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PubMed1996

A detoxication route for acetaldehyde: metabolism of diacetyl, acetoin, and 2,3-butanediol in liver homogenate and perfused liver of rats.

The metabolism of diacetyl (2,3-butanedione), acetoin (3-hydroxy-2-butanone), and 2,3-butanediol, which are metabolites of acetaldehyde was quantitatively investigated using rat liver homogenate, liver perfusion, and in vivo experiments. Diacetyl and acetoin were reduced to 2,3-butanediol in these experiments, but acetoin and 2,3-butanediol were scarcely oxidized to diacetyl, indicating that the reduction reaction to 2,3-butanediol from diacetyl occurs actively in rat liver. The formation of acetoin from diacetyl required either NADH or NADPH as a reductant, while the reduction of acetoin to 2,3-butanediol required NADH. Acetoin and 2,3-butanediol were more readily accumulated than diacetyl in brain tissue.

Acetaldehyde↗

PubMed1989

Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus.

In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used Tn5::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposon-harboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutorphus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of Tn5 were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a lambda L47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of Tn5 covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of Tn5::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.

Acetoin↗

PubMed1994

Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system.

E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.

Acetoin Dehydrogenase↗

PubMed1993

Biometric Study of Acetoin Production in Hanseniaspora guilliermondii and Kloeckera apiculata.

Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of Hanseniaspora guilliermondii and Kloeckera apiculata were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (P = 0.001) in acetoin production and K. apiculata exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with Saccharomyces cerevisiae seems promising for optimization of wine bouquet.

Journal Article↗

PubMed1993

Effect of Initial Oxygen Concentration on Diacetyl and Acetoin Production by Lactococcus lactis subsp. lactis biovar diacetylactis.

The production of aroma compounds (acetoin and diacetyl) in fresh unripened cheese by Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 was studied at 30 degrees C at different initial oxygen concentrations (0, 21, 50, and 100% of the medium saturation by oxygen). Regardless of the initial O(2) concentration, maximal production of these compounds was reached only after all the citrate was consumed. Diacetyl and acetoin production was 0.01 and 2.4 mM, respectively, at 0% oxygen. Maximum acetoin concentration reached 5.4 mM at 100% oxygen. Diacetyl production was increased by factors of 2, 6, and 18 at initial oxygen concentrations of 21, 50, and 100%, respectively. The diacetyl/acetoin concentration ratio increased linearly with initial oxygen concentration: it was eight times higher at 100% (3.3%) than at 0% oxygen (0.4%). The effect of oxygen on diacetyl and acetoin production was also shown with other lactococci. At 0% oxygen, specific activity of alpha-acetolactate synthetase (0.15 U/mg) and NADH oxidase (0.04 U/mg) was 3.6 and 5.4 times lower, respectively, than at 100% oxygen. The increasing alpha-acetolactate synthetase activity in the presence of oxygen would explain the higher production of diacetyl and acetoin. The NADH oxidase activity would replace the role of the lactate dehydrogenase, diacetyl reductase, and acetoin reductase in the reoxidation of NADH, allowing accumulation of these two aroma compounds.

Journal Article↗

PubMed1971

Diacetyl and acetoin production by Lactobacillus casei.

Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension.

Acetates↗

PubMed2001

Regulation of the acetoin catabolic pathway is controlled by sigma L in Bacillus subtilis.

Bacillus subtilis grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. The acoABCL operon encodes the E1alpha, E1beta, E2, and E3 subunits of the acetoin dehydrogenase complex in B. subtilis. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The acoR gene is located downstream from the acoABCL operon and encodes a positive regulator which stimulates the transcription of the operon. The product of acoR has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a -12, -24 promoter, and transcription is abolished in acoR and sigL mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an acoR'-'lacZ strain of B. subtilis showed that the expression of acoR is not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of acoR is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region of acoR was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of acoR is mediated by the binding of CcpA to the promoter region of acoR.

