title: Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris.
abstract: BACKGROUND
  Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris.
  
  
  RESULTS
  Adaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation.
  
  
  CONCLUSIONS
  Our data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.|Austrian Centre of Industrial Biotechnology (ACIB), 1190, Vienna, Austria.
authors.forename: Josef W
authors.initials: JW
authors.lastname: Moser
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), 1190, Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Prielhofer
authors.affiliation: University of Applied Sciences FH-Campus Wien, Bioengineering, Vienna, Austria.
authors.forename: Samuel M
authors.initials: SM
authors.lastname: Gerner
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), 1190, Vienna, Austria.|University of Applied Sciences FH-Campus Wien, Bioengineering, Vienna, Austria.
authors.forename: Alexandra B
authors.initials: AB
authors.lastname: Graf
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Iain B H
authors.initials: IB
authors.lastname: Wilson
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), 1190, Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria. martin.dragosits@boku.ac.at.
authors.forename: Martin
authors.identifier: http://orcid.org/0000-0003-2522-8469
authors.initials: M
authors.lastname: Dragosits
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D061249:Batch Cell Culture Techniques / Q000379:methods; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry*; D019020:Directed Molecular Evolution; D000432:Methanol / Q000378:metabolism*; D009154:Mutation; D010843:Pichia / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D010865:Pilot Projects; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000432:Methanol
keywords: Experimental evolution; Methanol; Pichia pastoris; Recombinant protein