Abstract
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.
Highlights
There is currently a strong interest in the development of new eukaryotic hosts for the secretion of heterologous proteins
S. cerevisiae is regarded as a non-optimal host for the large scale production of foreign proteins due to reduced biomass yield caused by aerobic alcohol fermentation
Expression and Secretion of the Rice ␣-Amylase Gene in Y. lipolytica—To direct rice ␣-amylase through the Y. lipolytica secretory pathway, four kinds of expression vectors, pXOM103, pXOX103, pXOP103, and pXOS103, were constructed in which the strong and inducible XPR2 promoter was used with the following configurations between the XPR2 promoter and terminator: 1) XPR2 prepro-region-rice ␣-amylase coding sequence, 2) rice ␣-amylase signal peptiderice ␣-amylase coding sequence, 3) XPR2 signal peptide-rice ␣-amylase coding sequence, and 4) XPR2 signal peptide-dipeptide stretch-rice ␣-amylase coding sequence
Summary
There is currently a strong interest in the development of new eukaryotic hosts for the secretion of heterologous proteins. S. cerevisiae is regarded as a non-optimal host for the large scale production of foreign proteins due to reduced biomass yield caused by aerobic alcohol fermentation. In many cases, it has been difficult for S. cerevisiae to secrete large quantities of proteins 40 kDa or larger. Hyperglycosylation of recombinant proteins is another concern Because of these problems, non-conventional yeasts such as Pichia pastoris [6, 7], Kluyveromyces lactis [8], and Hansenula polymorpha [9] have been explored as new hosts for foreign gene expression [5]. Since the promoter of XPR2 is strong and regulated by pH and nitrogen source, the XPR2 promoter has been used to express homologous and heterologous genes in Y. lipolytica (20 –22)
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