红球菌R04苯甲酸转运相关膜蛋白RHOGL009301的生理功能

Physiological function of membrane protein RHOGL009301 involved in transport of benzoate in Rhodococcus sp. R04

【目的】探究红球菌(Rhodococcus sp.)R04膜蛋白RHOGL009301的生理功能和突变菌株的代谢特性,确定该膜蛋白的生理功能与苯甲酸转运的关系。【方法】将RHOGL009301基因与绿色荧光蛋白基因在Rhodococcus erythropolis进行融合表达,Delta Vision观察该基因蛋白产物的定位。通过基因同源重组敲除RHOGL009301基因,并对比野生型菌株和缺陷型菌株在不同碳源培养下的生长情况。HPLC测定红球菌R04野生型菌株和缺陷型菌株代谢联苯和苯甲酸时细胞内外代谢物,分析不同生长条件下代谢物的浓度变化。【结果】RHOGL009301基因与绿色荧光蛋白基因在Rhodococcus erythropolis中实现共表达,并定位在细胞膜上。获得了RHOGL009301基因的缺陷型菌株R04ΔMP,与野生型菌株相比,缺陷型菌株在联苯和苯甲酸培养条件下的生物量明显降低,生长速度减慢。HPLC分析表明RHOGL009301基因的缺失抑制了苯甲酸的转运。【结论】膜蛋白RHOGL009301是苯甲酸代谢和转运相关的蛋白,基于序列同源性分析,该膜蛋白是一种新型的苯甲酸转运蛋白。

[Objective] The physiological function of membrane protein RHOGL009301 in Rhodococcus sp. R04 and the metabolic properties of the mutant strain were studied to determine the relationship between the physiological function of the membrane protein and the transport of benzoate. [Methods] The RHOGL009301 gene and the green fluorescent protein gene were fused for expressing in Rhodococcus erythropolis, and the location of RHOGL009301 was observed by Delta Vision. The RHOGL009301 gene was knocked out by homologous recombination, and the growth of wild strain and deficient strain in different carbon sources were compared. The internal and external metabolites of the wild strain and the deficient strain when grown on biphenyl and benzoate were measured by HPLC, and the changes of metabolite concentration in different growth conditions were analyzed. [Results] A fusion gene that contained RHOGL009301 gene and the green fluorescent protein gene was co-expressed in Rhodococcus erythropolis and localized on the cell membrane. The deficient strain R04ΔMP of RHOGL009301 gene was obtained. The biomass of the deficient strain was significantly reduced in biphenyl and benzoate culture, and its growth rate was slowed down. HPLC analysis showed that the deletion of RHOGL009301 gene inhibited the transport of benzoate. [Conclusion] RHOGL009301 membrane protein is one of the proteins involved in metabolism and transport of benzoate. Based on sequence homology analysis, we can conclude that the membrane protein is a novel benzoate transport protein.