Limits...
Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

Love KR, Politano TJ, Panagiotou V, Jiang B, Stadheim TA, Love JC - PLoS ONE (2012)

Bottom Line: Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly.We then developed a simple mathematical model describing the flux of folded protein through the ER.This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

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Related in: MedlinePlus

Characterization of relationships between intracellular and secreted proteins for single cells of P. pastoris.Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.
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pone-0037915-g003: Characterization of relationships between intracellular and secreted proteins for single cells of P. pastoris.Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.

Mentions: Strains producing eGFP under either pGAPDH or pAOX1 showed that nearly all cells produced folded protein, but only a subset was secreting it. Further comparison of the relative rates of secretion of eGFP to intracellular quantities in individual cells showed two distinct, co-existing subpopulations: one that secretes eGFP at detectable levels and another that is non-secreting (Figure 3A and 3B; File S1). Interestingly, both populations have similar amounts of folded intracellular eGFP, with the secreting population containing slightly more protein (p<0.001). The population that actively secreted protein did so at a rate moderately proportional to the amount of intracellular eGFP (pGAPDH, r = 0.18; pAOX1, r = 0.06). Induction of UPR by treating cells with the strong reducing agent dithiothreitol (DTT), known to specifically affect protein folding within the secretory pathway [32], led to a six-fold reduction in the median rate of eGFP secretion (data not shown). Nonetheless, the correlation between accumulation of folded protein and secretion in the actively secreting population was maintained (pGAPDH, r = 0.12). Furthermore, the non-secreting populations of both the untreated and DTT-treated cultures had similar amounts of intracellular eGFP, indicating that the UPR itself does not lead to an excess accumulation of folded protein inside the ER. The proportionality between intracellular levels of eGFP and rates of secretion—even under conditions of induced UPR—suggest that the flux of folded proteins out of the ER, and subsequently through the secretory pathway, is a rate-limiting process for productive export.


Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.

Love KR, Politano TJ, Panagiotou V, Jiang B, Stadheim TA, Love JC - PLoS ONE (2012)

Characterization of relationships between intracellular and secreted proteins for single cells of P. pastoris.Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369916&req=5

pone-0037915-g003: Characterization of relationships between intracellular and secreted proteins for single cells of P. pastoris.Density plots of the relative rates of eGFP secretion by single cells analyzed by microengraving with respect to the relative amount of intracellular eGFP determined by fluorescence microscopy for clones containing (A) one eGFP gene copy under pGAPDH or (B) two eGFP gene copies under pAOX1. Dashed line indicates the limit of detection for secreted eGFP in microengraving (background+2σ). The median amounts of internal eGFP for cells above and below this limit of detection are marked (X) and are significantly different (Mann-Whitney test, p≪0.001 for both pGAPDH and pAOX strains). (C) Density plot of the relative rates of secretion analyzed by microengraving for the glycosylated Fc fragment and eGFP produced simultaneously in single cells at two different loci using pGAPDH. Pearson's correlation coefficient for secretion of these two proteins as shown is 0.79.
Mentions: Strains producing eGFP under either pGAPDH or pAOX1 showed that nearly all cells produced folded protein, but only a subset was secreting it. Further comparison of the relative rates of secretion of eGFP to intracellular quantities in individual cells showed two distinct, co-existing subpopulations: one that secretes eGFP at detectable levels and another that is non-secreting (Figure 3A and 3B; File S1). Interestingly, both populations have similar amounts of folded intracellular eGFP, with the secreting population containing slightly more protein (p<0.001). The population that actively secreted protein did so at a rate moderately proportional to the amount of intracellular eGFP (pGAPDH, r = 0.18; pAOX1, r = 0.06). Induction of UPR by treating cells with the strong reducing agent dithiothreitol (DTT), known to specifically affect protein folding within the secretory pathway [32], led to a six-fold reduction in the median rate of eGFP secretion (data not shown). Nonetheless, the correlation between accumulation of folded protein and secretion in the actively secreting population was maintained (pGAPDH, r = 0.12). Furthermore, the non-secreting populations of both the untreated and DTT-treated cultures had similar amounts of intracellular eGFP, indicating that the UPR itself does not lead to an excess accumulation of folded protein inside the ER. The proportionality between intracellular levels of eGFP and rates of secretion—even under conditions of induced UPR—suggest that the flux of folded proteins out of the ER, and subsequently through the secretory pathway, is a rate-limiting process for productive export.

Bottom Line: Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly.We then developed a simple mathematical model describing the flux of folded protein through the ER.This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
Biopharmaceuticals represent the fastest growing sector of the global pharmaceutical industry. Cost-efficient production of these biologic drugs requires a robust host organism for generating high titers of protein during fermentation. Understanding key cellular processes that limit protein production and secretion is, therefore, essential for rational strain engineering. Here, with single-cell resolution, we systematically analysed the productivity of a series of Pichia pastoris strains that produce different proteins both constitutively and inducibly. We characterized each strain by qPCR, RT-qPCR, microengraving, and imaging cytometry. We then developed a simple mathematical model describing the flux of folded protein through the ER. This combination of single-cell measurements and computational modelling shows that protein trafficking through the secretory machinery is often the rate-limiting step in single-cell production, and strategies to enhance the overall capacity of protein secretion within hosts for the production of heterologous proteins may improve productivity.

Show MeSH
Related in: MedlinePlus