Heterologous Overexpression and Characterization of a Flavoprotein-CytochromecComplex Fructose Dehydrogenase of Gluconobacter japonicus NBRC3260

Abstract

ABSTRACTA heterotrimeric flavoprotein-cytochromeccomplex fructose dehydrogenase (FDH) ofGluconobacter japonicusNBRC3260 catalyzes the oxidation ofd-fructose to produce 5-keto-d-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. ThefdhSCLgenes encoding the FDH complex ofG. japonicusNBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames forfdhSCLencoding the small, cytochromec, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression offdhSCLwas conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene ofGluconobacter oxydansand aG. oxydansstrain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designatedTTGFDH andATGFDH. Membranes of the cells producing recombinantTTGFDH andATGFDH showed approximately 20 times and 100 times higher specific activity than those ofG. japonicusNBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly highd-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochromecsubunit is responsible not only for membrane anchoring but also for ubiquinone reduction.