title: Inactivation of a GAL4-like transcription factor improves cell fitness and product yield in glycoengineered Pichia pastoris strains.
abstract: With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA bo_jiang@merck.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Argyros
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: John
authors.initials: J
authors.lastname: Bukowski
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Nelson
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Nathan
authors.initials: N
authors.lastname: Sharkey
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Sehoon
authors.initials: S
authors.lastname: Kim
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Copeland
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.affiliation: Informatics and IT, Merck & Co., Boston, Massachusetts, USA.
authors.forename: Ronghua
authors.initials: R
authors.lastname: Chen
authors.affiliation: Informatics and IT, Merck & Co., Boston, Massachusetts, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhuang
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Natarajan
authors.initials: N
authors.lastname: Sethuraman
authors.affiliation: GlycoFi Inc., Discovery Biologics, Merck & Co., Lebanon, New Hampshire, USA.
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
mesh_terms: D004268:DNA-Binding Proteins / Q000235:genetics; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D060847:Metabolic Engineering; D050296:Microbial Viability; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D017385:Sequence Homology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014774:Virulence
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors