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The biotin metabolism of the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha (syn. Cupriavidus necator ), which is used for biopolymer production in industry, was investigated. A biotin auxotroph mutant lacking bioF was generated, and biotin depletion in the cells and the minimal biotin demand of a biotin-auxotrophic R. eutropha strain were determined. Three consecutive cultivations in biotin-free medium were necessary to prevent growth of the auxotrophic mutant, and 40 ng/ml biotin was sufficient to promote cell growth. Nevertheless, 200 ng/ml biotin was necessary to ensure growth comparable to that of the wild type, which is similar to the demand of biotin-auxotrophic mutants among other prokaryotic and eukaryotic microbes. A phenotypic complementation of the R. eutropha ΔbioF mutant was only achieved by homologous expression of bioF of R. eutropha or heterologous expression of bioF of Bacillus subtilis but not by bioF of Escherichia coli Together with the results from bioinformatic analysis of BioFs, this leads to the assumption that the intermediate of biotin synthesis in R. eutropha is pimeloyl-CoA instead of pimeloyl-acyl carrier protein (ACP) like in the Gram-positive B. subtilis Internal biotin content was enhanced by homologous expression of accB , whereas homologous expression of accB and accC2 in combination led to decreased biotin concentrations in the cells. Although a DNA-binding domain of the regulator protein BirA is missing, biotin synthesis seemed to be influenced by the amount of acceptor protein present. IMPORTANCE  Ralstonia eutropha is applied in industry for the production of biopolymers and serves as a research platform for the production of diverse fine chemicals. Due to its ability to grow on hydrogen and carbon dioxide as the sole carbon and energy source, R. eutropha is often utilized for metabolic engineering to convert inexpensive resources into value-added products. The understanding of the metabolic pathways in this bacterium is mandatory for further bioengineering of the strain and for the development of new strategies for biotechnological production.

Biotin Synthesis in Ralstonia eutropha H16 Utilizes Pimeloyl Coenzyme A and Can Be Regulated by the Amount of Acceptor Protein