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.     

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.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

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.     

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.     

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.     

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 atf1Delta atf2Delta 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.     

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

Aim: 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. Methods and Results: 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. Conclusions: 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. Significance and Impact of the Study: 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.     

Detection of spoilage-causing yeasts and bacteria in tchapalo,the Ivorian traditional sorghum beer

In this study, we aimed to analyse the spoilage potential of the isolated yeast, LAB and AAB species. Thus, 11 strains were inoculated at 0·3% (v/v) into a sterile filtered tchapalo and stored for 3 days at ambient temperature (27–30°C). All the tested strains grew well or remained stable except for Limosilactobacillus fermentum and Pediococcus acidilactici, which decreased throughout the storage time. A significant decrease of total soluble solids was observed only for Saccharomyces cerevisiae (from 7·8 to 5·8 °Brix) and Meyerozyma guilliermondii (from 7·8 to 5·5 °Brix). The tchapalo samples inoculated with the LAB strains Weissella paramesenteroides, P. acidilactici, L. fermentum and the yeast strain Candida tropicalis were judged similar to the control by the panellists. However, the strains of Lacticaseibacillus paracasei and Latilactobacillus curvatus (LAB), S. cerevisiae, M. guilliermondii and Kluyveromyces marxianus (yeasts) and Acetobacter pasteurianus and A. cerevisiae (AAB) induced the spoilage of the tchapalo appearance, smell and/or taste. In the spoiled tchapalo quantitative and qualitative modification of some volatile compounds (VOCs), such as lilac aldehyde, ethyl acetate, ethyl hexanoate, ethyl octanoate and phenethyl acetate, were observed. These results provide information about the microorganisms that need to be removed to extend the shelf life of tchapalo.     

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.     

Acetic acid removal from corn stover hydrolysate using ethyl acetate and the impact on Saccharomyces cerevisiae bioethanol fermentation

Acetic acid is introduced into cellulose conversion processes as a consequence of composition of lignocellulose feedstocks, causing significant inhibition of adapted, genetically modified and wild‐type S. cerevisiae in bioethanol fermentation. While adaptation or modification of yeast may reduce inhibition, the most effective approach is to remove the acetic acid prior to fermentation. This work addresses liquid–liquid extraction of acetic acid from biomass hydrolysate through a pathway that mitigates acetic acid inhibition while avoiding the negative effects of the extractant, which itself may exhibit inhibition. Candidate solvents were selected using simulation results from Aspen Plus?, based on their ability to extract acetic acid which was confirmed by experimentation. All solvents showed varying degrees of toxicity toward yeast, but the relative volatility of ethyl acetate enabled its use as simple vacuum evaporation could reduce small concentrations of aqueous ethyl acetate to minimally inhibitory levels. The toxicity threshold of ethyl acetate, in the presence of acetic acid, was found to be 10 g L^?1. The fermentation was enhanced by extracting 90% of the acetic acid using ethyl acetate, followed by vacuum evaporation to remove 88% removal of residual ethyl acetate along with 10% of the broth. NRRL Y‐1546 yeast was used to demonstrate a 13% increase in concentration, 14% in ethanol specific production rate, and 11% ethanol yield. This study demonstrated that extraction of acetic acid with ethyl acetate followed by evaporative removal of ethyl acetate from the raffinate phase has potential to significantly enhance ethanol fermentation in a corn stover bioethanol facility. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:929–937, 2016     

The chemical composition,antifungal, antioxidant and antimutagenicity properties of bioactive compounds from fungal endophytes associated with Thai orchids

