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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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Vacuole morphology changes in P. pastoris GS115 during methanol-induced high-level production of HBsAg. Time-dependent change in the percentage of cells containing large spherical vacuoles with autophagic bodies (AB, ), cells containing large spherical vacuole without autophagic bodies (○), cells containing irregular vacuoles (▴), cells with small vacuoles (Δ), cells without any visible vacuoles (), and apoptotic cells (□). The bar in the electron micrographs of representative cells corresponds to 500 nm.
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Figure 4: Vacuole morphology changes in P. pastoris GS115 during methanol-induced high-level production of HBsAg. Time-dependent change in the percentage of cells containing large spherical vacuoles with autophagic bodies (AB, ), cells containing large spherical vacuole without autophagic bodies (○), cells containing irregular vacuoles (▴), cells with small vacuoles (Δ), cells without any visible vacuoles (), and apoptotic cells (□). The bar in the electron micrographs of representative cells corresponds to 500 nm.

Mentions: Moreover, a strong increase of the vacuolar aspartyl protease APR1 (PEP4 in S. cerevisiae) was observed in the methanol fed-batch phase (Figure 1A, B, E and Additional file 1) suggesting the induction of vacuolar degradation pathways in addition to ERAD. An electron microscopic examination of the cells revealed a drastic change in vacuole morphology after the start of the methanol fed-batch phase (Figure 4). At the end of the glycerol batch phase, the majority of cells possessed large spherical vacuoles of which many contained auto- phagic bodies. The appearance of spherical vacuoles with autophagic bodies indicate the onset of nutrient limiting conditions and the accompanying recycling of cell material [36]. After the start of methanol feeding, the number of cells with spherical vacuoles declined strongly and, instead, cells with irregularly shaped vacuoles increased in number (Figure 4). A closer electron microscopic examination revealed invagination of vacuoles (Figure 5) as is typically observed during peroxisome degradation by microautophagy (micropexophagy) [37,38]. Micropexophagy requires high levels of ATP [39] most likely available in the methanol fed-batch phase through primarily dissimilatory methanol catabolism. Interestingly, vacuolar enclosure was mainly related to peroxisomes (for details see Figure 5) and not to HBsAg deposits suggesting that vacuolar degradation pathways were not induced by HBsAg accumulation but most likely by damaged peroxisomes. In other cases, activation of autophagic processes have been reported in S. cerevisiae and mammalian cells upon induction of ER stress through addition of reducing agents [40] and tunicamycin and thapsigargin [41], respectively. Moreover, the analysis of the interactome of a degradation-prone and secreted Fab fragment in P. pastoris revealed mainly proteasomal degradation but also degradation via vacuolar pathways [42]. Also, analysis of the effects of producing folded-state stability variants of human lysozyme on the activation of stress responsive pathways revealed a reverse correlation of protein stability versus activation of degradative processes such as ERAD and ER-phagy, apparent through enhanced expression of e.g. sec61 and e.g. atg1, respectively [12]. However, in our case invaginated vacuoles were either closely connected to peroxisomes (Figure 5) or otherwise did not show any clear connection to other organelles (e.g. mitochondria or ER). Autophagy of peroxisomes (pexophagy) has been previously reported for P. pastoris upon shifting from methanol to ethanol or glucose but not during growth on methanol. Our findings suggest that constitutive autophagic recycling of peroxisomes might be part of the house-keeping machinery of Pichia pastoris also under methanol growth conditions helping cells to deal with damage caused by reactive oxygen species created through methanol oxidation. Vacuolar enclosure of peroxisomes was already apparent in the middle of the production phase (Figures 4 and 5D) and increased further during the ongoing methanol fed-batch phase (Figures 4 and 5). Indeed, it has been shown for the methylotrophic yeast Hansenula polymorpha that constitutive pexophagy is vital during growth on methanol as mutant cells with defects in autophagy displayed reduced vitality [43] and damaged peroxisomes are rapidly subjected to autophagic degradation in H. polymorpha[44]. Thus, the strong increase in the vacuolar protease APR1 during the methanol fed-batch phase might not be related to HBsAg production but to vacuolar degradation of damaged peroxisomes.


