Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene

Mutants of Alcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element Tn5 into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The Tn5-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a lambda L47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) SalI fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.     

Three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity from Alcaligenes eutrophus

The existence of three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity was confirmed in Alicaligenes eutrophus. The fermentative alcohol dehydrogenase, which also exhibits acetaldehyde dehydrogenase activity, is one of these proteins. The other two proteins were purified from A. eutrophus N9A mutant AS4 grown on ethanol applying chromatography on DEAE-Sephacel and triazine-dye affinity media. Acetaldehyde dehydrogenase II, which amounts to about 14% of the total soluble protein in cells grown on ethanol, was purified to homogeneity. The relative molecular masses of the native enzyme and of the subunits were 195,000 or 56,000, respectively. This enzyme exhibits a high affinity for acetaldehyde (Km = 4 microM). Acetaldehyde dehydrogenase I amounts only to less than 1% of the total soluble protein. The relative molecular masses of the native enzyme and of the subunits were 185,000 and 52,000, respectively. This enzyme exhibits a low affinity for acetaldehyde (Km = 2.6 mM). Antibodies raised against acetaldehyde dehydrogenase II did not react with acetaldehyde dehydrogenase I. Two different strains, A. eutrophus N9A mutant AS1, which represents a different mutant type and can utilize both ethanol or 2,3-butanediol, and the type strain of A. eutrophus (TF93), which can utilize ethanol, form two acetaldehyde dehydrogenases during growth on ethanol, too. As in AS4, one of these enzymes from each strain amounted to a substantial portion of the total soluble protein in the cells. These major acetaldehyde dehydrogenases were purified from both strains; they resemble acetaldehyde dehydrogenase II isolated from AS4 in all relevant properties. Antibodies against the enzyme isolated from AS4 gave identical cross-reactions with the enzymes isolated from AS1 and TF93.     

A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties

A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.     

Exocellular products from Bifidobacterium adolescentis as immunomodifiers in the lymphoproliferative responses of mouse splenocytes

Abstract Pelobacter carbinolicus strain GraBd1 fermented methylacetoin, which is a good carbon source for growth ( μ = 0.16 h⁻¹) of this strict anaerobic bacterium, to acetone, acetate and ethanol (main products), acetoin, 2,3-butanediol and methylbutanediol (minor products). During growth on 2,3-butanediol, acetoin and methyl-acetoin the formation of a protein exhibiting acetoin: DCPIP oxidoreductase activity is induced. This enzyme amounts to a substantial portion of the soluble proteins. In vitro, it cleaves acetoin into acetate and acetaldehyde but reacts also with diacetyl or methylacetoin. We discussed four different models for the degradation of acetoin in the cells and came to the conclusion that in vivo the oxidative-thiolytic acetoin cleavage model is most probably realized in P. carbinolicus .     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.     

Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation

Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP⁺-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD⁺-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.Non-standard abbreviations AADC acetoacetate decarboxylase - AcaDH acetaldehyde dehydrogenase - BuaDH butyraldehyde dehydrogenase - CoA-TF acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase - NAD-ADH, NAD⁺ dependent alcohol dehydrogenase - NADP-ADH, NADP⁺ dependent alcohol dehydrogenase     

The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation

Summary Mutants of Escherichia coli resistant to chloroethanol or to chloroacetaldehyde were selected. Such mutants were found to lack the fermentative coenzyme A (CoA) linked acetaldehyde dehydrogenase activity. Most also lacked the associated fermentative enzyme alcohol dehydrogenase. Both types of mutants, those lacking acetaldehyde dehydrogenase alone or lacking both enzymes, mapped close to the regulatory adhC gene at 27 min on the E. coli genetic map. The previously described acd mutants which lack acetaldehyde dehydrogenase and which map at 63 min were shown to be pleiotropic, affecting respiration and growth on a variety of substrates. It therefore seems likely that the structural genes for both the acetaldehyde and alcohol dehydrogenases lie in the adhCE operon. This interpretation was confirmed by the isolation of temperature sensitive chloracetaldehyde-resistant mutants, some of which produced thermolabile acetaldehyde dehydrogenase and alcohol dehydrogenase and were also found to map at the adh locus. Reversion analysis indicated that mutants lacking one or both enzymes carried single mutations. The gene order in the adh region was determined by three point crosses to be trp - zch:: Tn10 - adh - galU- bglY - tyrT - chlC.     

Fermentation enzymes in strains of Paracoccus denitrificans

Various strains of Paracoccus denitrificans grown under conditions of unrestricted oxygen supply contained low but measurable activities of fermentation enzymes such as ethanol dehydrogenase and 2,3-butanediol dehydrogenase. However, when the bacteria were subsequently incubated for up to 22 h under restricted aeration conditions permitting respiration rates of only 10 or 6% of the maximum value to occur, the above enzymes increased in specific activities by 5- or 10-fold to 0.14 mol/min·mg protein. Lactate dehydrogenase was not detected. Six strains tested reacted almost alike.Cells grown anaerobically on fructose in the presence of limiting concentrations of KNO₃ contained specific activities of up to 0.41 (in case of ethanol dehydrogenase) and 0.56 (butanediol dehydrogenase) mol/min·mg protein. Lactate dehydrogenase was only formed at low activity (0.012 mol/min·mg protein) after a long period of incubation.Cells of P. denitrificans strain Stanier 381 grown anaerobically in the chemostat on fructose+KNO₃ with either fructose or nitrate as the limiting factor differed with respect to the specific enzyme activities, too. Ethanol dehydrogenase was high under conditions of nitrate limitation and low under fructose limitation. 2,3-Butanediol dehydrogenase, but not lactate dehydrogenase, was formed in moderate activities.     

Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis

The present work aims to block 2,3-butanediol synthesis in acetoin fermentation of Bacillus subtilis. First, we constructed a recombinant strain BS168D by deleting the 2,3-butanediol dehydrogenase gene bdhA of the B. subtilis168, and there was almost no 2,3-butanediol production in 20?g/L of glucose media. The acetoin yield of BS168D reached 6.61?g/L, which was about 1.5 times higher than that of the control B. subtilis168 (4.47?g/L). Then, when the glucose concentration was increased to 100?g/L, the acetoin yield reached 24.6?g/L, but 2.4?g/L of 2,3-butanediol was detected at the end of fermentation. The analysis of 2,3-butanediol chiral structure indicated that the main 2,3-butanediol production of BS168D was meso-2,3-butanediol, and the bdhA gene was only responsible for (2R,3R)-2,3-butanediol synthesis. Therefore, we speculated that there may exit another pathway relating to the meso-2,3-butanediol synthesis in the B. subtilis. In addition, the results of low oxygen condition fermentation showed that deletion of bdhA gene successfully blocked the reversible transformation between acetoin and 2,3-butanediol and eliminated the effect of dissolved oxygen on the transformation.     

Role of Saccharomyces cerevisiae Oxidoreductases Bdh1p and Ara1p in the Metabolism of Acetoin and 2,3-Butanediol

NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.Acetoin and 2,3-butanediol are minor products generated by Saccharomyces cerevisiae during alcohol fermentation. Their sensory impacts on wine are poorly documented. Acetoin may affect the wine bouquet, although its perception threshold in wine is relatively high, around 150 mg/liter (21, 31). On the other hand, 2,3-butanediol is odorless (33) and cannot be expected to appreciably affect the sensory quality of wine. However, the compound may contribute to the wine body (28).Acetaldehyde, pyruvate, and α-acetolactate are the main precursors of acetoin in S. cerevisiae. Acetoin can be formed from acetaldehyde and/or pyruvate through an anomalous reaction of pyruvate decarboxylase. Thus, although its main activity is to irreversibly decarboxylate pyruvate to acetaldehyde, it can also catalyze carbon-carbon bond formation, yielding acetoin from pyruvate and/or acetaldehyde (2, 4). In addition, α-acetolactate would produce acetoin through its nonenzymatic decarboxylation to diacetyl and subsequent reduction to acetoin through the action of several NADH- and NADPH-dependent oxidoreductases (12). However, the situation is more complex in wine fermentation, where other yeasts and bacteria display supplementary enzymatic activities capable of producing both acetoin and 2,3-butanediol (1, 27).We have previously characterized a butanediol dehydrogenase (Bdh1p) as a medium-chain dehydrogenase/reductase (MDR) that can reversibly transform R-acetoin and S-acetoin to (2R,3R)-2,3-butanediol and meso-2,3-butanediol, respectively, in a NAD(H)-dependent reaction (10). BDH2 is a gene adjacent to BDH1 whose uncharacterized protein product (Bdh2p) shares 51% sequence identity with Bdh1p. To evaluate the in vivo roles of Bdh1p and Bdh2p, we compared the levels of several extracellular metabolites in cultures of wild-type and deficient strains. The results show that, although Bdh1p is the main enzyme in 2,3-butanediol production [essentially the (2R,3R)-2,3-butanediol stereoisomer], some meso-2,3-butanediol is still produced by the bdh1Δ strains. We have characterized Ara1p as an oxidoreductase that can reduce racemic acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol in the presence of NADPH.Furthermore, we have overexpressed Bdh2p with a histidine tag at its carboxyl terminus and have shown it to be inactive toward acetoin and 2,3-butanediol. A microarray study indicated that BDH1 and BDH2 are reciprocally regulated under the conditions studied.     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C₅-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M _r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M _r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - M _r relative molecular mass - MTPA-Cl -methoxy--trifluoromethylphenyl acetic acid chloride - PEIC 1-phenylethylisocyanate     

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine''s sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.     

Isolation and properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose.

Following treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine, three mutants of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 that produced diacetyl and acetoin from glucose were isolated. The lactate dehydrogenase activity of these mutants was strongly attenuated, and the mutants produced less lactate than the parental strain. The kinetic properties of lactate dehydrogenase of strain CNRZ 483 and the mutants revealed differences in the affinity of the enzyme for pyruvate, NADH, and fructose-1,6-diphosphate. When cultured aerobically, strain CNRZ 483 transformed 2.3% of glucose to acetoin and produced no diacetyl or 2,3-butanediol. Under the same conditions, mutants 483L1, 483L2, and 483L3 transformed 42.0, 78.9, and 75.8%, respectively, of glucose to C4 compounds (diacetyl, acetoin, and 2,3-butanediol). Anaerobically, strain CNRZ 483 produced no C4 compounds, while mutants 483L1, 483L2, and 483L3 transformed 2.0, 37.0, and 25.8% of glucose to acetoin and 2,3-butanediol. In contrast to the parental strain, the NADH balance showed that the mutants regenerated most of the NAD via NADH oxidase under aerobic conditions and by ethanol production under anaerobic conditions.     

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.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Saccharomyces cerevisiae NAD(H)‐dependent 2,3‐butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3‐butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)‐dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu²²¹, Ile²²², and Ala²²³) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site‐directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. Biotechnol. Bioeng. 2009; 104: 381–389 © 2009 Wiley Periodicals, Inc.     

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.     

Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene

The completion of the Saccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD(+), but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2, 3-butanediol (K(m) = 14 mm, k(cat) = 78,000 min(-)(1)) and meso-butanediol (K(m) = 65 mm, k(cat) = 46,000 min(-)(1)) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K(m) = 4.5 mm, k(cat) = 98,000 min(-)(1)) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3. 5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R, 3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.