Engineering of the Pyruvate Dehydrogenase Bypass in Saccharomyces cerevisiae: Role of the Cytosolic Mg2+ and Mitochondrial K+ Acetaldehyde Dehydrogenases Ald6p and Ald4p in Acetate Formation during Alcoholic Fermentation

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate by Saccharomyces cerevisiae during alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressing ACS2 and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies of ALD6, encoding the cytosolic Mg²⁺-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion of ALD4, encoding the mitochondrial K⁺-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, the ald6 ald4 double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of the ALD family.     

Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae: role of the cytosolic Mg(2+) and mitochondrial K(+) acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate by Saccharomyces cerevisiae during alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressing ACS2 and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies of ALD6, encoding the cytosolic Mg(2+)-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion of ALD4, encoding the mitochondrial K(+)-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, the ald6 ald4 double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of the ALD family.     

Response of wine yeast (Saccharomyces cerevisiae) aldehyde dehydrogenases to acetaldehyde stress during Icewine fermentation

AIMS: We previously reported that the aldehyde dehydrogenase encoded by ALD3 but not ALD6 was responsible, in part, for the increased acetic acid found in Icewines based on the expression profile of these genes during fermentation. We have now completed the expression profile of the remaining yeast aldehyde dehydrogenase genes ALD2, ALD4 and ALD5 during these fermentations to determine their contribution to acetic acid production. The contribution of acetaldehyde stress as a signal to stimulate ALD expression during these fermentations was investigated for all ALD genes. The expression of glycerol-3-phosphate encoded by GPD2 was also followed during these fermentations to determine its role in addition to the role we already identified for GPD1 in the elevated glycerol produced during Icewine fermentation. METHODS AND RESULTS: Icewine juice (38.5 degrees Brix, 398 +/- 5 g l(-1) sugar), diluted Icewine juice (20.8 degrees Brix, 196 +/- 4 g l(-1) sugar) and the diluted juice with sugar levels equal to the original Icewine juice (36.6 degrees Brix, 395 +/- 6 g l(-1) sugar) were fermented in duplicate using the commercial wine yeast K1-V1116. Acetic acid and glycerol production increased 8.4- and 2.7-fold in the Icewine vs the diluted juice fermentation, respectively, accompanied by a fourfold transient increase in acetaldehyde in the Icewine condition during the first week. Both mitochondrial aldehyde dehydrogenases encoded by ALD4 and ALD5 were expressed, with ALD5 expression highest at the start of all fermentations and ALD4 expression increasing during the first week of each condition. ALD2, ALD4, ALD5 and GPD2 showed no differential expression between the three fermentation conditions indicating their lack of involvement in elevating acetic acid and glycerol in Icewine. When yeast fermenting the diluted fermentation was exposed to exogenous acetaldehyde, the transient spike in acetaldehyde increased the expression of ALD3 but this response alone was not sufficient to cause an increase in acetic acid. Expression of the other aldehyde dehydrogenases was unaffected by the acetaldehyde addition. CONCLUSIONS: The aldehyde dehydrogenases encoded by ALD2, ALD4 and ALD5 do not contribute to the elevated acetic acid production during Icewine fermentation. Expression of GPD2 was not upregulated in high sugar fermentations and does not reflect the elevated levels of glycerol found in these wines. Acetaldehyde at a concentration produced during Icewine fermentation stimulates the expression of ALD3, but has no impact on the expression of ALD2, -4, -5 and -6. Upregulation of ALD3 alone in the dilute fermentation is not sufficient to increase acetic acid in wine and requires additional responses found in cells under hyperosmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work confirms that increased acetic acid and glycerol production during Icewine fermentation follows upregulation of ALD3 and GPD1 respectively, but upregulation of ALD3 alone is not sufficient to increase acetic acid production. Additional responses of cells under osmotic stress are required to increase acetic acid in Icewine.     

Effects of GPD1 Overexpression in Saccharomyces cerevisiae Commercial Wine Yeast Strains Lacking ALD6 Genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP⁺-dependent Mg²⁺-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

Sources of NADPH in yeast vary with carbon source

Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984-13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1Delta ald6Delta mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1Delta idp2Delta mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1Delta ald6Delta mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Delta ald6Delta mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1Delta ald6Delta mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP+ rise dramatically in the zwf1Delta idp2Delta mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.     

Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

Alcohol induced hepatic fibrosis: role of acetaldehyde

Alcohol abuse is one of the major causes of liver fibrosis worldwide. Although the pathogenesis of liver fibrosis is a very complex phenomenon involving different molecular and biological mechanisms, several lines of evidence established that the first ethanol metabolite, acetaldehyde, plays a key role in the onset and maintenance of the fibrogenetic process. This review briefly summarizes the molecular mechanisms underlying acetaldehyde pro-fibrogenic effects. Liver fibrosis represents a general wound-healing response to a variety of insults. Although mortality due to alcohol abuse has been constantly decreasing in the past 20 years in Southern Europe and North America, in several Eastern-European countries and Great Britain Alcoholic Liver Disease (ALD) shows a sharply increasing trend [Bosetti, C., Levi, F., Lucchini, F., Zatonski, W.A., Negri, E., La, V.C., 2007. Worldwide mortality from cirrhosis: an update to 2002. J. Hepatol. 46, 827-839]. ALD has a complex pathogenesis, in which acetaldehyde (AcCHO), the major ethanol metabolite, plays a central role. Ethanol is mainly metabolized in the liver by two oxidative pathways. In the first one ethanol is oxidized to acetaldehyde by the cytoplasmic alcohol dehydrogenase enzyme (ADH), acetaldehyde is then oxidized to acetic acid by the mitochondrial acetaldehyde dehydrogenase (ALDH). The second pathway is inducible and involves the microsomal ethanol-oxidizing system (MEOS), in which the oxidation of ethanol to acetaldehyde and acetic acid also leads to generation of reactive oxygen species (ROS). Chronic ethanol consumption significantly inhibits mitochondrial ALDH activity while the rate of ethanol oxidation to acetaldehyde is even enhanced, resulting in a striking increase of tissue and plasma acetaldehyde levels [Lieber, C.S., 1997. Ethanol metabolism, cirrhosis and alcoholism. Clin. Chim. Acta 257, 59-84]. This review will focus on the molecular mechanisms by which acetaldehyde promote liver fibrosis.     

Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing,Low-Alcohol Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1_221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3°.A large number of quality wines produced by modern winemaking practices, which favor harvesting fully ripened grapes, frequently contain an excessive ethanol content. This tendency is observed in the majority of the world''s wine-producing areas, and reducing the alcohol levels in wines has become a major concern of the wine industry. Consequently, numerous attempts have been made to engineer Saccharomyces cerevisiae yeast strains with reduced ethanol yields, which would offer faster and less expensive biological alternatives to the current physical processes available for the production of low- and reduced-alcohol wines (29). The biological approaches used so far are all based on diverting sugar metabolism toward by-products other than ethanol by metabolic engineering strategies (7, 8, 18, 19, 21, 22, 30). However, these strategies have so far not satisfied the need to obtain a significant reduction in the ethanol yield without causing the accumulation of undesirable secondary products and/or without affecting yeast physiology. Among these various advances, an efficient strategy is based on the rerouting of the carbon flux toward the production of glycerol. This polyol is a relatively neutral compound from an olfactory perspective, and it is has been demonstrated previously to contribute positively to wine quality through enhanced sweetness and viscosity (27). In yeast, glycerol plays a major role as an osmolyte under osmotic stress conditions and also functions as an essential redox sink in the absence of oxygen, when the reoxidation of excess cytosolic NADH is required (1, 2, 42, 44, 46). This compound is formed by the reduction of dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase (encoded by GPD1 and GPD2), followed by dephosphorylation by glycerol-3-phosphatase, which exists as two isoforms: Gpp1 and Gpp2p (see Fig. ​Fig.1).1). By overexpressing GPD1 or GPD2, the production of glycerol has been greatly enhanced, making it possible to decrease the ethanol yield as the result of carbon diversion and reduced NADH availability for the alcohol dehydrogenase reaction. This strategy has been used previously to reduce the ethanol yields in wine and brewer''s yeast (4, 7, 23, 25, 26, 31). For wine, it was shown that these glycerol-overproducing strains have the potential to reduce the ethanol content by 1 to 2°. Nevertheless, major modifications in the production levels of other metabolites, in particular acetate and acetoin (23, 31), which are undesirable at high concentrations in wine, are generated. The production of acetate in glycerol-overproducing wine yeasts has been reduced to a normal level by the deletion of ALD6, coding for an acetaldehyde dehydrogenase (4, 30). The major problem of the accumulation of acetoin, which was shown to accumulate at several grams per liter in commercial GPD1 ald6 wine yeast strains (4), remains to be overcome. At usual concentrations in wine, which vary from undetectable levels to 80 mg/liter (32, 33, 40), this compound has no negative organoleptic influence. However, at concentrations higher than its threshold level (around 150 mg/liter [11]), acetoin can confer an unpleasant buttery flavor on wines. In contrast, the reduced form of acetoin, 2,3-butanediol (2,3-BD), has neutral sensory qualities (data not shown). It is found in wines at concentrations ranging from 0.2 to 3 g/liter (14, 41).Open in a separate windowFIG. 1.Schematic representation of metabolic pathways implicated in our design strategy for a low-alcohol yeast. GP, glycerol phosphatase, encoded by GPP1 and GPP2; GPDH, glycerol phosphate dehydrogenase, encoded by GPD1 and GPD2; PDC, pyruvate decarboxylase, encoded by PDC1, PDC5, and PDC6; ACDH, acetaldehyde dehydrogenase, encoded by ALD4, ALD5, and ALD6; ADH, alcohol dehydrogenase, encoded by ADH1; BDH, Bdh1, encoded by BDH1 (other BDHs exist; however, no other identified gene has been associated with BDH activity); ALS, acetolactate synthase, encoded by ILV2; DS, diacetyl synthetase; DR, diacetyl reductase; G3P, glycerol-3-phosphatase; DHAP, dihydroxyacetone phosphate; acetyl CoA, acetyl coenzyme A; TPP, thiamine PP_i.Bdh1, encoded by BDH1, is the only identified enzyme in yeast catalyzing the reduction of acetoin into 2,3-BD (12). This enzyme has strict stereospecificity for the OHs of carbons in R configuration and acts preferentially as a reductase rather than as a dehydrogenase (11, 12). It is essentially responsible for the formation of (2R,3R)-2,3-BD and part of meso-2,3-BD from (3R)-acetoin and (3S)-acetoin, respectively.The accumulation of acetoin in strains engineered for glycerol overproduction has been attributed to several factors (4). On one hand, it was assumed that the amount of Bdh1 is a rate-limiting factor in the conversion of acetoin into 2,3-BD. On the other hand, it is possible that the Bdh1 reaction is limited by the low level of available NADH since this coenzyme is preferentially used for glycerol synthesis in these strains.The aim of the present study was to investigate in detail the metabolic prerequisites for reducing accumulated acetoin in S. cerevisiae overproducing glycerol and exhibiting reduced acetate formation by promoting the conversion of acetoin into the compound 2,3-BD, which has neutral sensory characteristics. In this study, we first determined the role of Bdh1 in the reduction of acetoin into 2,3-BD during wine fermentation. Next, we studied the impact of the overproduction of NADH-dependent Bdh1 or an engineered NADPH-dependent form of Bdh1 in a model wine yeast, V5, overexpressing GPD1 and lacking ALD6 during fermentation in synthetic must with various sugar concentrations. The NADPH-dependent Bdh1 has been obtained previously by the replacement of three amino acids involved in the NADH binding domain, resulting in the complete reversal of the coenzyme specificity from NADH to NADPH (6). The effects on the growth and fermentation properties of the engineered strains and the levels of by-products and key aromatic compounds formed by the strains were determined.     

Regulatory mutations that allow the growth ofEscherichia coli on butanol as carbon source

Summary Starting withadhC mutants ofEscherichia coli in which alcohol dehydrogenase (ADH) and acetaldehyde CoA dehydrogenase (ACDH) are expressed constitutively at high levels, we selected mutants with still higher levels of both enzymes. Selection for growth on ethanol in the presence of inhibitors of ADH gave several mutants that had from 2- to 10-fold increases in the levels of both enzymes. These mutations were found to map far from theadhC locus at around 90 min. SuchadhR mutants were unable to grow on acetate or ethanol in certain media unless supplemented with extramanganese. This growth disability was suppressed by secondary mutations, one of which,aceX, increased sensitivity to several toxic metals and may perhaps derepress Mn transport. When theadhR mutation expressing the highest ADH and ACDH levels was present together withfadR andatoC mutations (allowing efficient catabolism of acetoacetyl-CoA) and with anaceX mutation, the resulting strains became capable of usingn-butanol as sole carbon and energy source. The use of butanol byE. coli illustrates the artificial evolution of a new catabolic pathway, in this case by the selection of four successive regulatory mutations (fadR, adhC, atoC, andadhR) together with the poorly definedaceX mutation. Each stage in the acquisition of this nove pathway confers the ability to use a new growth substrate: decanoic acid (fadR), ethanol (adhC), butyric acid (atoC), and butanol (adhR, when present withaceX).     

