Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG
Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG
复制标题
鞘氨醇单胞菌 WL 胶生产过程中核苷酸糖前体 UDP-葡萄糖生物合成途径的表征。
DOI：
10.1016/j.jbiotec.2019.06.005
复制
发表时间：
2019-08-20
影响因子：
4.1
通讯作者：
Zhu, Hu
中科院分区：
文献类型：
--
作者：
Li, Hui;Li, Jing;Zhu, Hu
阐明鞘氨醇单胞菌产生的鞘氨醇WL胶前体UDP-葡萄糖的可能生物合成途径。对WG、磷酸葡萄糖变位酶(PGM)和UDP-葡萄糖焦磷酸化酶(UGPase)进行了生物信息学分析，并在大肠杆菌BL21(DE3)中进行了表达和鉴定。PGM属于磷酸葡萄糖变位酶/磷酸甘露糖变位酶亚类，UGPase被预测为四聚体结构的UDP-葡萄糖焦磷酸酶。两种酶均以可溶性形式表达，纯化至接近均一的水平，活性分别为1159和796U/mg，折叠结构合理，分别以单体和四聚体形式存在。PGM的最适pH和温度分别为9.0和50℃，该蛋白质在pH 8.0和40~50℃的温度下稳定，UGPase的最适pH和温度分别为9.0和45℃，蛋白质在pH 8.0和30~55℃的温度下稳定，还进行了以葡萄糖-6-磷酸和UTP为底物的PGM和UGPase的小规模一锅生物合成UDP-葡萄糖，并通过高效液相色谱检测观察到UDP-葡萄糖的形成。证实了UDP-葡萄糖的体外生物合成途径。PGM和UGPase将是工业生产中提高WL胶得率的代谢工程的理想靶点。
To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 degrees C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 degrees C. The optimal pH and temperature of UGPase were 9.0 and 45 degrees C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 degrees C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.