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)

Vacuole morphology changes in P. pastoris GS115 during methanol-induced high-level production of HBsAg. Time-dependent change in the percentage of cells containing large spherical vacuoles with autophagic bodies (AB, ), cells containing large spherical vacuole without autophagic bodies (○), cells containing irregular vacuoles (▴), cells with small vacuoles (Δ), cells without any visible vacuoles (), and apoptotic cells (□). The bar in the electron micrographs of representative cells corresponds to 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Vacuole morphology changes in P. pastoris GS115 during methanol-induced high-level production of HBsAg. Time-dependent change in the percentage of cells containing large spherical vacuoles with autophagic bodies (AB, ), cells containing large spherical vacuole without autophagic bodies (○), cells containing irregular vacuoles (▴), cells with small vacuoles (Δ), cells without any visible vacuoles (), and apoptotic cells (□). The bar in the electron micrographs of representative cells corresponds to 500 nm.
Mentions: Moreover, a strong increase of the vacuolar aspartyl protease APR1 (PEP4 in S. cerevisiae) was observed in the methanol fed-batch phase (Figure 1A, B, E and Additional file 1) suggesting the induction of vacuolar degradation pathways in addition to ERAD. An electron microscopic examination of the cells revealed a drastic change in vacuole morphology after the start of the methanol fed-batch phase (Figure 4). At the end of the glycerol batch phase, the majority of cells possessed large spherical vacuoles of which many contained auto- phagic bodies. The appearance of spherical vacuoles with autophagic bodies indicate the onset of nutrient limiting conditions and the accompanying recycling of cell material [36]. After the start of methanol feeding, the number of cells with spherical vacuoles declined strongly and, instead, cells with irregularly shaped vacuoles increased in number (Figure 4). A closer electron microscopic examination revealed invagination of vacuoles (Figure 5) as is typically observed during peroxisome degradation by microautophagy (micropexophagy) [37,38]. Micropexophagy requires high levels of ATP [39] most likely available in the methanol fed-batch phase through primarily dissimilatory methanol catabolism. Interestingly, vacuolar enclosure was mainly related to peroxisomes (for details see Figure 5) and not to HBsAg deposits suggesting that vacuolar degradation pathways were not induced by HBsAg accumulation but most likely by damaged peroxisomes. In other cases, activation of autophagic processes have been reported in S. cerevisiae and mammalian cells upon induction of ER stress through addition of reducing agents [40] and tunicamycin and thapsigargin [41], respectively. Moreover, the analysis of the interactome of a degradation-prone and secreted Fab fragment in P. pastoris revealed mainly proteasomal degradation but also degradation via vacuolar pathways [42]. Also, analysis of the effects of producing folded-state stability variants of human lysozyme on the activation of stress responsive pathways revealed a reverse correlation of protein stability versus activation of degradative processes such as ERAD and ER-phagy, apparent through enhanced expression of e.g. sec61 and e.g. atg1, respectively [12]. However, in our case invaginated vacuoles were either closely connected to peroxisomes (Figure 5) or otherwise did not show any clear connection to other organelles (e.g. mitochondria or ER). Autophagy of peroxisomes (pexophagy) has been previously reported for P. pastoris upon shifting from methanol to ethanol or glucose but not during growth on methanol. Our findings suggest that constitutive autophagic recycling of peroxisomes might be part of the house-keeping machinery of Pichia pastoris also under methanol growth conditions helping cells to deal with damage caused by reactive oxygen species created through methanol oxidation. Vacuolar enclosure of peroxisomes was already apparent in the middle of the production phase (Figures 4 and 5D) and increased further during the ongoing methanol fed-batch phase (Figures 4 and 5). Indeed, it has been shown for the methylotrophic yeast Hansenula polymorpha that constitutive pexophagy is vital during growth on methanol as mutant cells with defects in autophagy displayed reduced vitality [43] and damaged peroxisomes are rapidly subjected to autophagic degradation in H. polymorpha[44]. Thus, the strong increase in the vacuolar protease APR1 during the methanol fed-batch phase might not be related to HBsAg production but to vacuolar degradation of damaged peroxisomes.

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