Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro

The alcohol‐O‐acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl‐coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol‐O‐acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl‐CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium‐chain‐length acyl‐CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.     

The role of the Saccharomyces cerevisiae lipoate protein ligase homologue,Lip3, in lipoic acid synthesis

The covalent attachment of lipoate to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in Saccharomyces cerevisiae. A third lipoate‐dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. Lipoate is synthesized by extraction of its precursor, octanoyl‐acyl carrier protein (ACP), from the pool of fatty acid biosynthetic intermediates. Alternatively, lipoate is salvaged from previously modified proteins or from growth medium by lipoate protein ligases (Lpls). The first Lpl to be characterized, LplA of Escherichia coli, catalyses two partial reactions: activation of the acyl chain by formation of acyl–AMP, followed by transfer of the acyl chain to lipoyl domains (LDs). There is a surprising diversity within the Lpl family of enzymes, several of which catalyse reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl‐ACP to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl–GCV to other LDs in an amido‐transfer reaction. Study of eukaryotic Lpls has lagged behind studies of the bacterial enzymes. We report that the Lip3 Lpl homologue of the yeast S. cerevisiae has octanoyl‐CoA–protein transferase activity, and discuss implications of this activity on the physiological role of Lip3 in lipoate synthesis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

DISTRIBUTION OF ESTERS PRODUCED DURING SUGAR FERMENTATION BETWEEN THE YEAST CELL AND THE MEDIUM

The distribution of the esters formed during sugar fermentations between the yeast cells and the medium was investigated in fermentations by 5 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum (carlsbergensis). The esters studied included the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₂ fatty acids. All of both acetates appeared in the medium. The proportion of the fatty acid ethyl esters transferred to the medium decreased with increasing chain length: all in the medium for ethyl caproate, 54–68% for ethyl caprylate, 8–17% for ethyl caprate, and all remaining in the yeast cell for ethyl laurate. A higher proportion of the esters formed appeared to remain in the cells of the S. uvarum strains than in cells of S. cerevisiae.     

Sake: Production and flavor

Abstract

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

Physiological and biochemical characteristics of the ethyl tiglate production pathway in the yeast Saprochaete suaveolens

A yeast identified as Saprochaete suaveolens was investigated for its capacity to produce a large panel of flavouring molecules. With a production of 32 compounds including 28 esters, S. suaveolens seems to be a good producer of fruity flavours and fragrances and especially of unsaturated esters, such as ethyl tiglate. Physiological and biochemical analyses were performed in this study in an attempt to comprehend the metabolic route to the formation of this compound. We show that the accumulation of ethyl tiglate by S. suaveolens is specifically induced by isoleucine. However, and contrary to S. cerevisiae, which harbours a classical Ehrlich pathway leading to the production of 2‐methylbutanol from isoleucine, our results provide phenotypic and enzymological evidence of ethyl tiglate biosynthesis in S. suaveolens through the catabolism of this amino acid by the β‐oxidation pathway, which generates tiglyl‐CoA as a probable intermediate. A kinetic analysis of this flavour molecule during growth of S. suaveolens on glucose and isoleucine showed a phase of production of ethyl tiglate that culminated concurrently with isoleucine exhaustion, followed by a disappearance of this compound, likely due to reassimilation by the yeast. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation of Long-chain Esters of Starch Using Fatty Acid Chlorides in the Absence of an Organic Solvent

An efficient method of synthesizing fatty acid esters of starch without organic solvents is described. The octanoate of starch was prepared by gelatinization of native starch with formic acid, followed by treatment with octanoyl chloride. Esterification was readily carried out under a stream of N₂ at a stirring rate of 500 min ^-1 at 25—130°C for a reaction duration of 20—120 min. Formic acid enabled esterification of starch by long-chain acyl chlorides with minimum degradation. The major factors affecting the esterification reaction were optimized. The results indicated that the octanoyl degree of substitution (DS₈) was increased with increase in fatty acid chloride concentration (from 3—12 eq. per anhydroglucose unit) and rise in temperature (25°C to 130°C). Higher octanoyl chloride concentrations and temperatures led to a reduction in yield due to acid hydrolysis of starch chains. A maximum DS₈ of 1.7 was reached after 40 min of reaction, and high concentrations of formic acid decreased the octanoyl substitution. On the other hand, higher concentration of octanoyl chloride, higher temperatures and longer reaction times increased the substitution of the long acyl chain into starch.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

The Influence of Inoculum Level on Fermentation and Flavour Compounds of White Wines Made from cv. Emir

In this study, the influence of the addition of a commercial wine yeast (Saccharomyces cerevisiae) at inocula of 1 × 10⁴ to 1 × 10⁷ cells/ml in Emir must was investigated with a focus on yeast growth, fermentation rate, ethyl alcohol and flavour compound formation. Spontaneous fermentation without inoculation was also performed. Higher peak counts were observed with higher amounts of S. cerevisiae yeast. Addition of various amounts of yeast led to the earlier disappearance of non‐Saccharomyces yeasts. The fermentation rate was improved with higher amounts of yeast, but ethanol production was not affected. Concentrations of higher alcohols increased with increasing inoculum levels, especially inoculum sizes of 1 × 10⁶ cells/ml and 1 × 10⁷ cells/ml. The amount of ethyl acetate was reduced with increased inoculum levels.     

