Membrane-bound, 2-keto-D-gluconate-yielding D-gluconate dehydrogenase from "Gluconobacter dioxyacetonicus" IFO 3271: molecular properties and gene disruption

Abstract

Most Gluconobacter species produce and accumulate 2-keto-d-gluconate (2KGA) and 5KGA simultaneously from d-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-delta-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, d-arabitol, and d-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.

FIG. 1.

Schematic representation of the gene fragment obtained in this study. Primers for PCR are indicated by arrows. Thick lines are DNA fragments obtained in this study. Hatched boxes represent gene fragments used for hybridization. The open triangle shows the position where the kanamycin or tetracycline resistance gene was inserted for mutation. ORF-5 is not complete.

FIG. 2.

SDS-PAGE analysis of purified FADH-GADH from “G. dioxyacetonicus” IFO 3271. Ten micrograms of purified FAD-SLDH was applied, and the gel was stained with Coomassie brilliant blue R-250. The arrows indicate the large, the cytochrome c, and the small subunits of FAD-GADH, from top to bottom.

FIG. 3.

PCR products to confirm gene disruption. PCR was performed with sets of primers, namely, GADH1-4 and GADH1-5 (A and C) and SldA-1 and SldA-2 (B), and genomic DNA from the wild-type (lanes 1) and mutant (lanes 2) strains. PCR products were analyzed by agarose gel electrophoresis. (A) gndL::Km strain; (B) sldA::Km strain; (C) gndL::Tc sldA::Km strain. Lanes M, λ DNA/StyI marker DNA.

FIG. 4.

2KGA and 5KGA production in culture medium by mutant strains. (A) Culture supernatant (4 μl) after cells were grown on G-GA medium for 30 h was analyzed by thin-layer chromatography as described in Materials and Methods Lanes: 1 to 4, 4 μl of a 100 mM standard solution (d-glucose [lane 1], NaGA [lane 2], 2KGA [lane 3], or 5KGA [lane 4]); 5 to 9, culture medium (before inoculation [lane 5] or with the wild type [lane 6], gndL::Km strain [lane 7], sldA::Km strain [lane 8], or gndL::Tc sldA::Km strain [lane 9]). (B) The culture supernatant was also quantified using 2KGR and 5KGR as described in Materials and Methods. Data were obtained from three independent experiments. ND, not detected.