Synthesis And Regulation Of Trehalose In The Psychrotolerant Yeast Guehomyces Pullulans17-1

It has been reported that80%of the biosphere has temperatures below5C. Forexample, much of the oceans, which cover some70%of the Earth’s surface, are at anaverage temperature of-1to5C. The low temperatures and low liquid wateravailability within such cold habitats make these regions extremely inhospitable tomost of forms of life. However, psychrophiles and psychrotolerants can grow well insuch cold environments. Therefore, it is of considerable significance to know how thepsychrophiles survive in their native environments.Recent results have shown that trehalose does not only primarily function as areserve carbohydrate, but also as a highly efficient protectant, enhancing theresistance of cellular components against adverse conditions such as high temperature,freezing, low dehydration, high osmotic pressure and high concentration of ethanol.In yeast, trehalose is synthesized in a two-step process. First,trehalose-6-phosphate (T-6-P) is formed from uridine diphosphate glucose (UDPG)and α-glucose-6-phosphate by T-6-P synthase (Tps1). Second, this compound isdephosphorylated to trehalose by T-6-P phosphatase (Tps2). Usually，T-6-P synthase(Tps1) is encoded by TPS1gene and T-6-P phosphatase (Tps2) is encoded by TPS2gene.In our previous studies, a psychrotolerant yeast Guehomyces pullulans17-1wasisolated from sea sediment in Antarctica. The psychrotolerant yeast G. pullulans17-1grows the best at15C. The TPS1gene of G. pullulans17-1was cloned from thegenomic DNA using the degenerated primers，inverse PCR and Genomic walking.The TPS1gene (accession number: JX046041) had an open reading frame of1590bpencoding a530amino acid protein with calculated molecular weight of59.8kDa.Besides, it was found that the promoter of the gene contained several stress responseelements (STRE) such as C4T and AG4, indicating that the gene expression might be regulated by stress shock. When the yeast cells grown at15 C for48h weretransferred to new medium and grown at10C,15C and25C, respectively, trehalosecontent, trehalose-6-phosphate synthase (Tps1) activity and the transcriptional level ofthe GPTPS1gene in the yeast cells grown at25C were higher than those of the yeastcells grown at10C and15C. However, trehalose content, Tps1activity and thetranscriptional level of the GPTPS1gene in the yeast cells grown at10C were lowerthan those of the yeast cells grown at15C and25C. These results may reveal thattrehalose play some role in thermotolerance of G. pullulans17-1, but could not playany role in cold tolerance of the yeast. This may also suggest that the psychrotolerantyeast is more sensitive to high temperature (above20°C) than to low temperature(below10°C), so that trehalose needs to be synthesized to protect the cells grown athigh temperature.At the same time, partial sequence of the TPS2gene was cloned from the G.pullulans17-1using the degenerated primers and Genomic walking. The partial TPS2gene (accession number: KC470041) had an open reading frame of2376bp encoding792amino acids.