Acetoin↗

PubMed2002

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae.

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.

Acetoin↗

PubMed1989

Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate.

Dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase were formed when Pelobacter carbinolicus strain GraBd1 was grown on acetoin. The specific activities of these enzymes amounted to 0.50 and 28.7 U/mg protein, respectively. The crude extract catalyzed the CoASH- and NAD+-dependent formation of acetyl-CoA from acetoin and methylacetoin. From ethylene glycol-grown cells these activities were absent. Crude extracts also exhibited acetoin: methyl viologen and acetoin: metronidazole oxidoreductase activity. As shown by reconstitution experiments methylviologen reduction was dependent on the presence of a light-brownish protein (Mr 220,000 +/- 10,000); metronidazole reduction was in addition dependent on the presence of a dark-brownish protein (Mr 4,900 +/- 800), which is probably a ferredoxin. However, both components were synthesized constitutively. We discussed a model for oxidative-thiolytic cleavage of acetoin which is analogous to the reaction of the pyruvate dehydrogenase enzyme complex rather than to pyruvate: ferredoxin oxidoreductase.

Acetoin↗

PubMed1992

Simple and sensitive determination of diacetyl and acetoin in biological samples and alcoholic drinks by gas chromatography with electron-capture detection.

Acetoin was quantitatively oxidized into diacetyl by Fe3+ in 1 M perchloric acid. The reaction of diacetyl with 4,5-dichloro-1,2-diaminobenzene afforded 6,7-dichloro-2,3-dimethylquinoxaline (DCDMQ), which was extracted by benzene containing aldrin (25 ng/ml) as an internal standard, and determined by gas chromatography with electron-capture detection. The method is very simple and sensitive. The detection limit of DCDMQ (either diacetyl or acetoin) was 10 fmol/microliters of the benzene extract, and the determination limit of DCDMQ (either diacetyl or acetoin) was 50 fmol/microliters of the extract. Both acetoin and diacetyl could be determined in 0.1 ml of normal human urine or blood, and both were found in rat liver, kidney and brain. The method was also applied to the determination of acetoin and diacetyl in alcoholic drinks.

Acetoin↗

PubMed2005

Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate-responsive LysR-type regulator AlsR in Vibrio cholerae.

AphA is a quorum sensing-regulated activator that initiates the virulence cascade in Vibrio cholerae by cooperating with the LysR-type regulator AphB at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphA, we carried out a microarray analysis and show here that AphA influences the expression of 15 genes not associated with the VPI. One set of genes strongly repressed by AphA is involved in the biosynthesis of acetoin, a product synthesized by a variety of bacteria that plays a role in preventing intracellular acidification and which is essential for the viability of V. cholerae in the presence of glucose. Also present in this operon are two putative signal transduction proteins with EAL and GGDEF domains that oppositely influence motility and biofilm formation. Gel mobility shift assays show that AphA binds to a site upstream of the first gene in the acetoin operon. Transcriptional lacZ fusions indicate that at low cell density AphA represses the expression of the acetoin genes up to 15-fold. Voges Proskauer tests confirm that deletion of AphA increases the production of acetoin under non-inducing conditions and also that the LysR-type regulator AlsR divergently transcribed from the operon is required for its production. This is the first report of a specific repressor protein involved in the transcriptional control of acetoin production as well as the co-regulation of these genes with those that influence motility and biofilm formation. The results here provide a model for the dual regulation of these processes by acetate and quorum sensing through AlsR and AphA.

Acetoin↗

PubMed1994

Identification and molecular characterization of the aco genes encoding the Pelobacter carbinolicus acetoin dehydrogenase enzyme system.

Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes.

Acetoin Dehydrogenase↗

PubMed1971

Metabolism of acetoin in mammalian liver slices and extracts. Interconversion with butane-2,3-diol and biacetyl.

