A novel platform for the production of nonhydroxylated gelatins based on the methylotrophic yeast Hansenula polymorpha.
The use of yeast as a host for heterologous expression of proteins that are normally derived from animal tissue is a promising way to ensure defined products that are devoid of potential harmful animal side products. Here we report on the production and secretion of a custom-designed gelatin, Hu3-His8, by the yeast Hansenula polymorpha. We observed that Hu3-His8 was poorly secreted by the heterologous Saccharomyces cerevisiae invertase secretion signal. In contrast, the S. cerevisiae mating factor alpha prepro sequence efficiently directed secretion into the culture medium. However, at higher copy numbers, intracellular accumulation of Hu3-His8 precursors occurred. Overproduction of Erv29p, a protein required for packaging of the glycosylated pro-alpha factor into COPII vesicles, did not improve gelatin secretion in the multicopy strain. Previously, H. polymorpha was reported to hydroxylate proline residues in gelatinous sequences. In contrast, we were unable to detect hydroxyprolines in the secreted Hu3-His8. Also, we failed to identify a gene encoding prolyl-4-hydroxylase in the H. polymorpha genome.
Geerlings
Torsten H
TH
Laboratory of Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.
de Boer
Arjo L
AL
Lunenborg
Marcel G J
MG
Veenhuis
Marten
M
van der Klei
Ida J
IJ
D005656:Fungal Proteins / Q000235:genetics; D005780:Gelatin / Q000737:chemistry / Q000378:metabolism*; D006909:Hydroxyproline / Q000032:analysis; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000235:genetics
D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005780:Gelatin; D011393:Procollagen-Proline Dioxygenase; D043324:beta-Fructofuranosidase; D006909:Hydroxyproline