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Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter

Paper ID Volume ID Publish Year Pages File Format Full-Text
33847 44993 2009 8 PDF Available
Title
Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter
Abstract

The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding a β-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h−1. It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/Δgas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.

Keywords
Pichia pastoris; Fed-batch cultivation; Formaldehyde dehydrogenase promoter; Rhizopus oryzae lipase; Unfolded protein response; BiP; Hac1; Gas1
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Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter
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Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 25, Issue 6, 15 September 2009, Pages 396–403
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