Formation Of Acetate Esters Research Articles

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation.

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.

Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation.

Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.

Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Differences in Enzymatic Properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum Alcohol Acetyltransferases and Their Impact on Aroma-Active Compounds Production.

Higher alcohols and acetate esters belong to the most important yeast secondary metabolites that significantly contribute to the overall flavor and aroma profile of fermented products. In Saccharomyces cerevisiae, esterification of higher alcohols is catalyzed mainly by the alcohol acetyltransferases encoded by genes ATF1 and ATF2. Previous investigation has shown other Saccharomyces species, e.g., S. kudriavzevii and S. uvarum, to vary in aroma-active higher alcohols and acetate esters formation when compared to S. cerevisiae. Here, we aimed to analyze the enzymes encoded by the ATF1 and ATF2 genes from S. kudriavzevii (SkATF1, SkATF2) and S. uvarum (SuATF1, SuATF2). The heterologous expression of the individual ATF1 and ATF2 genes in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. Particularly, an increase of 2-phenylethyl acetate production by the strains that harbored ATF1 and ATF2 genes from S. kudriavzevii and S. uvarum was observed. When grown with individual amino acids as the nitrogen source, the strain that harbored SkATF1 showed particularly high 2-phenylethyl acetate production and the strains with introduced SkATF2 or SuATF2 revealed increased production of isobutyl acetate, isoamyl acetate, and 2-phenylethyl acetate compared to the reference strains with endogenous ATF genes. The alcohol acetyltransferase activities of the individual Atf1 and Atf2 enzymes measured in the cell extracts of the S. cerevisiae atf1 atf2 iah1 triple-null strain were detected for all the measured substrates. This indicated that S. kudriavzevii and S. uvarum Atf enzymes had broad range substrate specificity as S. cerevisiae Atf enzymes. Individual Atf1 enzymes exhibited markedly different kinetic properties since SkAtf1p showed c. twofold higher and SuAtf1p c. threefold higher Km for isoamyl alcohol than ScAtf1p. Together these results indicated that the differences found among the three Saccharomyces species during the aroma-active acetate ester formation may be due, to some extent, to the distinct properties of Atf enzymes.

The Fungal Aroma Gene ATF1 Promotes Dispersal of Yeast Cells through Insect Vectors

Yeast cells produce various volatile metabolites that are key contributors to the pleasing fruity and flowery aroma of fermented beverages. Several of these fruity metabolites, including isoamyl acetate and ethyl acetate, are produced by a dedicated enzyme, the alcohol acetyl transferase Atf1. However, despite much research, the physiological role of acetate ester formation in yeast remains unknown. Using a combination of molecular biology, neurobiology, and behavioral tests, we demonstrate that deletion of ATF1 alters the olfactory response in the antennal lobe of fruit flies that feed on yeast cells. The flies are much less attracted to the mutant yeast cells, and this in turn results in reduced dispersal of the mutant yeast cells by the flies. Together, our results uncover the molecular details of an intriguing aroma-based communication and mutualism between microbes and their insect vectors. Similar mechanisms may exist in other microbes, including microbes on flowering plants and pathogens.

Increase of acetate ester-hydrolysing esterase activity in mixed cultures of Saccharomyces cerevisiae and Pichia anomala

To examine the efficacy of mixed cultures with Saccharomyces cerevisiae and Pichia anomala on flavour profiles of alcoholic beverages, a Pichia mutant with low levels of ethyl acetate that negatively impact on the sensory quality was isolated. A petite mutant isolated from P. anomala NBRC 10213 treated with ethidium bromide had the lower activity of ethyl acetate-hydrolysing esterase (EAHase) than the wild-type in crude extracts. In the fermentation tests of pure cultures, the P. anomala mutant produced less ethanol, acetate and ethyl acetate than the wild-type. In mixed cultures with S. cerevisiae, the P. anomala mutant died quicker and produced lower amounts of ethyl acetate than the wild-type. Mixed cultures of S. cerevisiae and P. anomala showed higher activities of EAHase than pure culture of S. cerevisiae throughout the fermentation periods. The transition to the formation of acetate esters was considerably analogous to the transition to the activity of acetate ester-hydrolysing esterase with little time lag. The P. anomala mutant was superior to the wild-type in flavour profiles. The higher ethyl acetate concentrations formed mainly by P. anomala in mixed cultures are the primary stimulus for the EAHase in S. cerevisiae and the activity of acetate ester-hydrolysing esterase is crucial to the formation of acetate esters in mixed cultures of S. cerevisiae and P. anomala. An application of non-Saccharomyces yeast, P. anomala to enhance the sensory quality in alcoholic beverage and a mechanism of the formation of acetate esters in mixed cultures with S. cerevisiae and P. anomala were offered.