1. [(14)C]Acetoin was enzymically synthesized from [(14)C]pyruvate with a pyruvate decarboxylase preparation. Its optical activity was [alpha](20) (d)-78 degrees . 2. Large amounts (1000-fold higher than physiological concentrations) of acetoin were incubated with rat liver mince. Acetoin disappeared but very little (14)CO(2) was evolved. A compound accumulated, which was purified and identified as butane-2,3-diol. Chromatography on borate-impregnated paper indicated the presence of both the erythro and threo forms. 3. Liver extracts capable of interconverting biacetyl, acetoin and butane-2,3-diol were obtained. These interconversions were catalysed by two different enzymes: acetoin dehydrogenase (EC 1.1.1.5) and butane-2,3-diol dehydrogenase (EC 1.1.1.4), previously identified in bacteria. Both required NAD(+) or NADP(+) as cofactors and were different from alcohol dehydrogenase. The equilibrium in both cases favoured the more reduced compound. 4. The activity of butane-2,3-diol dehydrogenase was decreased by dialysis against EDTA: the addition of Co(2+), Cu(2+), Zn(2+) and other bivalent metal ions restored activity. 5. Biacetyl reductase was resolved into multiple forms by CM-Sephadex chromatography and electrophoresis.

Alcohol Oxidoreductases↗

PubMed1965

Metabolism of poly-beta-hydroxybutyrate and acetoin in Bacillus cereus.

Kominek, Leo A. (University of Illinois, Urbana), and H. Orin Halvorson. Metabolism of poly-beta-hydroxybutyrate and acetoin in Bacillus cereus. J. Bacteriol. 90:1251-1259. 1965.-The synthesis of poly-beta-hydroxybutyrate (PHB) in Bacillus cereus strain T begins after the cessation of logarithmic growth. Its accumulation is preceded by the formation of acetoacetyl coenzyme A reductase, an enzyme used for its biosynthesis. Exogenous acetic acid present in the medium owing to incomplete glucose oxidation serves as the carbon source for polymer formation during the initial stages of its synthesis. Pyruvic acid is converted to acetoin by an enzyme system that is formed during vegetative growth. The formation of this enzyme system is dependent on a low pH in the medium. As the cells enter the sporulating stage, they lose the ability to form acetoin. The acetoin that accumulates is utilized via the 2,3-butanediol cycle which begins to function late in the sporulation stage. This cycle generates acetic acid which is used for PHB synthesis and is also oxidized to carbon dioxide. PHB accumulation reaches a maximum just prior to the formation of spores, and it is degraded during the process of sporulation. The effect of sporulation inhibitors and pH on PHB and acetoin metabolism are discussed.

Alcohol Oxidoreductases↗

PubMed1993

Purification and characterization of the E1 component of the Clostridium magnum acetoin dehydrogenase enzyme system.

In Clostridium magnum strain Wo Bd P1 the formation of the enzyme components of the acetoin dehydrogenase enzyme system E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase Ao:DCPIP OR), E2 (dihydrolipoamide acetyltransferase DHLTA) and E3 (dihydrolipoamide dehydrogenase DHLDH) were induced during growth on acetoin. Ao:DCPIP OR was purified from acetoin-grown cells in two steps by chromatography on DEAE-Sephacel and on Mono Q HR. Native Ao:DCPIP OR exhibited a M(r) of 138,000; it consisted of two different subunits of M(r) alpha 38,500 and M(r) beta 34,000, and it occurred most probably in a tetrameric alpha 2 beta 2 structure. The N-terminal amino acid sequences of the alpha- and beta-subunits revealed homologies to the N-termini of the corresponding subunits of Ao:DCPIP OR from Pelobacter carbinolicus and from Alcaligenes eutrophus; furthermore, the N-terminus of the beta-subunit exhibited homologies to the N-termini of beta-subunits from different 2-oxo acid dehydrogenases.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1996

Cloning and sequencing of a 40.6 kb segment in the 73 degrees-76 degrees region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization.

