Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays.
BACKGROUND
DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae.


RESULTS
By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts.


CONCLUSIONS
The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results.
Graf
Alexandra
A
Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria. alexandra.graf@boku.ac.at
Gasser
Brigitte
B
Dragosits
Martin
M
Sauer
Michael
M
Leparc
Germán G
GG
Tüchler
Thomas
T
Kreil
David P
DP
Mattanovich
Diethard
D
D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling / Q000379:methods; D005800:Genes, Fungal / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010843:Pichia / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity
D050976:Basic-Leucine Zipper Transcription Factors; D005656:Fungal Proteins; C091341:HAC1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004229:Dithiothreitol