title: Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth.
abstract: The demand for recombinant proteins both for biopharmaceutical and technical applications is rapidly growing, and therefore the need to establish highly productive expression systems is steadily increasing. Yeasts, such as Pichia pastoris, are among the widely used production platforms with a strong emphasis on secreted proteins. Protein secretion is a limiting factor of productivity. There is strong evidence that secretion is coupled to specific growth rate (µ) in yeast, being higher at higher µ. For maximum productivity and product titer, high specific secretion rates at low µ would be desired. At high secretion rates cultures contain a large fraction of cells in the G2 and M phases of cell cycle. Consequently, the cell design target of a high fraction of cells in G2 + M phase was achieved by constitutive overexpression of the cyclin gene CLB2. Together with predictive process modeling this reverse engineered production strain improved the space time yield (STY) of an antibody Fab fragment by 18% and the product titer by 53%. This concept was verified with another secreted protein, human trypsinogen.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria; telephone: +43476546569; fax: +4313697615; Austrian Centre of Industrial Biotechnology (ACIB GmbH),Vienna, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Buchetics
authors.forename: Martin
authors.initials: M
authors.lastname: Dragosits
authors.forename: Michael
authors.initials: M
authors.lastname: Maurer
authors.forename: Corinna
authors.initials: C
authors.lastname: Rebnegger
authors.forename: Danilo
authors.initials: D
authors.lastname: Porro
authors.forename: Michael
authors.initials: M
authors.lastname: Sauer
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gasser
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
mesh_terms: D002455:Cell Division; D019926:Cyclin B / Q000096:biosynthesis / Q000235:genetics; D016195:G2 Phase; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics; D014362:Trypsinogen / Q000235:genetics / Q000378:metabolism*
chemical_list: C093749:CLB2 protein, S cerevisiae; D019926:Cyclin B; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014362:Trypsinogen