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 α 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-α 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.

Hansenula polymorpha Pex20p is an oligomer that binds the peroxisomal targeting signal 2 (PTS2)

We have cloned and characterized the Hansenula polymorpha PEX20 gene. The HpPEX20 gene encodes a protein of 309 amino acids (HpPex20p) with a calculated molecular mass of ~35 kDa. In cells of an HpPEX20 disruption strain, PTS2 proteins were mislocalized to the cytosol, whereas PTS1 matrix protein import proceeded normally. Also, the PTS2 proteins amine oxidase and thiolase were normally assembled and active in these cells, suggesting HpPex20p is not involved in oligomerization/activation of these proteins. Localization studies revealed that HpPex20p is predominantly associated with peroxisomes. Using fluorescence correlation spectroscopy we determined the native molecular mass of purified HpPex20p and binding of a synthetic peptide containing a PTS2 sequence. The data revealed that purified HpPex20p forms oligomers, which specifically bind PTS2-containing peptides.

Hansenula polymorpha Tup1p is important for peroxisome degradation

In the yeast Hansenula polymorpha peroxisomes are selectively degraded upon a shift of cells from methanol to glucose-containing media. We identified the H. polymorpha TUP1 gene by functional complementation of the peroxisome degradation deficient mutant pdd2-4. Tup1 proteins function in transcriptional repression of specific sets of genes involved in various cellular processes. Our combined data indicate that H. polymorpha TUP1 is involved in regulation of the switch between peroxisome biogenesis and selective degradation. The initial DNA fragment that complemented H. polymorpha pdd2-4 contained a second gene, encoding H. polymorpha Vps4p. Deletion of the VPS4 gene did not affect selective peroxisome degradation.