Enzymes involved in vinyl acetate decomposition by Pseudomonas fluorescens PCM 2123 strain

Esterases are widely used in food processing industry, but there is little information concerning enzymes involved in decompositions of esters contributing to pollution of environment. Vinyl acetate (an ester of vinyl alcohol and acetic acid) is a representative of volatile organic compounds (VOCs) in decomposition, of which hydrolyses and oxidoreductases are mainly involved. Their activities under periodically changing conditions of environment are essential for the removal of dangerous VOCs. Esterase and alcohol/aldehyde dehydrogenase activities were determined in crude cell extract from Pseudomonas fluorescens PMC 2123 after vinyl acetate induction. All examined enzymes exhibit their highest activity at 30–35 °C and pH 7.0–7.5. Esterase preferably hydrolyzed ester bonds with short fatty chains without plain differences for C₂ or C₄. Comparison of Km values for alcohol and aldehyde dehydrogenases for acetaldehyde suggested that this metabolite was preferentially oxidized than reduced. Activity of alcohol dehydrogenase reducing acetaldehyde to ethanol suggested that one mechanism of defense against the elevated concentration of toxic acetaldehyde could be its temporary reduction to ethanol. Esterase activity was inhibited by phenylmethanesulfonyl fluoride, while β-mercaptoethanol, dithiothreitol, and ethylenediaminetetraacetic acid had no inhibitor effect. From among metal ions, only Mg²⁺ and Fe²⁺ stimulated the cleavage of ester bond.     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Growth and fermentation behaviour of Brettanomyces/Dekkera yeasts under different conditions of aerobiosis

Brettanomyces/Dekkera yeasts grow in wine and their presence is often associated with spoiling activity. In this report, we investigated on the influence of different conditions of aerobiosis on growth and fermentation behaviour of these spoilage yeasts in wine. Results showed that in all conditions tested the Brettanomyces strain consumed all sugars, taking wine fermentation to completion. Strict-anaerobic conditions limited the growth of Brettanomyces. Both anaerobiosis (using a fermentation trap) and strict anaerobiosis did not negatively affect the principal by-products of fermentation whereas semi-anaerobiosis caused an increase of acetic acid, acetaldehyde and ethyl acetate that negatively affected the fermentation profile of resulting products.     

Fermentation characteristics of hybrids between the cryophilic wine yeast Saccharomyces bayanus and the mesophilic wine yeast Saccharomyces cerevisiae

The cryophilic wine yeasts Saccharomyces bayanus YM-84 and YM-126 were used for hybridization with the mesophilic wine yeast Saccharomyces cerevisiae OC-2. All six hybrids were stable in tetrad analysis and pulsed field gel electrophoresis, even after twenty subcultures over two years. The fermentabilities of these hybrids at a low temperature of 7°C were superior to the mesophilic wine yeast and the same as the cryophilic wine yeasts. Conversely, their fermentabilities at the intermediate temperatures of 28 and 35°C were similar to the mesophilic wine yeast. For laboratory-scale wine-making using Koshu grape juice at 10°C, the fermentability of these hybrids was superior to the mesophilic wine yeast. They also produced higher amounts of malic acid and flavor compounds such as higher alcohols, β-phenylethyl alcohol, isoamyl acetate and β-phenylethyl acetate, and lower amounts of acetic acid than those of OC-2. These results suggest that the cryophilic wine yeast S. bayanus is useful for improving the low temperature fermentation ability of wine yeast strains.     

Glycerol Overproduction by Engineered Saccharomyces cerevisiae Wine Yeast Strains Leads to Substantial Changes in By-Product Formation and to a Stimulation of Fermentation Rate in Stationary Phase

Six commercial wine yeast strains and three nonindustrial strains (two laboratory strains and one haploid strain derived from a wine yeast strain) were engineered to produce large amounts of glycerol with a lower ethanol yield. Overexpression of the GPD1 gene, encoding a glycerol-3-phosphate dehydrogenase, resulted in a 1.5- to 2.5-fold increase in glycerol production and a slight decrease in ethanol formation under conditions simulating wine fermentation. All the strains overexpressing GPD1 produced a larger amount of succinate and acetate, with marked differences in the level of these compounds between industrial and nonindustrial engineered strains. Acetoin and 2,3-butanediol formation was enhanced with significant variation between strains and in relation to the level of glycerol produced. Wine strains overproducing glycerol at moderate levels (12 to 18 g/liter) reduced acetoin almost completely to 2,3-butanediol. A lower biomass concentration was attained by GPD1-overexpressing strains, probably due to high acetaldehyde production during the growth phase. Despite the reduction in cell numbers, complete sugar exhaustion was achieved during fermentation in a sugar-rich medium. Surprisingly, the engineered wine yeast strains exhibited a significant increase in the fermentation rate in the stationary phase, which reduced the time of fermentation.