Polyphosphates and exopolyphosphatase activities in the yeast Saccharomyces cerevisiae under overexpression of homologous and heterologous PPN1 genes

Abstract

The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.

The cadmium tolerance in Saccharomyces cerevisiae depends on inorganic polyphosphate

“…The parent strain CRY and the mutant strains CRN, CRX, and CNX were obtained from A. Kornberg . The strains with the pMB1/ PPN1 and pMB1/ PPX1 plasmids were designed as described . The description of the strains is shown in Table .…”

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

“…The strain CRN/pMB1_PPN1 Sc of S . cerevisiae overexpressing the polyphosphatase PPN1 [ 22 ] was maintained on an agarized YNB medium. The strain was grown at 29°C in flasks with 200 mL of YNB at 120 rpm for 24 h to obtain biomass.…”

“…The biomass was harvested by centrifugation at 3000 g and washed twice with cold distilled water. Spheroplasts were obtained as described [ 22 ] and treated at 0°C in a glass homogenizer with a Teflon pestle in 25 mM Tris-HCl (pH 7.2) containing 0.5 mM phenylmethylsulphonyl fluoride. The lysate was centrifuged at 5000 g 10 min.…”

“…Exopolyphosphatase and endopolyphosphatase activities were assayed at 30°C. Exopolyphosphatase activity was assayed by the rate of P i formation [ 22 ]. The amount of the enzyme forming 1 μmole of P i per 1 min was taken as a unit of enzyme activity (U).…”

Polyphosphatase PPN1 of <i>Saccharomyces cerevisiae</i> Is a Deoxyadenosine Triphosphate Phosphohydrolase

“…The ΔPPN1 mutant strain CRN of S. cerevisiae was obtained from N. Rao and A. Kornberg [15]. The strain CRN/pMB1_PPN1 Sc of S. cerevisiae overexpressing the polyphosphatase PPN1 was designed by Eldarov and co-authors earlier [20]. Yeast strains were grown in a synthetic minimal YNB medium containing (per 1 l) 1.7 g of bacto yeast nitrogen bases (Difсo, Detroit, USA), 20 g of glucose, by 20 mg of L-tryptophane, L-histidine, L-methionine and adenine, and 60 mg of L-leucine.…”