An Efficient Method Using Gluconacetobacter europaeus To Reduce an Unfavorable Flavor Compound, Acetoin, in Rice Vinegar Production

ABSTRACTGluconacetobacter 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 byG. europaeusKGMA0119 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. ThepyrEdisruptant designated KGMA0704 (ΔpyrE) showed 5-fluoroorotic acid resistance. KGMA0704 and thepyrEgene were used for further gene disruption experiments as a host cell and a selectable marker, respectively. Targeted disruption ofaldCorals, which encodes α-acetolactate decarboxylase or α-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 thepyrEmarker 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 targetedaldCoralsgene disruption. ThealdCdisruptant KGMA4004 and thealsdisruptant 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 thataldCdisruption is critical for acetoin reduction.G. europaeusKGMA4004 has clear application potential in the production of rice vinegar with less unfavorable flavor.