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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.

Vanz AL, Lünsdorf H, Adnan A, Nimtz M, Gurramkonda C, Khanna N, Rinas U - Microb. Cell Fact. (2012)

Bottom Line: However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Technical Chemistry-Life Science, Leibniz University of Hannover, Hannover, Germany.

ABSTRACT

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.

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Mitochondrial morphology and size changes during methanol-induced high-level production of the Hepatitis B surface antigen. Transmission electron micrographs of P. pastoris GS115 (A) at the end of the glycerol batch phase and (B) 112 hours after the onset of methanol feeding. White arrow heads indicate mitochondria. (C) Quantification of the average total mitochondrial area within the cross sectional average cell area. The vertical bars encompass individual cell variance, e.g. the lowest and highest total mitochondrial area observed in a single cell in each growth phase; (left column) at the end of the glycerol batch phase and (right column) 112 hours after the onset of methanol feeding.
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Figure 3: Mitochondrial morphology and size changes during methanol-induced high-level production of the Hepatitis B surface antigen. Transmission electron micrographs of P. pastoris GS115 (A) at the end of the glycerol batch phase and (B) 112 hours after the onset of methanol feeding. White arrow heads indicate mitochondria. (C) Quantification of the average total mitochondrial area within the cross sectional average cell area. The vertical bars encompass individual cell variance, e.g. the lowest and highest total mitochondrial area observed in a single cell in each growth phase; (left column) at the end of the glycerol batch phase and (right column) 112 hours after the onset of methanol feeding.

Mentions: Interestingly, AOX1 already accumulated to high levels during the adapation phase to methanol (Phase II), while the subsequent enzymes of the methanol dissimilation pathway reached their highest concentrations in the production phase (Phase III) or even later in the decline phase (Phase IV) (Figure 1A-C). Methanol is not only oxidized by Pichia pastoris in the dissimilation pathway for potential energy generation through reoxidation of NADH in the respiratory chain but can also be incorporated into biomass in the assimilation pathway. In this case, formaldehyde, which is formed through oxidation of methanol by AOX1 (or AOX2), is not further converted to carbon dioxide, but condensed with xylulose 5-phosphate (Xu5P), through the action of dihydroxyacetone synthase (DAS1), a special transketolase which converts formaldehyde and Xu5P into the central C3-compounds dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (GAP) [16] (Figure 2). In contrast to the strong increase of the enzymes from the methanol dissimilation pathway, the level of DAS1 increased only moderately during the production phase (Figures 1A, B and 2) suggesting that the majority of methanol is processed through the dissimilation pathway leading to the generation of NADH and carbon dioxide. In fact, a carbon mass balance analysis revealed that 70 – 80% of the methanol metabolized is converted into carbon dioxide in the methanol fed-batch phase (data not shown). Interestingly, the mitochondrial area within cellular cross sections increased significantly during the methanol fed-batch phase (Figure 3) probably as a result of the elevated NADH supply from methanol dissimilation and the enhanced energy demand for growth, production and cell maintenance (e.g. repair and recycling).


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.

Vanz AL, Lünsdorf H, Adnan A, Nimtz M, Gurramkonda C, Khanna N, Rinas U - Microb. Cell Fact. (2012)

Mitochondrial morphology and size changes during methanol-induced high-level production of the Hepatitis B surface antigen. Transmission electron micrographs of P. pastoris GS115 (A) at the end of the glycerol batch phase and (B) 112 hours after the onset of methanol feeding. White arrow heads indicate mitochondria. (C) Quantification of the average total mitochondrial area within the cross sectional average cell area. The vertical bars encompass individual cell variance, e.g. the lowest and highest total mitochondrial area observed in a single cell in each growth phase; (left column) at the end of the glycerol batch phase and (right column) 112 hours after the onset of methanol feeding.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539919&req=5

Figure 3: Mitochondrial morphology and size changes during methanol-induced high-level production of the Hepatitis B surface antigen. Transmission electron micrographs of P. pastoris GS115 (A) at the end of the glycerol batch phase and (B) 112 hours after the onset of methanol feeding. White arrow heads indicate mitochondria. (C) Quantification of the average total mitochondrial area within the cross sectional average cell area. The vertical bars encompass individual cell variance, e.g. the lowest and highest total mitochondrial area observed in a single cell in each growth phase; (left column) at the end of the glycerol batch phase and (right column) 112 hours after the onset of methanol feeding.
Mentions: Interestingly, AOX1 already accumulated to high levels during the adapation phase to methanol (Phase II), while the subsequent enzymes of the methanol dissimilation pathway reached their highest concentrations in the production phase (Phase III) or even later in the decline phase (Phase IV) (Figure 1A-C). Methanol is not only oxidized by Pichia pastoris in the dissimilation pathway for potential energy generation through reoxidation of NADH in the respiratory chain but can also be incorporated into biomass in the assimilation pathway. In this case, formaldehyde, which is formed through oxidation of methanol by AOX1 (or AOX2), is not further converted to carbon dioxide, but condensed with xylulose 5-phosphate (Xu5P), through the action of dihydroxyacetone synthase (DAS1), a special transketolase which converts formaldehyde and Xu5P into the central C3-compounds dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (GAP) [16] (Figure 2). In contrast to the strong increase of the enzymes from the methanol dissimilation pathway, the level of DAS1 increased only moderately during the production phase (Figures 1A, B and 2) suggesting that the majority of methanol is processed through the dissimilation pathway leading to the generation of NADH and carbon dioxide. In fact, a carbon mass balance analysis revealed that 70 – 80% of the methanol metabolized is converted into carbon dioxide in the methanol fed-batch phase (data not shown). Interestingly, the mitochondrial area within cellular cross sections increased significantly during the methanol fed-batch phase (Figure 3) probably as a result of the elevated NADH supply from methanol dissimilation and the enhanced energy demand for growth, production and cell maintenance (e.g. repair and recycling).

Bottom Line: However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Technical Chemistry-Life Science, Leibniz University of Hannover, Hannover, Germany.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus