Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Δ atf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.     

The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways

The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations. A number of these volatile esters, e.g. ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine. Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer. Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells. This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction. Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression. Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue beta-L-alanine, showing that the effect of the nitrogen source did not depend on its metabolism. In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress. These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations. In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast.     

Production of volatile organic sulfur compounds (VOSCs) by basidiomycetous yeasts

Thirty-seven basidiomycetous yeasts belonging to 30 species of seven genera were grown on media containing l-cysteine or l-methionine as sole nitrogen sources with the objective of evaluating volatile organic sulfur compound (VOSC) production. The headspace of yeast cultures was analyzed by the solid-phase microextraction (SPME) sampling method, and volatile compounds were quantified and identified by GC-MS techniques. Ten strains assimilating L-methionine produced the following VOSCs: 3-(methylthio)-1-propanol, methanethiol, S-methyl thioacetate, dimethyl disulfide, dimethyl trisulfide, allyl methyl sulphide and 4,5-dihydro-3(2H)-thiophenone. Production was <1 mgl(-1) except for 3-(methylthio)-1-propanol of which between 40 and 400 mgl(-1) was synthesized. Higher alcohols (isobutyl alcohol, isoamyl alcohol and active amyl alcohol) and esters (ethyl acetate, ethyl propionate, n-propyl acetate, isobutyl acetate, n-propyl propionate, n-butyl acetate, isoamyl acetate, amyl acetate, isoamyl propionate, amyl propionate and 2-phenylmethyl acetate) were also sporadically produced. This is the first report of VOSCs production by basidiomycetous yeasts. Consequently, basidiomycetous yeasts may be considered an interesting new group of microbial VOSCs producers for the flavor industry.     

Pregnenolone esterification in Saccharomyces cerevisiae. A potential detoxification mechanism.

While studying the effect of steroids on the growth of the yeast Saccharomyces cerevisiae, we found that pregnenolone was converted into the acetate ester. This reaction was identified as a transfer of the acetyl group from acetyl-CoA to the 3beta-hydroxyl group of pregnenolone. The corresponding enzyme, acetyl-CoA:pregnenolone acetyltransferase (APAT) is specific for Delta5- or Delta4-3beta-hydroxysteroids and short-chain acyl-CoAs. The apparent Km for pregnenolone is approximately 0.5 microm. The protein associated with APAT activity was partially purified and finally isolated from an SDS/polyacrylamide gel. Tryptic peptides were generated and N-terminally sequenced. Two peptide sequences allowed the identification of an open reading frame (YGR177c, in the S. cerevisiae genome database) translating into a 62-kDa protein of hitherto unknown function. This protein encoded by a gene known as ATF2 displays 37% identity with an alcohol acetyltransferase encoded by the yeast gene ATF1. Disruption of ATF2 led to the complete elimination of APAT activity and consequently abolished the esterification of pregnenolone. In addition, a toxic effect of pregnenolone linked to the disruption of ATF2 was observed. Pregnenolone toxicity is more pronounced when the atf2-Delta mutation is introduced in a yeast strain devoid of the ATP-binding cassette transporters, PDR5 and SNQ2. Our results suggest that Atf2p (APAT) plays an active role in the detoxification of 3beta-hydroxysteroids in association with the efflux pumps Pdr5p and Snq2p.     

Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides. Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium. In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA. We chose the production of isoamyl acetate as a model system. Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value. Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate. The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli. This genetic engineered E. coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium. In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production. Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production. The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA. Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production. The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production.     

Modulating aroma compounds during wine fermentation by manipulating carnitine acetyltransferases in Saccharomyces cerevisiae

The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

High throughput,colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.     

A study on volatile organic compounds (VOCs) produced by tropical ascomycetous yeasts

As a part of a program aiming at the selection of strains which might be of interest as sources of natural flavouring molecules, the production of volatile organic compounds (VOCs) by 98 ascomycetous yeast strains (representative of 40 species belonging to 12 genera) isolated from tropical environments was investigated. Volatiles produced were sampled by means of headspace solid-phase microextraction (SPME) and the compounds were analysed and identified by gas chromatography–mass spectroscopy (GC–MS). The VOCs produced were found to be alcohols (amyl alcohol and isoamyl alcohol), aldehydes (2-methyl-2-hexenal and 2-isopropyl-5-methyl-2-hexenal) and esters (ethyl isobutyrate, isobutyl acetate, isoamyl acetate, 2-methylbutyl acetate, ethyl isovalerate, isoamyl propionate and phenylmethyl acetate). Differences in VOC profiles were used to cluster the yeast strains into 25 VOC phenotypes. The different frequency of VOC phenotypes in three specific habitats was correlated to the divergent environmental conditions, possibly affecting the selection of specific yeasts. From a biotechnological viewpoint, this study reveals the potentiality of ascomycetous yeasts isolated from tropical environments as a promising source of VOCs relevant in food and fragrance industry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cider fermentation with three Williopsis saturnus yeast strains and volatile changes

Williopsis saturnus var. subsufficiens NCYC 2728, W. saturnus var. saturnus NCYC 22 and W. saturnus var. mrakii NCYC 500 were used to carry out cider fermentation to assess their impact on the volatile composition of cider. The changes of yeast cell population, °Brix and pH were similar among the three yeasts. Strain NCYC 500 grew best, with the highest cell population of 1.14 × 108 CFU ml−1, followed by strains NCYC 2728 and NCYC 22 (8 × 107 CFU ml−1 and 3.19 × 107 CFU ml−1 respectively). Esters were the most abundant volatiles produced, followed by alcohols. Among the esters, ethyl acetate, 2-phenylethyl acetate, isoamyl acetate, cis-3-hexenyl acetate and hexyl acetate were the major volatiles. The major alcohols were ethanol, isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol. The three Williopsis yeasts transformed volatile compounds during cider fermentation with significant variations in terms of volatile production and degradation. This study implied that fermentation with Williopsis yeasts could result in cider with a more complex yet fruity aroma.     