The Saccharomyces cerevisiae alcohol acetyl transferase Atf1p is localized in lipid particles.

The yeast alcohol acetyl transferase I, Atf1p, is responsible for the major part of volatile acetate ester production in fermenting Saccharomyces cerevisiae cells. Some of these esters, such as ethyl acetate and isoamyl acetate, are important for the fruity flavours of wine, beer and other fermented beverages. In order to reveal the subcellular localization of Atf1p and further unravel the possible physiological role of this protein, ATF1::GFP fusion constructs were overexpressed in brewer's yeast. The transformant strain showed a significant increase in acetate ester formation, similar to that of an ATF1 overexpression strain, indicating that the Atf1p-GFP fusion protein was active. UV fluorescence microscopy revealed that the fusion protein was localized in small, sphere-like organelles. These organelles could be selectively stained by the fluorescent dye Nile red, indicating that they contained high amounts of neutral lipids and/or sterols, a specific characteristic of yeast lipid particles. Purification of lipid particles from wild type and ATF1 deletion cells confirmed that the Atf1p-GFP fusion protein was located in these organelles. Furthermore, a clear alcohol acetyl transferase activity could be measured in the purified lipid particles of both wild type and transformed cells. The localization of Atf1p in lipid particles may indicate that Atf1p has a specific role in the lipid and/or sterol metabolism that takes place in these particles.

Inhibition of acetate ester biosynthesis in banana (Musa sapientum L.) fruit pulp under anaerobic conditions.

The effect of anaerobic conditions on acetate ester biosynthesis in ripened banana pulp was investigated. Incubation of the pulp in less than 1% O(2) resulted in a significant reduction in the formation of ethyl acetate. Regardless of the presence of a large amount of endogenous ethanol and the remaining exogenous isobutyl alcohol after complete anaerobic incubation with the pulp, the production of acetate ester decreased. The effect of addition of pyruvate, isobutyl alcohol, acetate, and methyl hexanoate on acetate ester formation in 100% N(2) was also investigated. The addition of pyruvate and isobutyl alcohol to the pulp gave lower acetate esters in N(2) than in air, whereas the pulp incubated with acetate and isobutyl alcohol produced more acetate ester in both conditions. Therefore, the lack of acetyl CoA, or more precisely acetate, in the tissue is the main reason for the inhibition of acetate ester formation under anaerobic conditions. The activity of beta-oxidation measured by incubation with methyl hexanoate was detected only in the samples incubated in air. The formation of acetyl CoA, derived from pyruvate through mitochondria and through beta-oxidation, was inhibited by anaerobic conditions, which suggests that mitochondrial activity and/or beta-oxidation are essential for ester biosynthesis.

Pathway of Acetate Ester Formation in Banana Fruit Pulp

For the determination of acetate ester formation, banana pulp slices or pulp cells suspension were incubated with pyruvate, acetate or citrate, alcohol, and co-factors. When the various concentrations of pyruvate were added to the pulp slices, higher concentrations showed more acetate ester production. Addition of ATP, CoA and Mg to the pulp cells also induced the formation of acetate esters. Substrates and co-factors accelerating TCA cycle enhanced the ester formation in the cell suspension. Results indicate that acid part of acetate esters was mainly derived from pyruvate via acetate in banana fruit although small amount of acetyl CoA was also formed from citrate.

