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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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Simplified carbon metabolic network. Identified enzymes from pathways involved in glycerol and methanol metabolism, from the glycolytic and pentose phosphate pathway, and TCA cycle and their changes in response to the shift from growth on glycerol to growth and HBsAg production on methanol. The red arrow (↑) indicates an increasing and the green arrow (↓) a decreasing amount of the enzyme in the methanol fed-batch phase. An orange dash (−) indicates no significant change. One arrow indicates small (0.6-1 log2 change), two arrows strong (1–4 log2 change) and three arrows very strong changes (> 4 log2 change). Abbreviations (enzymes): AOX1, alcohol oxidase 1; CTA1, catalase; PMP20, peroxiredoxin; FLD1, formaldehyde dehydrogenase; FGH1, S-formylglutathione hydrolase; FDH1, NAD(+)-dependent formate dehydrogenase; ADH, alcohol dehydrogenase; DAS1, transketolase (dihydroxyacetone synthase); TPI1, triose phosphate isomerase; FBA1, fructose 1,6-bisphosphate aldolase; TDH1, glyceraldehyde-3-phosphate dehydrogenase; PGK1, 3-phosphoglycerate kinase; GMP1, tetrameric phosphoglycerate mutase; ENO2, enolase; 6PGD, 6-phosphogluconate dehydrogenase; RKI1, 1-ribose-5-phosphate ketol-isomerase; ACO1, aconitase; IDH2, subunit of mitochondrial NAD(+)-dependent isocitrate dehydrogenase; FUM1, fumarase; MDH1, mitochondrial malate dehydrogenase.
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Figure 2: Simplified carbon metabolic network. Identified enzymes from pathways involved in glycerol and methanol metabolism, from the glycolytic and pentose phosphate pathway, and TCA cycle and their changes in response to the shift from growth on glycerol to growth and HBsAg production on methanol. The red arrow (↑) indicates an increasing and the green arrow (↓) a decreasing amount of the enzyme in the methanol fed-batch phase. An orange dash (−) indicates no significant change. One arrow indicates small (0.6-1 log2 change), two arrows strong (1–4 log2 change) and three arrows very strong changes (> 4 log2 change). Abbreviations (enzymes): AOX1, alcohol oxidase 1; CTA1, catalase; PMP20, peroxiredoxin; FLD1, formaldehyde dehydrogenase; FGH1, S-formylglutathione hydrolase; FDH1, NAD(+)-dependent formate dehydrogenase; ADH, alcohol dehydrogenase; DAS1, transketolase (dihydroxyacetone synthase); TPI1, triose phosphate isomerase; FBA1, fructose 1,6-bisphosphate aldolase; TDH1, glyceraldehyde-3-phosphate dehydrogenase; PGK1, 3-phosphoglycerate kinase; GMP1, tetrameric phosphoglycerate mutase; ENO2, enolase; 6PGD, 6-phosphogluconate dehydrogenase; RKI1, 1-ribose-5-phosphate ketol-isomerase; ACO1, aconitase; IDH2, subunit of mitochondrial NAD(+)-dependent isocitrate dehydrogenase; FUM1, fumarase; MDH1, mitochondrial malate dehydrogenase.

Mentions: In particular, proteins required for methanol utilization which were only present in minor amounts during growth on glycerol started to dominate the proteome after the onset of the methanol feeding phase. Surprisingly, a major protein which accumulated during growth on methanol was identified as alcohol oxidase 1 (AOX1) (Figures 1A-C, 2 and Additional file 2). This HBsAg producing strain was first classified as Muts[4], hence the presence of aox1 and the accumulation of AOX1 unexpected. A comprehensive analysis verified the formation of AOX1 as well as the presence of the intact encoding gene aox1 (for details please refer to Additional file 2). Thus, high level recombinant protein production may lead to impaired growth on methanol despite the presence of functional aox1.


