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.

中文翻译：

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真养产碱杆菌 H16 中的生物素合成利用庚二酰辅酶 A，并且可以通过受体蛋白的量进行调节。


研究了用于工业生物聚合物生产的革兰氏阴性兼性化能自养细菌Ralstonia eutropica （同名Cupriavidus necator ）的生物素代谢。产生了缺乏bioF的生物素营养缺陷型突变体，并测定了细胞中生物素的消耗和生物素营养缺陷型富养罗尔斯通氏菌菌株的最小生物素需求。为了防止营养缺陷型突变体的生长，需要在无生物素培养基中连续培养3次，并且40ng/ml生物素足以促进细胞生长。然而，需要 200 ng/ml 生物素才能确保与野生型相当的生长，这与其他原核和真核微生物中生物素营养缺陷型突变体的需求相似。富养罗尔斯通氏菌ΔbioF突变体的表型互补只能通过富养罗尔斯通氏菌的bioF同源表达或枯草芽孢杆菌的bioF异源表达来实现，而不能通过大肠杆菌的bioF来实现。结合BioFs的生物信息学分析结果，这导致了这样的假设：富养罗伯茨菌中生物素合成的中间体是庚二酰辅酶A，而不是革兰氏阳性枯草芽孢杆菌中的庚二酰酰基载体蛋白（ACP）。 accB的同源表达增强了内部生物素含量，而accB和accC2的同源表达组合导致细胞中生物素浓度降低。 尽管调节蛋白 BirA 的 DNA 结合域缺失，但生物素合成似乎受到受体蛋白存在量的影响。