Acetate ester formation in wine by mixed cultures in laboratory fermentations

Two non- Saccharomyces wine yeast strains, Hanseniaspora guilliermondii 11104 and Pichia anomala 10590, selected as good producers of acetate esters when grown on synthetic microbiological medium, have been tested in wine fermentations as mixed cultures together with Saccharomyces cerevisiae. Wines produced using mixed cultures showed levels of acetaldehyde, acetic acid, glycerol and total higher alcohols within the ranges described for wine, whereas an increase in acetate ester concentrations was found. Ethyl acetate was the main ester produced, and isoamyl acetate and 2-phenylethyl acetate made up the next largest group of ester compounds in the wines analysed. H. guilliermondii 11104 was found to be a strong producer of 2-phenylethyl acetate in both pure and mixed cultures whereas S. cerevisiae was the best producer of ethyl esters. Mixed cultures did not influence ethyl ester levels at all.

Studies on acetate ester production by non- Saccharomyces wine yeasts

A double coupling bioreactor system was used to fast screen yeast strains for the production of acetate esters. Eleven yeast strains were used belonging to the genera Candida, Hanseniaspora, Metschnikowia, Pichia, Schizosaccharomyces and Zygosacharomyces, mainly isolated from grapes and wine, and two wine Saccharomyces cerevisiae strains. The acetate ester forming activities of yeast strains belonging to the genera Hanseniaspora ( Hanseniaspora guilliermondii and H. uvarum) and Pichia ( Pichia anomala) showed different substrate specificities and were able to produce ethyl acetate, geranyl acetate, isoamyl acetate and 2-phenylethyl acetate. The influence of aeration culture conditions on the formation of acetate esters by non- Saccharomyces wine yeast and S. cerevisiae was examined by growing the yeasts on synthetic microbiological medium. S. cerevisiae produced low levels of acetate esters when the cells were cultured under highly aeration conditions, while, under the same conditions, H. guilliermondii 11104 and P. anomala 10590 were found to be strong producers of 2-phenylethyl acetate and isoamyl acetate, respectively.

Flavor formation and cell physiology during the production of alcohol-free beer with immobilized Saccharomyces cerevisiae

Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor operating in downflow. This ensures a highly controllable system with optimal reactor design. In the present study, we report on changes in the physiology of immobilized yeast cells in the reactor. During production, a simultaneous increase in the activity of alcohol acetyl transferase, and formation of the esters ethyl acetate and isoamyl acetate were observed. In addition, the amount of unsaturated fatty acids decreased significantly. Since these phenomena coincided with a significant decrease in growth rate, we conclude that the anaerobic conditions, and the absence of substantial levels of unsaturated fatty acids in wort limit cell growth during production and stimulate formation of acetate esters. Low temperatures (2°C) appeared to suppress production of α-acetolactate whereas at 12°C, substantial levels were obtained. An optimal and constant flavor profile of the alcohol-free beer can be achieved by introduction of regular aerobic periods to stimulate yeast growth. Temperature can be used to control the rate of growth as well as the rate of flavor formation.

Investigating Ester Metabolism: Characterization of the ATF1 Gene in Saccharomyces Cerevisiae

Esters, which are produced during fermentation, are important contributors to the flavor of beer and whisky. Many different esters are formed during wort fermentations, however, most research has centered on the production of acetate esters. The mechanisms that regulate ester metabolism are not well characterized but it has been known for many years that ester levels produced during fermentation are influenced by a variety of factors. Nevertheless, it is unclear how these environmental factors influence yeast metabolism and, accordingly, ester synthesis. The enzyme alcohol acetyltransferase has been implicated in the formation of acetate esters and, as a result, most work has concentrated on the role of this enzyme. However, alcohol acetyltransferase is an extremely unstable protein when it has been purified; consequently, it is difficult to study. In this work, we have developed an in vivo system to study the expression of the gene encoding alcohol acetyltransferase, ATF1. A 3.3 kb BamHI/XbaI fragment of ATF1, containing 1.9 kb of native promoter sequence, was fused to the Escherichia coli lacZ gene, and expression of ATF1 was measured under different conditions. Expression of ATF1 correlated with the production of ethyl acetate during fermentation. There was no clear relationship between ATF1 expression and isoamyl acetate production.

