EXPRESSION, SECRETION, AND PROCESSING OF RICE ALPHA-AMYLASE IN THE YEAST YARROWIA-LIPOLYTICA

The gene encoding rice a-amylase in Oryza sativa was expressed in the yeast Yarrowia lipolytica, which is a potential host system for hetero logous protein expression, For efficient secretion, the strong and ind ucible XPR2 promoter was used in the construction of four kinds of exp ression vectors with the following configurations between the XPR2 pro moter and terminator: 1) XPR2 prepro-reson-rice alpha-amylase coding s equence, 2) rice cy-amylase signal peptide-rice alpha-amylase coding s equence, 3) XPR2 signal peptide-rice cu-amylase coding sequence, and 4 ) XPR2 signal peptide dipeptide stretchrice cu-amylase coding sequence , Secretion of active recombinant rice cu-amylase into the culture med ium was achieved only in the first two cases, demonstrating that the X PR2 signal peptide is not sufficient to direct the secretion of hetero logous protein, Furthermore, our study shows that the XPR2 prepro-regi on causes imprecise processing (after Pro(150)-Ala(151) or Val(135)-Le u(136) instead of Lys(156)-Arg(l57)) and leads to N-terminal amino aci d sequences that differ from that of native rice cu-amylase. Secondary structure analysis proposed that the structural form in the vicinity of the KEX2-like endopeptidase processing site in the XPR2 pro-region might play a critical role in the processing of heterologous proteins, These results suggest that the XPR2 pro-region is dispensable for obt aining the precise N-terminal amino acid in heterologous protein secre tion, In contrast, utilizing the rice Lu-amylase signal peptide was su fficient in directing secretion of recombinant protein with the expect ed N-terminal sequence, indicating that the signal peptide of rice cu- amylase was effectively recognized and processed by the Y. lipolytica secretory pathway.