title: Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks.
abstract: Although successfully used for heterologous gene expression for more than twenty years, general knowledge about all factors influencing protein expression by Pichia pastoris is still lacking. For high titers of protein clones are optimized individually for each target protein. Optimization efforts in this study were focused on the DNA level, evaluating a set of 48 different individual synthetic genes (TrCBH2) coding for the same protein sequence of a Trichoderma reesei cellulase in combination with three different promoter sequences: PGAP (constitutive) and the synthetic AOX1 promoter variants PDeS (derepressed) and PEn (enhanced, inducible). Expression of active secreted enzyme varied from undetectable to ∼300% of the best known gene, as determined by secreted enzyme activity analyses of supernatants from 96 well plate and bioreactor cultivations. Finally, the best optimized gene and new promoters were combined to engineer highly productive P. pastoris CBH2 expression strains. Although no methanol was used for induction a final titer of more than 18g/l of secreted protein was produced under controlled conditions in small scale bioreactor cultivations after 60-70h of growth limiting glycerol feed. This is the highest concentration of secreted enzyme in P. pastoris published so far and single parts of the expression cassette could be independently optimized showing additive effects for improvements in protein production by P. pastoris.
authors.affiliation: ACIB GmbH Graz University of Technology, Institute of Molecular Biotechnology, NAWI Graz, Petersgasse 14, A8010 Graz, Austria.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mellitzer
authors.affiliation: ACIB GmbH Graz University of Technology, Institute of Molecular Biotechnology, NAWI Graz, Petersgasse 14, A8010 Graz, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Ruth
authors.affiliation: DNA2.0 Inc., Menlo Park, CA 94025, USA.
authors.forename: Claes
authors.initials: C
authors.lastname: Gustafsson
authors.affiliation: DNA2.0 Inc., Menlo Park, CA 94025, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Welch
authors.affiliation: Center for Medical Research, Graz, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Grünberger
authors.affiliation: VTU Technology GmbH, Grambach, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Weis
authors.affiliation: VTU Technology GmbH, Grambach, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Purkarthofer
authors.affiliation: ACIB GmbH Graz University of Technology, Institute of Molecular Biotechnology, NAWI Graz, Petersgasse 14, A8010 Graz, Austria. Electronic address: anton.glieder@acib.at.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
mesh_terms: D019149:Bioreactors; D002480:Cellulase / Q000096:biosynthesis* / Q000378:metabolism; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D000432:Methanol / Q000737:chemistry; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
chemical_list: D002480:Cellulase; D000432:Methanol
keywords: Cellulase secretion; Gene dosage; Pichia pastoris; Synonymous codon optimization; Synthetic promoters