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Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
23490 43443 2013 8 PDF Available
Title
Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris
Abstract

•We characterized the AOX1 promoter and peroxisome biogenesis in different fermentation process conditions.•The activity of AOX1 promoter and peroxisome biogenesis was highly subject to the availability of methanol and oxygen molecules.•The AOX1 promoter was most active under the methanol-limited condition (ML).•Peroxisome biogenesis was highly dependent on methanol consumption.

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 86 h of induction in the ML than in the OL (0.026 vs 0.020 mg g−1 h−1). However, the oxygen-limited condition was a robust process suitable for longer induction (180 h) 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.

Keywords
ML, methanol-limited at normoxic condition; SML, switched feeding of carbon sources under carbon-limited at normoxy; OL, oxygen-limited condition; qp, specific productivity of a monoclonal antibody (mg g−1 h−1); qm, specific consumption rate of methanol (
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Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 166, Issue 4, 20 July 2013, Pages 174–181
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering