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

David Resina, Michael Maurer, Oriol Cos, Carolina Arnau, Marc Carnicer, Hans Marx, Brigitte Gasser, Francisco Valero, Diethard Mattanovich, Pau Ferrer

Research output: Indexed journal article Articlepeer-review

48 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)396-403
Number of pages8
JournalNew Biotechnology
Volume25
Issue number6
DOIs
Publication statusPublished - 15 Sept 2009
Externally publishedYes

Keywords

  • BiP
  • Fed-batch cultivation
  • Formaldehyde dehydrogenase promoter
  • Gas1
  • Hac1
  • Pichia pastoris
  • Rhizopus oryzae lipase
  • Unfolded protein response

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