title: Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization.
abstract: BACKGROUND
  The classic AOX1 replacement approach is still one of the most often used techniques for expression of recombinant proteins in the methylotrophic yeast Pichia pastoris. Although this approach is largely successful, it frequently delivers clones with unpredicted production characteristics and a work-intense screening process is required to find the strain with desired productivity.
  
  
  RESULTS
  In this project 845 P. pastoris clones, transformed with a GFP expression cassette, were analyzed for their methanol-utilization (Mut)-phenotypes, GFP gene expression levels and gene copy numbers. Several groups of strains with irregular features were identified. Such features include GFP expression that is markedly higher or lower than expected based on gene copy number as well as strains that grew under selective conditions but where the GFP gene cassette and its expression could not be detected. From these classes of strains 31 characteristic clones were selected and their genomes sequenced. By correlating the assembled genome data with the experimental phenotypes novel insights were obtained. These comprise a clear connection between productivity and cassette-to-cassette orientation in the genome, the occurrence of false-positive clones due to a secondary recombination event, and lower total productivity due to the presence of untransformed cells within the isolates were discovered. To cope with some of these problems, the original vector was optimized by replacing the AOX1 terminator, preventing the occurrence of false-positive clones due to the secondary recombination event.
  
  
  CONCLUSIONS
  Standard methods for transformation of P. pastoris led to a multitude of unintended and sometimes detrimental integration events, lowering total productivity. By documenting the connections between productivity and integration event we obtained a deeper understanding of the genetics of mutation in P. pastoris. These findings and the derived improved mutagenesis and transformation procedures and tools will help other scientists working on recombinant protein production in P. pastoris and similar non-conventional yeasts.
authors.affiliation: Fermentation Engineering, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany.|Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, 33615, Bielefeld, Germany.
authors.forename: Jan-Philipp
authors.initials: JP
authors.lastname: Schwarzhans
authors.affiliation: Genome Research of Industrial Microorganisms, CeBiTec, Bielefeld University, Universitätsstr. 27, 33615, Bielefeld, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Wibberg
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, 33615, Bielefeld, Germany.
authors.forename: Anika
authors.initials: A
authors.lastname: Winkler
authors.affiliation: Fermentation Engineering, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Luttermann
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstr. 27, 33615, Bielefeld, Germany.|CeBiTec, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.affiliation: Fermentation Engineering, Bielefeld University, Universitätsstr. 25, 33615, Bielefeld, Germany. karl.friehs@uni-bielefeld.de.|CeBiTec, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany. karl.friehs@uni-bielefeld.de.
authors.forename: Karl
authors.initials: K
authors.lastname: Friehs
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D042931:Aldehyde Oxidase
keywords: AOX1 promoter; Expression cassette orientation; False-positive; Genome sequencing; Insertion locus; Non-conventional yeast; Pichia pastoris; Recombinant protein production