Increasing pentose phosphate pathway flux enhances recombinant protein production in Pichia pastoris.

Justyna Nocon,Hannes Rußmayer,Brigitte Gasser,Teresa Mairinger,Matthias Steiger,Diethard Mattanovich,Jonas Hohlweg,Stephan Hann

doi:10.1007/s00253-016-7363-5

Abstract

Production of heterologous proteins in Pichia pastoris (syn. Komagataella sp.) has been shown to exert a metabolic burden on the host metabolism. This burden is associated with metabolite drain, which redirects nucleotides and amino acids from primary metabolism. On the other hand, recombinant protein production affects energy and redox homeostasis of the host cell. In a previous study, we have demonstrated that overexpression of single genes of the oxidative pentose phosphate pathway (PPP) had a positive influence on recombinant production of cytosolic human superoxide dismutase (hSOD). In this study, different combinations of these genes belonging to the oxidative PPP were generated and analyzed. Thereby, a 3.8-fold increase of hSOD production was detected when glucose-6-phosphate dehydrogenase (ZWF1) and 6-gluconolactonase (SOL3) were simultaneously overexpressed, while the combinations of other genes from PPP had no positive effect on protein production. By measuring isotopologue patterns of 13C-labelled metabolites, we could detect an upshift in the flux ratio of PPP to glycolysis upon ZWF1 and SOL3 co-overexpression, as well as increased levels of 6-phosphogluconate. The substantial improvement of hSOD production by ZWF1 and SOL3 co-overexpression appeared to be connected to an increase in PPP flux. In conclusion, we show that overexpression of SOL3 together with ZWF1 enhanced both the PPP flux ratio and hSOD accumulation, providing evidence that in P. pastoris Sol3 limits the flux through PPP and recombinant protein production.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7363-5) contains supplementary material, which is available to authorized users.

Highlights

Production of heterologous proteins has long been shown to exert a metabolic burden on the producing host cell (Bentley et al 1990; Glick 1995)
We showed recently that in P. pastoris ZWF1 is upregulated while SOL3 expression is not changed in glucose-limited conditions or on methanol media (Prielhofer et al 2015), both conditions that lead to an increase of phosphate pathway (PPP) flux (Baumann et al 2010; Russmayer et al 2015a)
Based on predictions from a genome scale metabolic model, we demonstrated recently that the overexpression of single genes of the pentose phosphate pathway can have a positive effect on production of recombinant proteins (Nocon et al 2014)

Summary

Introduction

Production of heterologous proteins has long been shown to exert a metabolic burden on the producing host cell (Bentley et al 1990; Glick 1995). This phenomenon has been observed in all main classes of host organisms (bacteria, yeasts, mammalian cells), typically leading to a reduction of the maximum specific growth rate and of biomass yield. It is part of a stress reaction to protein overproduction (for a review see Mattanovich et al (2004)). Decreased biomass production in protein producing P. pastoris strains has often been observed (e.g., (Dragosits et al 2009; Heyland et al 2010; Jorda et al 2012)

Methods

Results

Conclusion