LIPID METABOLISM AND THE REGULATION OF VOLATILE ESTER SYNTHESIS IN SACCHAROMYCES CEREVISIAE

The specific rates at which ethyl acetate and iso-amyl acetate are produced by yeast increase markedly at that point in fermentation where syntheses of lipids (i.e. saturated fatty acids and squalene) stop. An increase in the acetyl-CoA: CoASH ratio, or a reduced availability of substrates (fatty acyl-CoAs) for acyl transferases, are possible reasons for such a stimulation of ester synthesis. Increased rates of ester production are not sustained but contribute significantly (ca. 30% for ethyl acetate) to the total concentration of acetate esters in beer fermented from 1·040 all-malt wort. Addition of linoleic acid (50 mg litre^?1) suppresses the induction of ester synthesis and reduces overall formation of both ethyl and iso-amyl acetates by ca. 80%. Possibly, linoleic acid exerts these effects either by directly inhibiting the activity of an ester-synthesising enzyme, or by allowing alternative use of acetyl-CoA for the synthesis of saturated fatty acids.     

ESTERASES OF BAKER'S YEAST. II. SUBSTRATE SPECIFICITIES TOWARDS ESTERS FORMED DURING SUGAR FERMENTATIONS

Ethyl esters of fatty acids (C₂–C₁₂), isoamyl acetate and 2-phenethyl acetate were studied as substrates for yeast esterases and compared with the synthetic substrates, p-nitrophenyl esters and β-naphthyl esters. Intact yeast, the 55% and 55–75% ammonium sulphate precipitate of centrifuged yeast homogenate, and partly purified esterases were used for the determination of the hydrolysation activity towards the esters. The results showed that the yeast esterases prefer to hydrolyse the ethyl esters with acyl chain length of C₅ to C₁₂. The acetate esters, ethyl acetate, isoamyl acetate and 2-phenethyl acetate are only very slowly hydrolysed or remain unaffected. The substrate specificity of different esterases varies and can be used for their characterisation. Investigating pH optimum curves using intact yeast and a crude esterase preparation and different substrates confirmed the earlier result that there are esterases on both sides of the plasma membrane. The specificity of intracellular and periplasmically located esterases is, however, different.     

PRODUCTION OF ESTERS BY DIFFERENT YEAST STRAINS IN SUGAR FERMENTATIONS

The production of the acetates of isoamyl alcohol and phenethyl alcohol and the ethyl esters of the C₆-C₁₀ fatty acids was investigated in semiaerobic sugar fermentations by 56 strains of Saccharomyces cerevisiae and 3 strains of S. uvarum. The S. cerevisiae yeasts generally produced more esters than the S. uvarum yeasts. Isoamyl acetate was the main component in the ester fractions examined and others in decreasing order, were ethyl caprylate, ethyl caproate, ethyl caprate and phenethyl acetate.     

Fermentation Characteristics and Aromatic Profile of Plum Wines Produced with Indigenous Microbiota and Pure Cultures of Selected Yeast

The aim of this study was to assess and compare fermentation characteristics and aromatic profile of plum wines produced with indigenous microbiota and pure cultures of different selected yeast. Experiments were carried out with plum (Prunus domestica L.) varieties of different fruit ripening times (?a?anska rana, ?a?anska lepotica, and Po?ega?a). Wine fermentations were conducted by the activity of indigenous microbiota, commercially available Saccharomyces cerevisiae and Saccharomyces bayanus yeast strains and joint activity of Schizosaccharomyces pombe and S. cerevisiae (sequential inoculation). Statistically significant differences in fermentative characteristics and the content of certain volatile compounds were observed as a result of metabolic activity of various indigenous and/or selected yeasts during fermentation of plum pomace. Minimal duration of fermentation (4 to 5 d) and fastest ethanol production rate (from 12.3 to 15.5 g/L/d) were the characteristics of the studied S. cerevisiae strains. Isobutanol, 3‐methyl‐1‐butanol, 1‐heptanol, and 1‐octanol were the most prevalent higher alcohols in the tested plum wine samples. The predominant ester in plum wines was ethyl acetate, ethyl lactate, amyl acetate, isoamyl acetate, and ethyl palmitate, esters responsible for the floral and fruity olfactory tones, were also present in large amounts. Also, the use of S. cerevisiae strains resulted in the production of plum wines with better sensory characteristics than ones produced with other investigated yeasts. Obtained results are significant since there is limited data on the compounds responsible for the unique flavor of plum wine, as well as on the impact of different yeast starter cultures application on the overall quality of fruit wines.     

