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Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures

Paper ID Volume ID Publish Year Pages File Format Full-Text
25218 43561 2007 11 PDF Available
Title
Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures
Abstract

In the methylotrophic yeast Pichia pastoris, alcohol oxidase (AOX) is a key enzyme involved in the dissimilation of methanol. Heterologous proteins are usually expressed under the control of the AOX1 promoter, which drives the expression of alcohol oxidase 1 in the wild-type strain. This study investigates the regulation of the alcohol oxidase enzyme of a recombinant P. pastoris Mut+ strain in cultures on glycerol and methanol as sole carbon sources and in mixed substrate cultures on both substrates. The aim was to have a better insight in the transition from growth on glycerol to growth on methanol, which is a key step in standard high cell density P. pastoris cultures for the production of foreign proteins. Nutrient shifts in chemostat cultures showed that after growth on glycerol use of mixed feeds of glycerol and methanol allowed faster induction of alcohol oxidase and faster adaptation of cellular metabolism than with a feed containing methanol as sole carbon source.The results of this study showed also how critical it is to avoid transient methanol accumulation during P. pastoris cultures operated at low residual methanol concentrations. Indeed, pulse experiments during chemostat cultures showed that sudden increase in methanol concentrations in cultures performed under methanol-limited or dual methanol and glycerol-limited growth conditions leads to wash-out of the culture because of too high consumption rate of methanol, which leads to excretion of toxic intermediates. High rate of methanol consumption was due to high specific AOX activities observed at low residual methanol concentrations.

Keywords
Pichia pastoris; Alcohol oxidase; Pulse; Mixed substrate; Calorimetry
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Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 130, Issue 3, 30 June 2007, Pages 236–246
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us