title: Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.
abstract: RESULTS
  We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation.
  
  
  CONCLUSIONS
  Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Bryn
authors.initials: B
authors.lastname: Edwards-Jones
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Rochelle
authors.initials: R
authors.lastname: Aw
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Geraint R
authors.initials: GR
authors.lastname: Barton
authors.affiliation: Department of Surgery & Cancer, Faculty of Medicine, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Tredwell
authors.affiliation: Department of Surgery & Cancer, Faculty of Medicine, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Jacob G
authors.initials: JG
authors.lastname: Bundy
authors.affiliation: Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, United Kingdom; Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Leak
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics*; D061249:Batch Cell Culture Techniques; D003470:Culture Media; D056915:DNA Copy Number Variations; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010843:Pichia / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D015202:Protein Engineering; D019076:Transgenes; D014357:Trypsin / Q000096:biosynthesis / Q000235:genetics*; D056811:Unfolded Protein Response
chemical_list: D003470:Culture Media; C489303:PRSS1 protein, human; D014357:Trypsin; D000432:Methanol