Aerobic production of isoamyl acetate by overexpression of the yeast alcohol acetyl-transferases AFT1 and AFT2 in Escherichia coli and using low-cost fermentation ingredients

Isoamyl acetate, produced via fermentation, is a natural flavor chemical with applications in the food industry. Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) can catalyze the esterification of isoamyl alcohol with acetyl coenzyme A. The respective genes were cloned and expressed in an appropriate ack-pta(-) strain of Escherichia coli. The engineered strains produce isoamyl acetate when isoamyl alcohol is added to the culture medium. Aerobic shake flask experiments examined isoamyl acetate production over various growth times, temperatures, and initial optical densities. The strain carrying the pBAD-ATF1 plasmid exhibited a high molar ester yield from glucose (1.13) after 48 h of aerobic growth at 25 degrees C. Low-cost media components, such as fusel oil, sorghum glucose and corn steep liquor, were found to give a high yield of isoamyl acetate. High-cell-density gave an increased isoamyl acetate yield of 0.18 g/g of glucose consumed.     

Flavour formation in fungi: characterisation of KlAtf,the <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> orthologue of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferases Atf1 and Atf2

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. In the brewers’ yeast Saccharomyces cerevisiae, the major part of these esters is formed by two alcohol acetyltransferases, Atf1 and Atf2. In this paper, the existence of orthologues of these S. cerevisiae alcohol acetyltransferases in several ascomycetous fungi was investigated. Bioinformatic analysis of sequenced fungal genomes revealed the presence of multiple orthologues. The Saccharomyces sensu stricto yeasts all have two genes coding for orthologues. More distantly related fungi like Saccharomyces castelii, Candida glabrata, Kluyveromyces waltii and Kluyveromyces lactis have only one orthologue in their genome. The homology between the identified proteins and the S. cerevisiae alcohol acetyltransferases suggests a role for these orthologues in the aroma-active ester formation. To verify this, the K. lactis orthologue KlAtf was cloned and expressed in S. cerevisiae. Gas chromatographic analysis of small-scale fermentations with the transformant strains showed that, while S. cerevisiae ATF1 overexpression resulted in a substantial increase in acetate ester levels, S. cerevisiae ATF2 and K. lactis ATF overexpression only caused a moderate increase in acetate esters. This study is the first report of the presence of an ester synthesis gene in K. lactis.     

Parameters Affecting Ethyl Ester Production by Saccharomyces cerevisiae during Fermentation

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.     

Enhanced acetate ester production of Chinese liquor yeast by overexpressing ATF1 through precise and seamless insertion of PGK1 promoter

As the most important group in the flavor profiles of Chinese liquor, ester aroma chemicals are responsible for the highly desired fruity odors. Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis in Saccharomyces cerevisiae. In this study, we overexpressed ATF1 in Chinese liquor yeast through precise and seamless insertion of PGK1 promoter (PGK1p) via a novel fusion PCR-mediated strategy. After two-step integration, PGK1p was embedded in the 5′-terminal of ATF1 exactly without introduction of any extraneous DNA sequence. In the liquid fermentation of corn hydrolysate, both mRNA level and AATase activity of ATF1 in mutant were pronounced higher than the parental strain. Meanwhile, productivity of ethyl acetate increased from 25.04 to 78.76 mg/l. The self-cloning strain without any heterologous sequences residual in its genome would contribute to further commercialization of favorable organoleptic characteristics in Chinese liquor.     

Formation of Volatile Esters in Strawberries

Aliphatic alcohols including methyl, ethyl, isopropyl, npropyl, isobutyl, nbutyl, isoamyl, namyl and hexyl alcohol were converted to their acetate, propionate, nbutyrate, isovalerate and caproate esters during the incubation with strawberry fruit tissue. Formate, isobutyrate and n-valerate esters were formed when alcohols were incubated together with these fatty acids and strawberry.

Seventy esters were formed from various combinations of alcohols and acids by means of incubation with strawberry.

No ester formation was observed when strawberry was homogenized.     

Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate

Although the bacterium E. coli is chosen as the host in many bioprocesses, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that could have otherwise been used for the desired product. The production of the ester isoamyl acetate from acetyl-CoA by ATF2, a yeast alcohol acetyl transferase, was used as a model system to demonstrate the beneficial effects of reducing acetate production. All strains tested for ester production also overexpressed panK, a native E. coli gene that previous studies have shown to increase free intracellular CoA levels when fed with pantothenic acid. A recombinant E. coli strain with a deletion in ackA-pta produces less acetate and more isoamyl acetate than the wild-type E. coli strain. When both acetate-producing pathways were deleted, the acetate production was greatly reduced. However, pyruvate began to accumulate, so that the overall ester production remained largely unchanged. To produce more ester, a previously established strategy of increasing the flux from pyruvate to acetyl-CoA was adopted by overexpressing pyruvate dehydrogenase. The ester production was then 80% higher in the poxB, ackA-pta strain (0.18 mM) than that found in the single ackA-pta mutant (0.10 mM), which also overexpressed PDH.