Behaviour of ethyl caproate during the production and distillation of ethyl caproate‐rich rice shochu

To increase the popularity of rice shochu, a process was developed to produce ethyl caproate‐rich rice shochu by adding a cultured broth of a caproic acid‐producing bacterial (CAPB) consortium to the fermentation. When the CAPB consortium containing fermented mash was subjected to vacuum distillation, the distillation efficiency of ethyl caproate was up to ~300%, while the distillation efficiencies of the other flavour compounds and ethanol were <100%. The behaviour of ethyl caproate during the production and distillation of an ethyl caproate‐rich rice shochu was investigated and the results showed that ethyl caproate was synthesized by Saccharomyces cerevisiae during the shochu production process, and that some of the ethyl caproate synthesized was secreted into the medium. Ethyl caproate in the medium evaporated easily and was transferred to the distillate when distilled. The increase in ethyl caproate after distillation was mainly derived from the release of the intracellular ethyl caproate, with negligible input from the chemical esterification of caproic acid and ethanol. During vacuum distillation, although few yeast cells were disrupted, secretion of the intracellular ethyl caproate was more efficient owing to the increase in temperature, allowing the distillation efficiency of ethyl caproate to be >100%. Copyright © 2016 The Institute of Brewing & Distilling     

MICROSOMAL-ASSOCIATED GLYCEROPHOSPHATE ACYLTRANSFERASE ACTIVITY IN GERMINATING SOYBEANS

Acyl CoA:sn-glycero-3-phosphate O-acyltransferase (glycerophosphate acyltransferase, EC 2.3.1.15) catalyzes the acylation of glycero-3-phosphate to form lysophosphatidic acid. This enzyme catalyzes the first committed step in the biosynthesis of phospholipids and acylglycerols in soybeans. Glycerophosphate acyltransferase was predominatly associated with the microsomal fraction of germinating soybeans. The pH optimum for the reaction was 7.0. The enzyme exhibited saturation kinetics for glycero-3-phosphate (K_m of 0.2 mM) and palmitoyl CoA (K_m of 6.4 μM). A variety of acyl CoA derivatives served as substrates for the enzyme. Palmitoyl CoA was the most effective acyl CoA substrate. The addition of the nonionic detergent Triton X-100 inhibited glycerophosphate acyltransferase activity. Thioreactive agents inhibited the enzyme indicating that a sulphydryl group is essential for activity. The activation energy for the reaction was 8.8 Kcal per mole. The microsomal enzyme was reasonably stable to temperatures up to 70° C.     

Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis

To identify genes responsible for acetaldehyde tolerance, genome‐wide screening was performed using a collection of haploid Saccharomyces cerevisiae strains deleted in single genes. The screen identified 49 genes whose deletion conferred acetaldehyde sensitivity, and these were termed the genes required for acetaldehyde tolerance. We focused on six of these genes required for acetaldehyde tolerance, ZWF1, GND1, RPE1, TKL1 and TAL1, which encode enzymes in the pentose phosphate pathway (PPP), and OAR1, which encodes for NADPH‐dependent 3‐oxoacyl‐(acyl‐carrier‐protein) reductase. These genes were not only responsible for acetaldehyde tolerance but also turned out to be induced by acetaldehyde. Moreover, the content of oleic acid was remarkably increased in yeast cells under acetaldehyde stress, and supplementation of oleic acid into the media partially alleviated acetaldehyde stress‐induced growth inhibition of strains disrupted in the genes required for acetaldehyde tolerance and OLE1. Taken together, our data suggest that the supply of NADPH and the process of fatty acid biosynthesis are the key factors in acetaldehyde tolerance in the yeast, and that oleic acid plays an important role in acetaldehyde tolerance. Copyright © 2008 John Wiley & Sons, Ltd.     

