2,5-Diketo-D-Gluconate Hyperproducing Gluconobacter sphaericus SJF2-1 with Reporting Multiple Genes Encoding the Membrane-Associated Flavoprotein-Cytochrome c Complexed Dehydrogenases

Abstract

Gluconobacter sphaericus has not yet been used in biotransformation studies. In this study, G. sphaericus SJF2-1, which produces a diffusible pigment, was isolated from grape. The spent culture medium became dark black when the cells were grown in medium containing glucose and then autoclaved. This bacterium produced 2,5-diketo-D-gluconate (2,5-DKG) from D-glucose and D-gluconate. When 5% D-glucose was used, the conversion efficiency was approximately 52.4% in a flask culture. 2,5-DKG is a precursor of 2-keto-L-gulonic acid, which is a key intermediate in the industrial production of L-ascorbic acid. The complete genome sequence of G. sphaericus SJF2-1 was determined for the first time in the G. sphaericus species. The total size was 3,198,086 bp, with 2867 protein-coding sequences; one chromosome and six plasmids were identified. From the genome of SJF2-1, multiple genes homologous to those involved in the conversion of D-glucose to 2,5-DKG were identified. In particular, six different genes encoding membrane-associated flavoprotein-cytochrome c complexed dehydrogenase were identified and divided into two different lineages. This study suggests the potential of G. sphaericus SJF2-1 to mass-produce 2,5-DKG and other D-glucose oxidation products.

Keywords: 2,5-diketo-D-gluconate; D-gluconate; D-glucose biotransformation; Gluconobacter sphaericus; L-ascorbic acid; membrane-associated flavoprotein-cytochrome c complexed dehydrogenase.

Figure 1

Biotransformation of D-glucose to 2-KLG via formation of 2,5-DKG, and synthesis of L-ascorbate from 2-KLG by chemical reactions.

Figure 2

Culture properties of SJF2-1. (a) Production of a diffusible light brown pigment by SJF2-1 grown on YPD agar. (b) Cells under a phase-contrast microscope. Bar, 1 µm. (c) A black-colored product formed by autoclaving the YPD culture solution of SJF2-1. (d) Growth inhibition of Fusarium moniliforme var. subglutinans (white mycelia) by SJF2-1 on YPD agar.

Figure 3

Analyses of D-glucose and sugar acid formed by Gluconobacter sphaericus SJF2-1. (a) TLC analysis (1: D-glucose 40 µg, 2: D-gluconate 400 µg, 3: 2-KG 40 µg, 4: 5-KG 40 µg, 5: YPP + 1% D-glucose, 6: YPP + 1% D-gluconate, 7: YPP + 1% 2-KG, 8: YPP + 1% 5-KG); (b) LC/MS analysis of product formed by D-glucose; (c) HPLC chromatograms of standards in red and culture supernatants in black with indicated substrate.

Figure 4

Time course of D-glucose fermentation by Gluconobacter sphaericus SJF2-1. Cells were cultured in 50 mL of YPP + 5% D-glucose medium as described in Materials and Methods. Sampling times are indicated. The results are from three independent experiments.

Figure 5

Genome comparison of Gluconobacter sphaericus SJF2-1 (reference) with indicated Gluconobacter strains conducted using RAST (outside tracks), and GC skew and GC content plots of SJF2-1 (inside). Tracks from the Gluconobacter strains represent pairwise BLAST comparisons between the ORFs of each strain to the ORFs of the SJF2-1 genome, with percentages of identity represented with different colors shown in the legend.

Figure 6

D-glucose oxidation pathway to 2,5-DKG and homologous genes encoding enzymes of the catalytic steps by SJF2-1. The genes in the arrows have been functionally demonstrated in different species, and the SJF2-1 genes in the boxes are homologous to the respective genes in the arrows. Gene clusters with the highest homology are shown in bold. Numbers in parentheses indicate the percentage of amino acid sequence identity compared to the genes in the arrows.

Figure 7

Rooted phylogenic tree of the subunits of the membrane-associated flavoprotein-cytochrome c complexed dehydrogenases using the Maximum likelihood algorithm. Trees are scaled by the amino acid distance between proteins. Bootstrap values with 1000 rapid bootstrap replicates are shown. The protein IDs of the gene clusters are shown. (a) Small subunit; (b) flavoprotein (large subunit); (c) cytochrome c subunit. GDase and KGDase for gluconate 2-dehydrogenase and 2-keto-D-gluconate dehydrogenase families, respectively.