title: Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris.
abstract: Production of recombinant proteins is affected by process conditions, where transcriptional regulation of Pichia pastoris alcohol oxidase 1 (PpAOX1) promoter has been a key factor to influence expression levels of proteins of interest. Here, we demonstrate that the AOX1 promoter and peroxisome biogenesis are regulated based on different process conditions. Two types of GFP-fusion proteins, Ub-R-GFP (short-lived GFP in the cytosol) and GFP-SKL (peroxisomal targeting GFP), were successfully used to characterize the time-course of the AOX1 promoter and peroxisome biogenesis, respectively. The activity of the AOX1 promoter and peroxisome biogenesis was highly subjected to different fermentation process conditions - methanol-limited condition at normoxy (ML), switched feeding of carbon sources (e.g., glucose and methanol) under carbon-limited condition at normoxy (SML), and oxygen-limited (OL) condition. The AOX1 promoter was most active under the ML, but less active under the OL. Peroxisome biogenesis showed a high dependency on methanol consumption. In addition, the proliferation of peroxisomes was inhibited in a medium containing glucose and stimulated in the methanol phase under a carbon-limited fed-batch culture condition. The specific productivity of a monoclonal antibody (qp) under the AOX1 promoter was higher at 86h of induction in the ML than in the OL (0.026 vs 0.020mgg(-1)h(-1)). However, the oxygen-limited condition was a robust process suitable for longer induction (180h) due to high cell fitness. Our study suggests that the maximal production of a recombinant protein is highly dependent on methanol consumption rate that is affected by the availability of methanol and oxygen molecules.
authors.affiliation: GlycoFi, Biologics Discovery, Merck & Co., Inc, 16 Cavendish Ct., Lebanon, NH 03766, USA.
authors.forename: Sehoon
authors.initials: S
authors.lastname: Kim
authors.forename: Shannon
authors.initials: S
authors.lastname: Warburton
authors.forename: Istvan
authors.initials: I
authors.lastname: Boldogh
authors.forename: Cecilia
authors.initials: C
authors.lastname: Svensson
authors.forename: Liza
authors.initials: L
authors.lastname: Pon
authors.forename: Marc
authors.initials: M
authors.lastname: d'Anjou
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Byung-Kwon
authors.initials: BK
authors.lastname: Choi
mesh_terms: D042931:Aldehyde Oxidase / Q000235:genetics*; D061249:Batch Cell Culture Techniques / Q000379:methods; D019149:Bioreactors; D002478:Cells, Cultured; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D000432:Methanol / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D042931:Aldehyde Oxidase; D005947:Glucose; D010100:Oxygen; D000432:Methanol
keywords: AOX1 promoter; ML; OL; Peroxisome biogenesis; Pichia pastoris; Process conditions; SML; methanol-limited at normoxic condition; oxygen-limited condition; q(m); q(p); specific consumption rate of methanol (mgg(−1)h(−1)); specific productivity of a monoclonal antibody (mgg(−1)h(−1)); switched feeding of carbon sources under carbon-limited at normoxy