title: Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions.
abstract: BACKGROUND
  It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes.
  
  
  RESULTS
  Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25 degrees C to 20 degrees C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced.
  
  
  CONCLUSIONS
  Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.
authors.affiliation: University of Natural Resources and Applied Life Sciences Vienna, Department of Biotechnology, Institute of Applied Microbiology, Vienna, Austria. brigitte.gasser@boku.ac.at <brigitte.gasser@boku.ac.at>
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.forename: Michael
authors.initials: M
authors.lastname: Maurer
authors.forename: Jari
authors.initials: J
authors.lastname: Rautio
authors.forename: Michael
authors.initials: M
authors.lastname: Sauer
authors.forename: Anamitra
authors.initials: A
authors.lastname: Bhattacharyya
authors.forename: Markku
authors.initials: M
authors.lastname: Saloheimo
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D005821:Genetic Techniques; D023281:Genomics / Q000379:methods; D008954:Models, Biological; D015233:Models, Statistical; D010843:Pichia / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014158:Transcription, Genetic
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D005656:Fungal Proteins; C091341:HAC1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins