title: Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris.
abstract: The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.
authors.affiliation: Department of Chemical, Biological, and Environmental Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès) 08193, Catalonia, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Cámara
authors.affiliation: Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, A-1190 Vienna, Austria.
authors.forename: Nils
authors.initials: N
authors.lastname: Landes
authors.affiliation: Department of Chemical, Biological, and Environmental Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès) 08193, Catalonia, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Albiol
authors.affiliation: Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, A-1190 Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.affiliation: Department of Biotechnology, BOKU - University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology, A-1190 Vienna, Austria.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Department of Chemical, Biological, and Environmental Engineering, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès) 08193, Catalonia, Spain.
authors.forename: Pau
authors.initials: P
authors.lastname: Ferrer
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D000432:Methanol / Q000378:metabolism*; D020675:Peroxisomes / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D025341:Principal Component Analysis; D011401:Promoter Regions, Genetic; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic; D059467:Transcriptome
chemical_list: D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D008049:Lipase; D000432:Methanol