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Autotrophic Production of Stable-Isotope-Labeled Arginine in Ralstonia eutropha Strain H16

机译:Ralstonia eutropha H16菌株中稳定同位素标记的精氨酸的自养生产。

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With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene ( cphA ) of Synechocystis sp. strain PCC6308 under the control of the strong P_( cbbL ) promoter of the R. eutropha H16 cbb_(c) operon (coding for autotrophic CO_(2) fixation). In batch cultures growing on H_(2) and CO_(2) as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with ~(13)CO_(2) and ~(15)NH_(4)Cl, and the ~(13)C/~(15)N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [~(13)C/~(15)N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.
机译:为了提高工业规模生产的稳定同位素(SI)标记的精氨酸,我们开发了一种系统,用于在自养营养条件下在富营养的Ralstonia eutropha重组菌株中异源生产含精氨酸的聚合物蓝霉素。我们构建了基于蓝藻属藻蓝藻合成酶基因(cphA)的表达质粒。菌株PCC6308受富营养芽孢杆菌H16 cbb_(c)操纵子的强P_(cbbL)启动子控制(编码自养CO_(2)固定)。在分别以H_(2)和CO_(2)作为唯一能源和碳源生长的分批培养物中,细胞的蓝霉素含量达到细胞干重(CDW)的5.5%。然而,在没有选择的情况下(即在无抗生素的培养基中),质粒的损失导致产量的大幅降低。因此,我们设计了一种新型的成瘾系统,适合在自养石条件下使用。基于加氢酶转录因子HoxA,该系统介导了石自养培养期间cphA的稳定表达,而无需使用抗生素。蓝霉素的最大产量为CDW的7.1%。为了测试我们的表达系统在实际生产条件下的标记效率,将细胞在10升规模的发酵液中培养,发酵液中加入〜(13)CO_(2)和〜(15)NH_(4)Cl,以及〜(13 C /〜(15)N标记的蓝霉素随后用0.1 M HCl处理萃取;每次补料分批发酵获得2.5至5 g [〜(13)C /〜(15)N]精氨酸,对应于98.8%至99.4%的同位素富集。

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