Some plant‐derived bioactive compounds produced by fungal endophytes have been proven to have antimicrobial and antioxidant activities. In this study, endophytic fungi were isolated from 20 orchid samples collected in northern Thailand from 12 genera of orchids. In total, 97 isolates were isolated from the leaves (44.3%), stems (40.2%) and flowers (15.5%) of the orchid samples. The antifungal activity was investigated of the endophytic isolates against the plant pathogenic fungi. The results showed that 13 endophytic isolates provided antifungal activities against Fusarium sp., Colletotrichum sp. and Curvularia sp. The endophyte CK F05‐5, which was isolated from the flower part of Dendrobium lindleyi, was chosen for further testing because it the highest level of antifungal activity against Fusarium sp. The isolate CK F05‐5 was identified as Fusarium oxysporum on the basis of its ITS sequences of 5.8 s rRNA, and phytochemical analysis revealed the presence of coumarins. The ethyl acetate extract of CK F05‐5 was examined for its total phenolic content and antioxidant activity using Folin–Ciocalteu's reagent and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging assay, respectively. The phenolic content was 160.51 mg of GAE/g of extract, and the free radical scavenging activity was 89.61 µg/ml at the half maximal inhibitory concentration (IC₅₀). The antimutagenic potential of the ethyl acetate extract of CK F05‐5 against Trp‐P‐1 mutagenic substances was determined using the Ames test which revealed that the extract of CK F05‐5 at 10 mg/plate had the highest antimutagenic activity against Trp‐P‐1 (51.2%) and 39.6% for strains TA98 and TA100, respectively. The active compounds present in the acetate extract of CK F05‐5 were examined using GC‐MS analysis, which displayed the presence of gibepyrone A, pyrrolo [1, 2‐a] pyrazine‐1, 4‐dione, hexahydro‐3‐(2‐methylpropyl) and indoleacetic acid as major components. Based on the results, this endophytic fungus contains various bioactive components that have various biological activities. This useful information could help in producing potentially valuable and novel pharmaceutical products.     

Sex pheromone of a North American population of the spotted tentiform leafminer, Phyllonorycter blancardella

The major sex pheromone compound of the spotted tentiform leafminer, Phyllonorycter blancardella (F.) (Lepidoptera: Gracillariidae), from Ontario, Canada, was identified as (10E)‐dodecen‐1‐yl acetate (E10‐12:Ac) using chemical analysis and field trapping experiments. The minor compounds (10E)‐dodecen‐1‐ol (E10‐12:OH) (4.6%), dodecan‐1‐ol (12:OH) (2.3%), and (10Z)‐dodecen‐1‐yl acetate (Z10‐12:Ac) 1.6% were also identified. The dienic acetate (4E,10E)‐dodecadien‐1‐yl acetate (E4,E10‐12:Ac), a compound reported to be attractive to P. blancardella, was not found in the glands of this population. A two‐component blend of the major and one of each the three minor compounds, in ratios similar to those found in the sex pheromone gland, did not increase the attractiveness of traps baited with synthetic pheromone. The minor compounds E10‐12:OH and 12:OH were not attractive to P. blancardella when tested individually. Z10‐12:Ac was attractive to P. blancardella, although traps baited with this compound captured only 2% of the moths that were captured in traps baited with the main compound. A four‐component blend of the major and each of the three minor compounds (100 : 1 : 1 : 1) was not more attractive than the major compound alone. The related species Phyllonorycter mespilella was captured in traps baited with E10‐12:Ac.     

Controlled formation of volatile components in cider making using a combination of Saccharomyces cerevisiae and Hanseniaspora valbyensis yeast species

The effect of pure and mixed fermentation by Saccharomyces cerevisiae and Hanseniaspora valbyensis on the formation of major volatile components in cider was investigated. When the interaction between yeast strains of S. cerevisiae and H. valbyensis was studied, it was found that the two strains each affected the cell growth of the other upon inoculation of S. cerevisiae during growth of H. valbyensis. The effects of pure and mixed cultures of S. cerevisiae and H. valbyensis on alcohol fermentation and major volatile compound formation in cider were assessed. S. cerevisiae showed a conversion of sugar to alcohol of 11.5%, while H. valbyensis produced alcohol with a conversion not exceeding 6%. Higher concentrations of ethyl acetate and phenethyl acetate were obtained with H. valbyensis, and higher concentrations of isoamyl alcohol and isobutyl were formed by S. cerevisiae. Consequently, a combination of these two yeast species in sequential fermentation was used to increase the concentration of ethyl esters by 7.41–20.96%, and to decrease the alcohol concentration by 25.06–51.38%. Efficient control of the formation of volatile compounds was achieved by adjusting the inoculation time of the two yeasts.     

Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with <Emphasis Type="Italic">OPI1</Emphasis> deletion

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.     

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.     

One‐pot synthesis of (R)‐1‐(pyridin‐4‐yl)ethyl acetate using tandem catalyst prepared by co‐immobilization of palladium and lipase on mesoporous foam: Optimization and kinetic modeling

The synthesis of (R )‐1‐(pyridin‐4‐yl)ethyl acetate was achieved over tandem palladium‐lipase catalyst with 100% selectivity using 4‐acetyl pyridine as a reactant. The 2% w /w palladium and lipase catalyst was successfully co‐immobilized in the microenvironment of the mesocellular foam and characterized by various techniques. The palladium metal from catalyst hydrogenated 4‐acetyl pyridine to form 1‐(pyridin‐4‐yl)ethanol. The generated intermediate product then underwent kinetic resolution over lipase and selectively gave (R )‐1‐(pyridin‐4‐ yl)ethyl acetate. The catalytic conditions were then studied for optimal performance of both steps. The reaction conditions were optimized to 50 °C and toluene as a solvent. Both chemical and enzymatic kinetic models of the reaction were developed for a given set of reaction conditions and kinetic parameters were predicted. At optimal conditions, the obtained selectivity of intermediate (1‐(pyridin‐4‐yl)ethanol) was 51.38%. The final product yield of ((R )‐1‐(pyridin‐4‐yl)ethyl acetate) was 48.62%.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey: Influence of aeration and inhibition of yeast growth by ethyl acetate

The ability of the yeast Kluyveromyces marxianus to convert lactose into ethyl acetate offers good opportunities for the economical reuse of whey. The formation of ethyl acetate as a bulk product depends on aerobic conditions. Aeration of the bioreactor results in discharge of the volatile ester with the exhaust gas that allows its process‐integrated recovery. The influence of aeration (varied from 10 to 50 L/h) was investigated during batch cultivation of K. marxianus DSM 5422 in 0.6 L whey‐borne medium using a stirred reactor. With lower aeration rates, the ester accumulated in the bioreactor and reached higher concentrations in the culture medium and the off gas. A high ester concentration in the gas phase is considered beneficial for ester recovery from the gas, while a high ester concentration in the medium inhibited yeast growth and slowed down the process. To further investigate this effect, the inhibition of growth by ethyl acetate was studied in a sealed cultivation system. Here, increasing ester concentrations caused a nearly linear decrease of the growth rate with complete inhibition at concentrations greater than 17 g/L ethyl acetate. Both the cultivation process and the growth rate depending on ethyl acetate were described by mathematical models. The simulated processes agreed well with the measured data.     

Synthesis of optically active ethyl 4-chloro-3-hydroxybutanoate by microbial reduction

 A total of 400 yeast strains were examined for the ability to reduce ethyl 4-chloroacetoacetate (COBE) to ethyl 4-chloro-3-hydroxybutyrate (CHBE) by using acetone-dried cells in the presence of a coenzyme-recycling system in water/n-butyl acetate. We discovered some yeast strains that reduced COBE to (S)-CHBE. Heating of acetone-dried cells of the selected yeast strains increased the optical purity of the product. There may be several enzymes that can reduce COBE stereoselectively in the same yeast cells. The cultured broth of Candida magnoliae accumulated 90 g/l (S)-CHBE (96.6% enantiomeric excess, e.e.) in the presence of glucose, NADP and glucose dehydrogenase in n-butyl acetate. When these cells were heated, the stereoselectivity of the reduction increased to 99% e.e. (S)-CHBE is one of the useful chiral building blocks applicable to the synthesis of some pharmaceuticals. We expect that the cheap and industrial production of this important chiral compound will follow the discovery of this yeast strain. Received: 9 September 1998 / Received last revision: 17 February 1999 / Accepted: 5 March 1999