discussed in the chapter whose low basal promoter activity is strongly enhanced by the addition of glucocorticoids to yeast cells expressing the glucocorticoid receptor.
This chapter presents the procedure for high-efficiency transformation of yeast by electroporation. The protocol are designed by adapting the principles of bacterial electroporarion to transformarion of Saccharomyces cerevisiae, taking care, in addition, to provide continuous osmotic support of the electrically compromised cells. Preparation of cells in advance of electroporation involves four 5-min centrifugations. There is minimal preincubation with DNA and no carder nucleic acid, and the pulse itself takes only a moment. Subsequent outgrowth is not required, and plating by spreading is sufficient to provide maximal efficiency. A prerequisite for molecular biological manipulation of any organism is a reliable and efficient means for introducing exogenous DNA into the cell. Yet each of the techniques in general use for transforming yeast—namely, lithium acetate transformation and spheroplast transformation, suffers from significant limitations. Lithium acetate transformation, although relatively fast and simple, provides only a low efficiency of DNA transfer (∼l03 colonies/μg of episomal plasmid). Spheroplast transformation, while more efficient (∼1–5 × 104 colonies/μg), is complicated and time consuming.
Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C. This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes. S. cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response). Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C. However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.
Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.
The synthesis of membrane phospholipids in Saccharomyces cerevisiae is a highly regulated process. The regulation of this essential metabolic pathway is exerted at the level of transcription, control of enzyme subunit levels, and allosteric modulation of enzyme activity. However, the major form of regulation, which accounts for the coordinated regulation of the system, is determined at the level of transcript abundance. This coordinated response to inositol requires a highly conserved cis-acting promoter element (5' CATGTGAAAT 3') designated the UAS(INO) element. The UAS(INO) element serves as binding site for a heterodimeric helix-loop-helix complex (Ino2p:Ino4p), which is required to activate transcription in the absence of inositol. In addition, a specific negative regulatory gene, OPII is required for repression of transcription in response to inositol supplementation. Although this regulatory cascade was initially defined for control of phospholipid biosynthetic gene expression, it also responsible for the regulation of several other unrelated yeast genes. Consequently, the presence of inositol in the growth medium appears to influence the expression of many yeast genes by a common regulatory pathway.
The yeast Saccharomyces cerevisiae contains two acetyl-CoA synthetase genes, ACS1 and ACS2. While ACS1 transcription is glucose repressible, ACS2 shows coregulation with structural genes of fatty acid biosynthesis. The ACS2 upstream region contains an ICRE (inositol/choline-responsive element) as an activating sequence and requires the regulatory genes INO2 and INO4 for maximal expression. We demonstrate in vitro binding of the heterodimeric activator protein Ino2p/Ino4p to the ACS2 promoter. In addition, the pleiotropic transcription factor Abf1p also binds to the ACS2 control region. The identification of ACS2 activating elements also found upstream of ACC1, FAS1 and FAS2 suggests a role of this acetyl-CoA synthetase isoenzyme for the generation of the acetyl-CoA pool required for fatty acid biosynthesis.
Influence of vitamins on yeast cell morphology
- Takeda
Takeda, M. and Tsukahara, T.: Influence of vitamins on yeast cell morphology. J. Brew. Sot. Jpn., 57, 1036-1041 (1962).
Vitamin requirement of yeast in the culture at low temperature
- Takeda
IO. Takeda, M. and Tsukahara, T.: Vitamin requirement of yeast in the culture at low temperature. J. Brew. Sot. Jpn., 57, 1109-1111 (1962).
Accumulation of neutral lipids in Saccharomyces carlsbergensis by myo-inositol deficiency and its mechanism
- Hayashi