BACKGROUND: 2,5-Furandicarboxylic acid (FDCA) is a promising building block for biobased recyclable polymers and a platform for other potential biobased chemicals. The common route of its production is by oxidation of sugar-derived 5-hydroxymethylfurfural (HMF). Several reports on biocatalytic oxidation using whole microbial cells or enzymes have been reported, which offers potentially a greener alternative compared to the chemical process. HMF oxidases and aryl alcohol oxidases are the only enzymes able to catalyse the complete oxidation to FDCA, however at low concentrations and are subject to inhibition by the FFCA (5-formylfuran-2-carboxylic acid) intermediate. The present report presents a study on the oxidation of FFCA to FDCA using the obligately aerobic bacterium Gluconobacter oxydans and identification of the enzymes catalyzing the reaction. RESULTS: Screening of three different strains showed G. oxydans DSM 50049 to possess the highest FFCA oxidation efficiency. Optimal reaction conditions for obtaining 100% conversion of 10 g/L (71 mM) FFCA to FDCA at 100% reaction yield were at pH 5, 30 °C and using 200 mg wwt /mL cells harvested at mild-exponential phase. In a reaction run at a 1 L scale using a total of 15 g/L (107 mM) FFCA supplied in a fed-batch mode, FDCA was obtained at a yield of 90% in 8.5 h. The product was recovered at 82% overall yield and 99% purity using a simple recovery process. Screening of several oxidoreductase enzymes from the gene sequences identified in the bacterial genome revealed two proteins annotated as membrane-bound aldehyde dehydrogenase (MALDH) and coniferyl aldehyde dehydrogenase (CALDH) to be the enzymes catalyzing the oxidization of FFCA. CONCLUSION: The study shows G. oxydans DSM 50049 and its enzymes to be promising biocatalysts for use in the FDCA production process from biomass. The high reaction rate and yield motivate further studies on characterization of the identified enzymes exhibiting the FFCA oxidizing activity, which can be used to construct an enzyme cascade together e.g. with HMF oxidase or aryl alcohol oxidase for one-pot production of FDCA from 5-HMF.