Pichia pastoris 'just in time' alternative respiration

1 Institute for Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, A-8010 Graz, Austria 2 Institute for Molecular Biosciences, Karl-Franzens University, Universitaetsplatz 2, A-8010 Graz, Austria Correspondence Anton Glieder a.glieder{at}tugraz.at Alternative oxidases (Aox or Aod) are present in the mitochondria of plants, fungi and many types of yeast. These enzymes transfer electrons from the ubiquinol pool directly to oxygen without contributing to the proton transfer across the mitochondrial membrane. Alternative oxidases are involved in stress responses, programmed cell death and maintenance of the cellular redox balance. The alternative oxidase gene of the methylotrophic yeast Pichia pastoris was isolated and cloned to study its regulation and the effects of deregulation of the alternative respiration by overexpression or disruption of the gene. Both disruption and overexpression had negative effects on the biomass yield; however, the growth rate and substrate uptake rate of the strain overexpressing the alternative oxidase were slightly increased. These effects were even more pronounced when higher glucose concentrations were used. The occurrence of free intracellular radicals and cell death phenomena was investigated using dihydrorhodamine 123 and the TUNEL test. The results suggest a major contribution of the alternative oxidase to P. pastoris cell viability. The negative effects of deregulated alternative respiration clearly indicated the importance of precise regulation of the alternative oxidase in this yeast. Abbreviations: CRR, cyanide-resistant respiration; ROS, reactive oxygen species; SHAM, salicylhydroxamic acid; TUNEL, TdT-mediated dUTP nick end labelling The GenBank/EMBL/DDBJ accession number for the AOD gene from P. pastoris is DQ465985.