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)

Simplified carbon metabolic network. Identified enzymes from pathways involved in glycerol and methanol metabolism, from the glycolytic and pentose phosphate pathway, and TCA cycle and their changes in response to the shift from growth on glycerol to growth and HBsAg production on methanol. The red arrow (↑) indicates an increasing and the green arrow (↓) a decreasing amount of the enzyme in the methanol fed-batch phase. An orange dash (−) indicates no significant change. One arrow indicates small (0.6-1 log2 change), two arrows strong (1–4 log2 change) and three arrows very strong changes (> 4 log2 change). Abbreviations (enzymes): AOX1, alcohol oxidase 1; CTA1, catalase; PMP20, peroxiredoxin; FLD1, formaldehyde dehydrogenase; FGH1, S-formylglutathione hydrolase; FDH1, NAD(+)-dependent formate dehydrogenase; ADH, alcohol dehydrogenase; DAS1, transketolase (dihydroxyacetone synthase); TPI1, triose phosphate isomerase; FBA1, fructose 1,6-bisphosphate aldolase; TDH1, glyceraldehyde-3-phosphate dehydrogenase; PGK1, 3-phosphoglycerate kinase; GMP1, tetrameric phosphoglycerate mutase; ENO2, enolase; 6PGD, 6-phosphogluconate dehydrogenase; RKI1, 1-ribose-5-phosphate ketol-isomerase; ACO1, aconitase; IDH2, subunit of mitochondrial NAD(+)-dependent isocitrate dehydrogenase; FUM1, fumarase; MDH1, mitochondrial malate dehydrogenase.
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Related In: Results  -  Collection

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Figure 2: Simplified carbon metabolic network. Identified enzymes from pathways involved in glycerol and methanol metabolism, from the glycolytic and pentose phosphate pathway, and TCA cycle and their changes in response to the shift from growth on glycerol to growth and HBsAg production on methanol. The red arrow (↑) indicates an increasing and the green arrow (↓) a decreasing amount of the enzyme in the methanol fed-batch phase. An orange dash (−) indicates no significant change. One arrow indicates small (0.6-1 log2 change), two arrows strong (1–4 log2 change) and three arrows very strong changes (> 4 log2 change). Abbreviations (enzymes): AOX1, alcohol oxidase 1; CTA1, catalase; PMP20, peroxiredoxin; FLD1, formaldehyde dehydrogenase; FGH1, S-formylglutathione hydrolase; FDH1, NAD(+)-dependent formate dehydrogenase; ADH, alcohol dehydrogenase; DAS1, transketolase (dihydroxyacetone synthase); TPI1, triose phosphate isomerase; FBA1, fructose 1,6-bisphosphate aldolase; TDH1, glyceraldehyde-3-phosphate dehydrogenase; PGK1, 3-phosphoglycerate kinase; GMP1, tetrameric phosphoglycerate mutase; ENO2, enolase; 6PGD, 6-phosphogluconate dehydrogenase; RKI1, 1-ribose-5-phosphate ketol-isomerase; ACO1, aconitase; IDH2, subunit of mitochondrial NAD(+)-dependent isocitrate dehydrogenase; FUM1, fumarase; MDH1, mitochondrial malate dehydrogenase.
Mentions: In particular, proteins required for methanol utilization which were only present in minor amounts during growth on glycerol started to dominate the proteome after the onset of the methanol feeding phase. Surprisingly, a major protein which accumulated during growth on methanol was identified as alcohol oxidase 1 (AOX1) (Figures 1A-C, 2 and Additional file 2). This HBsAg producing strain was first classified as Muts[4], hence the presence of aox1 and the accumulation of AOX1 unexpected. A comprehensive analysis verified the formation of AOX1 as well as the presence of the intact encoding gene aox1 (for details please refer to Additional file 2). Thus, high level recombinant protein production may lead to impaired growth on methanol despite the presence of functional aox1.

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