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

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 (13)C-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.

Purification of cell culture-derived influenza virus A/Puerto Rico/8/34 by membrane-based immobilized metal affinity chromatography.

The presented study focuses on the feasibility of immobilized metal affinity chromatography for purification of Madin Darby canine kidney cell culture-derived influenza virus particles. Therefore, influenza virus A/Puerto Rico/8/34 was screened for adsorption to different transition metal ions attached to iminodiacetic acid. Subsequently, capturing of the same virus strain using zinc-modified iminodiacetic acid membrane adsorbers was characterized regarding viral recoveries, host cell nucleic acid and total protein depletion as well as zinc-ion-leaching. In addition, the effect of the imidazole proton pump on virus stability was studied based on the hemagglutination activity. During adsorption in the presence of 1M sodium chloride the majority of virus particles were recovered in the product (64% hemagglutination activity). Host cell nucleic acid and total protein content were reduced to approximately 7 and 26%, respectively. This inexpensive and rapid method was applied reproducibly for influenza virus A/Puerto Rico/8/34 preparations on the laboratory scale. However, preliminary results with other virus strains indicated clearly a strong strain dependency for viral adsorption.