Gene cloning, expression, and characterization of trehalose-6-phosphate synthase from Pleurotus ostreatus.

Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) catalyzes the first step in trehalose synthesis, which involves transfer of glucose from uridine diphosphate glucose (UDPG) to glucose 6-phosphate (G6P) to form trehalose-6-phosphate. To determine the gene and enzymatic characteristics of TPS in Pleurotus ostreatus, we cloned and sequenced the cDNA of PoTPS1, which contains a 1665 bp open reading frame that encodes a 554-amino acid protein with a predicted molecular weight of 62.01 kDa. This gene was expressed in Escherichia coli BL21 and then the recombinant protein was purified and characterized. Results showed that the optimum pH and temperature for the recombinant PoTPS1 were 7.4 and 30 °C, respectively; the Km value against G6P and UDPG were 0.14 and 0.17 mM, respectively, and the Vmax and Kcat values were 91.86 nkat/g and 5.89 s-1 , respectively. Trehalose content was as high as 158.88 mg g-1 dry weight after heat treatment at 40 °C for 15 h, which was consistent with highest TPS1 activity at that time point. This result indicated that PoTPS1 was responsible for trehalose synthesis in P. ostreatus.