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Abstract Background Historically, complex media are used for the cultivation of Gluconobacter oxydans in industry and research. Using complex media has different drawbacks like higher costs for downstream processing and significant variations in fermentation performances. Synthetic media can overcome those drawbacks, lead to reproducible fermentation performances. However, the development of a synthetic medium is time and labour consuming. Detailed knowledge about auxotrophies and metabolic requirements of G. oxydans is necessary. In this work, we use a systematic approach applying the in-house developed μRAMOS technology to identify auxotrophies and develop a defined minimal medium for cultivation of G. oxydans fdh, improving the production process of the natural sweetener 5-ketofructose. Results A rich, defined synthetic medium, consisting of 48 components, including vitamins, amino acids and trace elements, was used as a basis for medium development. In a comprehensive series of experiments, component groups and single media components were individually omitted from or supplemented to the medium and analysed regarding their performance. Main components like salts and trace elements were necessary for the growth of G. oxydans fdh, whereas nucleotides were shown to be non-essential. Moreover, results indicated that the amino acids isoleucine, glutamate and glycine and the vitamins nicotinic acid, pantothenic acid and p-aminobenzoic acid are necessary for the growth of G. oxydans fdh. The glutamate concentration was increased three-fold, functioning as a precursor for amino acid synthesis. Finally, a defined minimal medium called ‘Gluconobacter minimal medium’ was developed. The performance of this medium was tested in comparison with commonly used media for Gluconobacter. Similar/competitive results regarding cultivation time, yield and productivity were obtained. Moreover, the application of the medium in a fed-batch fermentation process was successfully demonstrated. Conclusion The systematic investigation of a wide range of media components allowed the successful development of the Gluconobacter minimal medium. This chemically defined medium contains only 14 ingredients, customised for the cultivation of G. oxydans fdh and 5-ketofructose production. This enables a more straightforward process development regarding upstream and downstream processing. Moreover, metabolic demands of G. oxydans were identified, which further can be used in media or strain development for different processes.