Expression, secretion, and processing of rice alpha-amylase in the yeast Yarrowia lipolytica.
The gene encoding rice alpha-amylase in Oryza sativa was expressed in the yeast Yarrowia lipolytica, which is a potential host system for heterologous protein expression. For efficient secretion, the strong and inducible XPR2 promoter was used in the construction of four kinds of expression vectors with the following configurations between the XPR2 promoter and terminator: 1) XPR2 prepro-region-rice alpha-amylase coding sequence, 2) rice alpha-amylase signal peptide-rice alpha-amylase coding sequence, 3) XPR2 signal peptide-rice alpha-amylase coding sequence, and 4) XPR2 signal peptide-dipeptide stretch-rice alpha-amylase coding sequence. Secretion of active recombinant rice alpha-amylase into the culture medium was achieved only in the first two cases, demonstrating that the XPR2 signal peptide is not sufficient to direct the secretion of heterologous protein. Furthermore, our study shows that the XPR2 prepro-region causes imprecise processing (after Pro150-Ala151 or Val135-Leu136 instead of Lys156-Arg157) and leads to N-terminal amino acid sequences that differ from that of native rice alpha-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 obtaining the precise N-terminal amino acid in heterologous protein secretion. In contrast, utilizing the rice alpha-amylase signal peptide was sufficient in directing secretion of recombinant protein with the expected N-terminal sequence, indicating that the signal peptide of rice alpha-amylase was effectively recognized and processed by the Y. lipolytica secretory pathway.
Park
C S
CS
Biochemical Engineering Program, Department of Chemical Engineering and Material Science, University of California, Davis, California 95616, USA.
Chang
C C
CC
Kim
J Y
JY
Ogrydziak
D M
DM
Ryu
D D
DD
D003001:Cloning, Molecular; D015870:Gene Expression; D012275:Oryza / Q000201:enzymology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics; D015003:Yeasts / Q000235:genetics; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases