cGluconacetobacter europaeus, one of the microorganisms most commonly used for vinegar production, produces the unfavorable flavor compound acetoin. Since acetoin reduction is important for rice vinegar production, a genetic approach was attempted to reduce acetoin produced by G. europaeus KGMA0119 using specific gene knockout without introducing exogenous antibiotic resistance genes. A uracil-auxotrophic mutant with deletion of the orotate phosphoribosyltransferase gene (pyrE) was first isolated by positive selection using 5-fluoroorotic acid. The pyrE disruptant designated KGMA0704 (⌬pyrE) showed 5-fluoroorotic acid resistance. KGMA0704 and the pyrE gene were used for further gene disruption experiments as a host cell and a selectable marker, respectively. Targeted disruption of aldC or als, which encodes ␣-acetolactate decarboxylase or ␣-acetolactate synthase, was attempted in KGMA0704. The disruption of these genes was expected to result in a decrease in acetoin levels. A disruption vector harboring the pyrE marker within the targeted gene was constructed for double-crossover recombination. The cells of KGMA0704 were transformed with the exogenous DNA using electroporation, and genotypic analyses of the transformants revealed the unique occurrence of targeted aldC or als gene disruption. The aldC disruptant KGMA4004 and the als disruptant KGMA5315 were cultivated, and the amount of acetoin was monitored. The acetoin level in KGMA4004 culture was significantly reduced to 0.009% (wt/vol) compared with KGMA0119 (0.042% [wt/vol]), whereas that of KGMA5315 was not affected (0.037% [wt/vol]). This indicates that aldC disruption is critical for acetoin reduction. G. europaeus KGMA4004 has clear application potential in the production of rice vinegar with less unfavorable flavor.

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Using BaciUlus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pK. value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.Various thermostable alcohol dehydrogenases (ADH-Ts) have been analyzed for the industrial production of alcohol (2, 26, 38), including chiral alcohol (20). Bacillus stearothermophilus NCA1503 was found to produce an ADH-T amounting to 1 to 2% of soluble cell protein. This strain produced ethanol from sucrose or glucose as a carbon source under anaerobic conditions at high temperatures (2,27). Two types of ADH have been isolated from B. stearothermophilus NCA1503 and DSM2334 (33). ADH-T from NCA1503 showed enzymatic, structural, and immunological properties different from those of the ADH from DSM2334. The ADH from DSM2334 is active with primary alcohols, including methanol, and the rate-limiting step is NADH release as seen with horse liver ADH (3, 33). In contrast, substrate inhibition is not observed for ADH-T with any alcohols, and the enzyme-NADH dissociation is not considered to be a ratelimiting step (33). The gene for ADH from DSM2334 has been cloned in Escherichia coli (8). In this work, we cloned the ADH-T gene (adhT) from B. stearothermophilus NCA1503 in Bacillus subtilis.The ADH reaction mechanism was originally studied with horse liver ADH by X-ray crystallographic analysis and kinetic studies (3, 9, 36). Catalysis by horse liver ADH occurs by a proton release system involving a zinc atom, a water molecule, and serine and histidine residues. By comparing amino acid sequences of ADH-T and other ADHs, the catalytic system of ADH-T was inferred.We report here the molecular cloning and nucleotide sequencing of the ADH-T gene, adhT, from B. stearothermophilus NCA1503, a comparison of the deduced amino acid sequence...

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c Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant. B acterial cultures can function as attractants for insects (1).Fruit flies, such as Drosophila melanogaster, are recognized as an index for unsanitary conditions in urban areas because the flies infest damaged and overripe fruits and rotten foods. One of the important challenges is to keep the numbers of flies low, particularly in the food industries. A closely related species, the spottedwing drosophila (SWD) (Drosophila suzukii), which is native to southeastern Asia, is a newly emerging invasive pest for softskinned fruits such as blueberries, strawberries, and peaches in North America and Europe (2-4). Because the SWD breaks the skin of maturing healthy fruits using a serrated ovipositor to oviposit, unlike other Drosophila species, the fly causes significant damage to soft-skinned fruits (2). The damage caused by the SWD promotes microbial decay in the fruits, which results in a secondary infestation of other Drosophila species. The damaged fruits are likely to be rejected at the processing plant or export terminal (5). Although fruits are protected with sprays of chemical insecticides once the SWD is detected, growers risk the rejection ...