Biotin synthesis in Ralstonia eutropha H16 utilizes pimeloyl-CoA and can be regulated by the amount of acceptor protein.

The biotin metabolism of the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha (syn. Cupriavidus necator ), that is used for production in industry, was investigated. A biotin auxotroph mutant lacking bioF was generated, and biotin depletion in the cells as well as the minimal biotin demand of a biotin auxotroph R. eutropha strain were determined. Three consecutive cultivations in biotin free medium were necessary to prevent growth of the auxotroph mutant, and 40 ng/ml biotin were sufficient to promote cell growth. Nevertheless, 200 ng/ml biotin were necessary to ensure growth comparable to that of the wildtype which is similar to the demand of biotin auxotroph 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-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 sole carbon and energy source, R. eutropha is often utilized for metabolic engineering to convert cheap resources into value added products. The understanding of the metabolic pathways in this bacterium is mandatory for further bio-engineering of the strain and for the development of new strategies for biotechnological productions.