Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: Catabolic adaptation, stress responses, and autophagic processes
Creators: name: Vanz, Ana Leticia
name: Lünsdorf, Heinrich
name: Adnan, Ahmad
name: Nimtz, Manfred
name: Gurramkonda, Chandrasekhar
name: Khanna, Navin
name: Rinas, Ursula
Publisher: name: London : BioMed Central Ltd.
Year: 2012
Subjects: subject: Aox1 promoter
subject: Autophagy
subject: Carbon metabolism
subject: ER stress
subject: Pichia pastoris
subject: Proteome
subject: alcohol oxidase
subject: glycerol
subject: hepatitis B surface antigen
subject: methanol
subject: peroxiredoxin
subject: reactive oxygen metabolite
subject: alcohol metabolism
subject: alcohol oxidation
subject: article
subject: autophagy
subject: catabolism
subject: cell count
subject: cell size
subject: cell structure
subject: cell vacuole
subject: endoplasmic reticulum
subject: endoplasmic reticulum associated degradation
subject: fed batch culture
subject: fungus growth
subject: mitochondrion
subject: nonhuman
subject: oxidative stress
subject: protein degradation
subject: protein unfolding
subject: upregulation
subject: Endoplasmic Reticulum-Associated Degradation
subject: Fungal Proteins
subject: Glycerol
subject: Hepatitis B Surface Antigens
subject: Methanol
subject: Molecular Chaperones
subject: Oxidative Stress
subject: Peroxiredoxins
subject: Pichia
subject: Proteasome Endopeptidase Complex
subject: Protein Disulfide-Isomerases
subject: Reactive Oxygen Species
subject: Recombinant Proteins
subject: Unfolded Protein Response
subject: Vacuoles
subject: Eukaryota
subject: Hepatitis B virus
subject: Dewey Decimal Classification::500 | Naturwissenschaften
subject: Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften, Biologie

description: Background: Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process.Results: The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation.Conclusions: In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.