In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 40.6 kb chromosome segment, which contains the tre locus, has been cloned and sequenced. This region (40 601 bp; 73 degrees-76 degrees on the genetic map) contains 38 complete ORFs and one partial one. Three ORFs, the closest to the hsdC locus, correspond to the treP, treA and treR genes encoding enzyme IITre, trehalose-6-phosphate hydrolase and the repressor of the tre operon, respectively. A homology search for the products deduced from the 39 ORFs revealed that 23 exhibit significant similarity to known proteins, e.g. proteins involved in acetoin utilization, deoxyribonuclease, methyladenine glycosidase, hydroxyisobutyrate dehydrogenase, multidrug resistance proteins, protein phosphatase, cyclic-nucleotide phosphodiesterase, 5'-nucleotidase and NADP(H)-flavin oxidoreductase. Based on the gene organization and the results of the homology search, it is predicted that YfjG, YfjH, YfjI, YfjJ and YfjK form an acetoin dehydrogenase system (acetoin regulatory protein, and acetoin dehydrogenase components/subunits E3, E2, E1 beta and E1 alpha respectively). yfkN, an extremely large ORF comprising 4386 nucleotides, seems to correspond to the fusion of the genes for 2',3'-cyclic-nucleotide 2'-phosphodiesterase and 5'-nucleotidase precursor.

Acetoin↗

PubMed1975

Effect of substrates on acetoin production by Torulopsis colliculosa and Enterobacter species.

Under optimal conditions, Torulopsis colliculosa NRRL 172 and Enterobacter B-87 (ATCC 27613) produced 50 to 500 mg of acetoin per g of substrate. Whereas cane molasses, gur, glucose, and sucrose were suitable substrates for acetoin production, lactose and mannitol supported very good growth but yielded little or no acetoin. Production of acetoin increased with increases in the concentration of glucose, yeast extract, and peptone. Combination of substrates and intermittent feeding of substrate failed to increase the yields.

Acetoin↗

PubMed2005

Interactions between acetoin, a plant volatile, and pheromone in Rhynchophorus palmarum: behavioral and olfactory neuron responses.

Aggregation of Rhynchophorus palmarum weevils on host plants is mediated by a male pheromone (rhynchophorol: R) and host-plant volatiles (PVs) acting in synergy. Synthetic PV blends synergizing pheromone contain acetoin (A) and ethyl acetate (EtAc). R, A, and EtAc are detected by specialized olfactory receptor neurons (ORNs). In addition, particular types of ORNs are tuned to both A and R. To specify the role played by acetoin in pheromone perception, we recorded the responses of ORNs to 100 ng of A or R presented either separately or mixed. Behavioral responses to R, A, and EtAc were studied in a four-armed olfactometer and by field trapping. We screened 59 R-, A-, and AR-tuned ORNs by recording specific responses to odors presented either separately or mixed. Stimulations by blends elicited complex response profiles from the three ORN types: some gave synergistic responses, others were inhibited, and the remainder responded as though both odors were detected independently. Several gave either a weak or no response to a first stimulation by R, but responded clearly to a second stimulation after an intercalary stimulation by A. In the olfactometer, both sexes were more attracted to a blend of A + R (1 + 0.01 ng/sec) than to pure compounds, whereas EtAc did not enhance response to R. Pheromone-baited traps (1 mg/day) containing PV blends (650 mg/day) based on an ethanol/EtAc blend (1:1), plus either 5 or 10% A, or a more complex reference blend, or sugarcane (natural pheromone synergist), caught similar numbers of weevils and about twice as many insects as a control ethanol/EtAc blend. Traps with only pheromone caught about 10 times fewer insects. Behavioral results support the role of acetoin as a pheromone synergist for R. palmarum, and electrophysiological data provide evidence of modulation of peripheral sensory responses to pheromone by acetoin. Sexual dimorphism was observed neither at the ORN nor at the behavioral levels.

Acetoin↗