FRC Pushchino Center for Biological Research, Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino, Moscow region, 142290, Russia.
Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prosp. 33-2, Moscow, 119071, Russia.
Folia Microbiol (Praha). 2019 Nov;64(6):867-873. doi: 10.1007/s12223-019-00702-6. Epub 2019 Apr 1.
Inorganic polyphosphate is involved in architecture and functioning of yeast cell wall. The strain of Saccharomyces cerevisiae constitutively overexpressing acid phosphatase Pho5 was constructed for studying the Pho5 properties and its possible participation in polyphosphate metabolism. The parent strain was transformed by the vector carrying the PHO5 gene under a strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae. The culture liquid and biomass of transformant strain contained approximately equal total acid phosphatase activity. The levels of acid phosphatase activity associated with the cell wall and culture liquid increased in the transformant strain compared to the parent strain ~ 10- and 20-fold, respectively. The Pho5 preparation (specific activity of 46 U/mg protein and yield of 95 U/L) was obtained from culture liquid of overproducing strain. The overproducing strain had no changes in polyphosphate level. The activity of Pho5 with long-chained polyP was negligible. We concluded that Pho5 is not involved in polyphosphate metabolism. Purified Pho5 showed a similar activity with p-nitrophenylphosphate, ATP, ADP, glycerophosphate, and glucose-6-phosphate. The substrate specificity of Pho5 and its extracellular localization suggest its function: the hydrolysis of organic compounds with phosphoester bonds at phosphate limitation.
无机多聚磷酸盐参与酵母细胞壁的结构和功能。为了研究 Pho5 的特性及其在多聚磷酸盐代谢中的可能参与，构建了一株组成型过表达酸性磷酸酶 Pho5 的酿酒酵母菌株。该亲本菌株通过携带 PHO5 基因的载体转化，该载体受酿酒酵母 3-磷酸甘油醛脱氢酶的强组成型启动子控制。转化株的培养液和生物质中总酸性磷酸酶活性大致相等。与亲本菌株相比，转化株的细胞壁和培养液中酸性磷酸酶活性分别增加了约 10 倍和 20 倍。从过表达菌株的培养液中获得了 Pho5 制剂（比活 46 U/mg 蛋白，得率 95 U/L）。过表达菌株的多聚磷酸盐水平没有变化。Pho5 对长链多聚磷酸盐的活性可以忽略不计。我们得出结论，Pho5 不参与多聚磷酸盐代谢。纯化的 Pho5 对 p-硝基苯膦酸酯、ATP、ADP、甘油磷酸和葡萄糖-6-磷酸表现出相似的活性。Pho5 的底物特异性及其细胞外定位表明其功能：在磷酸盐限制时水解具有磷酸酯键的有机化合物。