Ester biosynthesis in “Rome” apples subjected to low-oxygen atmospheres

Abstract The relationship between acetate ester-forming activity of acetyl CoA alcohol transferase (ACAT), non-ethylene volatile emission, and flesh volatile content of “Rome” apples ( Malus domestica Borkh.) after removal from 9 months storage in low-oxygen environments was investigated. Apple samples held at 0–1°C were removed from refrigerated air (RA) or controlled-atmosphere (CA) storage at 1.0 or 0.5% v/v O 2 /1% CO 2 and placed in ambient laboratory conditions. Every three days fruit flesh and headspace were analyzed for volatile compounds using capillary gas chromatography. Acetate ester forming activity was assayed spectrophotometrically on partially-purified extracts of cortical tissue. Patterns of acetate ester formation depended upon storage environment and alcohol moiety precursor. Ethyl acetate content was always higher in the headspace and flesh of RA fruit, regardless of time after storage. Propyl acetate concentrations in CA stored apple flesh were higher until post-storage day 12 when they decreased. Headspace propyl acetate levels were higher in RA controls until days 9–15 when more emanated from CA fruit. Butyl acetate concentrations were lower in flesh and headspace of CA fruit until day 15, when RA fruit headspace levels decreased. Headspace and flesh concentrations of 2-methyl-1-butyl acetate were higher in CA apples. Acetate ester-forming activity was detectable at day 0 in 1.0% oxygen-stored fruits, but not in 0.5% oxygen-stored fruits. By post-storage day 9, ACAT activity in CA apples reached maximal levels, only to decrease by day 15. RA-stored apples had more ACAT activity at day 0, but did not substantially increase in activity like the 0.5% oxygen-stored apples removed from CA storage. It is unknown whether the ACAT protein is reactivated by exposure to ambient oxygen or synthesized de novo.

Control of acetate ester formation during alcohol fermentation with immobilized yeast

Abstract The effect of sugar composition on the formation of acetate esters using immobilized yeast was investigated. When the immobilized yeast was incubated in maltose medium lacking unsaturated fatty acids, the production of ethyl acetate and isoamyl acetate was poor when compared to glucose medium, although in maltose medium the production of acetyl-CoA was less than in glucose medium. Ester production was stimulated using the immobilized yeast and wort treated with glucoamylase to hydrolyze maltose. With this method, acetate esters were produced at normal levels compared to those of beer fermented in the conventional manner.

Ester Formation by Brewers’ Yeast during Sugar Fermentation

Formation of acetate esters by brewers’ yeast during sugar fermentation was investigated in relation to alcohol acetyltransferase activity influenced by the fatty acid composition of the yeast cell membrane. Glucose gave more acetate esters with a higher activity of alcohol acetyltransferase than the other carbohydrates. When maltose was fermented, the activity of alcohol acetyltransferase bound to the cell membrane was suppressed by unsaturated fatty acids accumulated in the cell membrane and the formation of acetate esters was greatly reduced without insufficient fermentation. On the other hand, when fructose was fermented, the ester formation was reduced with a decrease in the enzyme activity and the formation of higher alcohols through insufficient

Ester formation by brewers' yeast during sugar fermentation.

Formation of acetate esters by brewers’ yeast during sugar fermentation was investigated in relation to alcohol acetyltransferase activity influenced by the fatty acid composition of the yeast cell membrane. Glucose gave more acetate esters with a higher activity of alcohol acetyltransferase than the other carbohydrates. When maltose was fermented, the activity of alcohol acetyltransferase bound to the cell membrane was suppressed by unsaturated fatty acids accumulated in the cell membrane and the formation of acetate esters was greatly reduced without insufficient fermentation. On the other hand, when fructose was fermented, the ester formation was reduced with a decrease in the enzyme activity and the formation of higher alcohols through insufficient

Acetyl-CoA of brewers' yeast and formation of acetate esters.

Acetyl-CoA of brewers' yeast and formation of acetate esters.

Cellular Fatty Acid and Ester Formation by Brewers′ Yeast

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 in-corporated 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.

Cellular fatty acid and ester formation by brewers' yeast.

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.