Production of Dairy-Based,Natural Sulphur Flavor Concentrate by Yeast Fermentation

Production of sulphur flavor concentrate by yeast fermentation was investigated using dairy media (milk and cream) spiked with L-methionine. The yeasts studied included so-called dairy yeasts Candida kefyr, Kluyveromyces marxianus, Debaryomyces hansenii, Geotrichum candidum, and Yarrowia lipolytica as well as wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus. Methionol was produced as the predominant volatile sulphur flavor compound. Other volatile sulphur flavor compounds produced were dimethyl disulphide, S-methyl thioacetate, 3-methylthio-1-propanoic acid, methional, 3-methylthio-1-propene, and ethyl 3-methylthio-1-propanoate. In addition, numerous other volatile flavor compounds were produced, including ethanol, branched-chain alcohols, aromatic alcohol, ethyl esters, branched-chain esters, branched-chain fatty acids, and branched-chain aldehydes. The sensory attribute of a methionol sulphur flavor concentrate in cream is concentration-dependent, ranging from a savory-slight potato note to a strong potato-savory note to a cooked cheese-potato note.     

Fermentative behavior of Saccharomyces strains during microvinification of raspberry juice (Rubus idaeus L.)

Sixteen different strains of Saccharomyces cerevisiae and Saccharomyces bayanus were evaluated in the production of raspberry fruit wine. Raspberry juice sugar concentrations were adjusted to 16°Brix with a sucrose solution, and batch fermentations were performed at 22 °C. Various kinetic parameters, such as the conversion factors of the substrates into ethanol (Y_p/s), biomass (Y_x/s), glycerol (Y_g/s) and acetic acid (Y_ac/s), the volumetric productivity of ethanol (Q_p), the biomass productivity (P_x), and the fermentation efficiency (E_f) were calculated. Volatile compounds (alcohols, ethyl esters, acetates of higher alcohols and volatile fatty acids) were determined by gas chromatography (GC-FID). The highest values for the E_f, Y_p/s, Y_g/s, and Y_x/s parameters were obtained when strains commonly used in the fuel ethanol industry (S. cerevisiae PE-2, BG, SA, CAT-1, and VR-1) were used to ferment raspberry juice. S. cerevisiae strain UFLA FW 15, isolated from fruit, displayed similar results. Twenty-one volatile compounds were identified in raspberry wines. The highest concentrations of total volatile compounds were found in wines produced with S. cerevisiae strains UFLA FW 15 (87,435 μg/L), CAT-1 (80,317.01 μg/L), VR-1 (67,573.99 μg/L) and S. bayanus CBS 1505 (71,660.32 μg/L). The highest concentrations of ethyl esters were 454.33 μg/L, 440.33 μg/L and 438 μg/L for S. cerevisiae strains UFLA FW 15, VR-1 and BG, respectively. Similar to concentrations of ethyl esters, the highest concentrations of acetates (1927.67 μg/L) and higher alcohols (83,996.33 μg/L) were produced in raspberry wine from S. cerevisiae UFLA FW 15. The maximum concentration of volatile fatty acids was found in raspberry wine produced by S. cerevisiae strain VR-1. We conclude that S. cerevisiae strain UFLA FW 15 fermented raspberry juice and produced a fruit wine with low concentrations of acids and high concentrations of acetates, higher alcohols and ethyl esters.     

STUDIES ON YEAST ESTERASE

An esterase (carboxylic acid hydrolase EC 3 1.1.1.) has been prepared from Saccharomyces cerevisiae. This esterase is active at pH 4.4 and, though it is unstable in solution, it can be maintained in an active form either by lyophilization or by coupling to an affinity gel. At pH 4.4, yeast esterase can hydrolyse between 20 and 40% of the esters commonly present in beer, and it is capable of synthesizing ethyl acetate from ethanol and acetic acid in a simple buffered solution without the provision of a co-factor. Different yeast strains yield different amounts of esterase, and there appears to be a positive correlation between the ability of the yeast to form esterase and the level of esters present in fermentations accomplished by that yeast.     

Bioconversion of heptanal to heptanol by Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely known for its catalytic activity on substrates such as aldehyde and ketone. Interestingly, the activity of S. cerevisiae on heptanal (C₆H₁₃CHO), in spite of its being a very common aldehyde, has not been explored. The main objective of this study was therefore to investigate the bioconversion of heptanal, using a strain of the yeast S. cerevisiae. Bioconversion parameters such as incubation period, pH, concentration of substrate, yeast and maltose were also optimized. The study revealed heptanol as the major product. The optimum conditions for biotransformation were found to be: 3 days incubation; pH 7.0; heptanal concentration 0.15 ml/100 ml medium; and S. cerevisiae concentration of 0.15 g/100 ml medium. Reduction in maltose content (to 0.3 g maltose/100 ml medium) showed increased conversion of heptanal. Heptanoic acid and 2‐hydroxyheptanoic acid were obtained as two minor co‐products. The overall study showed that S. cerevisiae converted heptanal to heptanol by a yield of 68.9 ± 1.1% w/w under optimum conditions. Copyright © 2010 John Wiley & Sons, Ltd.