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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Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median kERAD≥ksec (purple), 2) where median ksec≪ksec in Condition 1 (light pink), and 3) where median ksec≪ksec in Condition 1 and median kERAD>kERAD and/or kexp<kexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing tsec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from Figure 4C. The stress-induced population (dark pink) was generated by reducing kexp or decreasing tERAD to obtain a similar median level of protein in the ER as the original population.
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pone-0037915-g005: Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median kERAD≥ksec (purple), 2) where median ksec≪ksec in Condition 1 (light pink), and 3) where median ksec≪ksec in Condition 1 and median kERAD>kERAD and/or kexp<kexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing tsec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from Figure 4C. The stress-induced population (dark pink) was generated by reducing kexp or decreasing tERAD to obtain a similar median level of protein in the ER as the original population.

Mentions: Our data here also indicates that secretion is a binary phenotype for individual cells: eGFP-producing cells co-exist as two distinct populations of cells with one actively secreting and one essentially “off” population (Figure 3). We previously reported that cells can switch dynamically between these two states of secretion [23]. Here, we have proposed a simple model to explain the steady-state distribution of folded proteins trafficking through the ER (Eqs. 2–4). This model provides mechanistic insight into how cells may transition between secreting and non-secreting states. Considering an initial population of secreting cells (purple, Figure 5) similar to those we observed experimentally (where the median rate of ERAD is greater than the median rate of secretion), it is expected that increasing stores of folded protein in the ER leads to the saturation of the available capacity for secretion and a subsequent decline in ksec. If kERAD is constant during this secretory decline, our model suggests that an accumulation of intracellular protein should occur, as incoming folded proteins are neither efficiently secreted from the ER nor degraded (light pink, Figure 5). A concomitant increase in kERAD and/or decrease in kexp is required to reduce residual folded protein and recover a distribution of cells with an intracellular protein level that is similar to that in the secreting populations (dark pink, Figure 5). A consequence of this upregulated activity, however, is that the median rate of secretion also decreases. This outcome is comparable with our experimental results, where the median amount of protein inside the non-secreting populations of pGAPDH or pAOX1 eGFP-producing strains is similar to that found in secreting populations (Figure 3). In fact, non-secreting populations of cells in strains using either promoter contain statistically lower amounts of intracellular eGFP. This observation is consistent with a mechanism in which cells under stress switch to an “off” state of secretion wherein excess folded protein is depleted from the ER and the flux of incoming proteins slows prior to restarting secretion.


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)

Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median kERAD≥ksec (purple), 2) where median ksec≪ksec in Condition 1 (light pink), and 3) where median ksec≪ksec in Condition 1 and median kERAD>kERAD and/or kexp<kexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing tsec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from Figure 4C. The stress-induced population (dark pink) was generated by reducing kexp or decreasing tERAD to obtain a similar median level of protein in the ER as the original population.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369916&req=5

pone-0037915-g005: Effects of altering relative rates of secretion and degradation on modeled distribution of intracellular and secreted protein.Density plot of the relative rates of protein secretion by single cells against the relative amount of intracellular protein for model data sets under three conditions: 1) where median kERAD≥ksec (purple), 2) where median ksec≪ksec in Condition 1 (light pink), and 3) where median ksec≪ksec in Condition 1 and median kERAD>kERAD and/or kexp<kexp in Condition 1 (dark pink). The median amount of intracellular protein for populations in Conditions 1 and 3 are marked (X). The secretion-inhibited population (light pink) was generated by increasing tsec to 400 min (standard deviation 40 min) while keeping all other parameters the same as the initial population (purple) derived from Figure 4C. The stress-induced population (dark pink) was generated by reducing kexp or decreasing tERAD to obtain a similar median level of protein in the ER as the original population.
Mentions: Our data here also indicates that secretion is a binary phenotype for individual cells: eGFP-producing cells co-exist as two distinct populations of cells with one actively secreting and one essentially “off” population (Figure 3). We previously reported that cells can switch dynamically between these two states of secretion [23]. Here, we have proposed a simple model to explain the steady-state distribution of folded proteins trafficking through the ER (Eqs. 2–4). This model provides mechanistic insight into how cells may transition between secreting and non-secreting states. Considering an initial population of secreting cells (purple, Figure 5) similar to those we observed experimentally (where the median rate of ERAD is greater than the median rate of secretion), it is expected that increasing stores of folded protein in the ER leads to the saturation of the available capacity for secretion and a subsequent decline in ksec. If kERAD is constant during this secretory decline, our model suggests that an accumulation of intracellular protein should occur, as incoming folded proteins are neither efficiently secreted from the ER nor degraded (light pink, Figure 5). A concomitant increase in kERAD and/or decrease in kexp is required to reduce residual folded protein and recover a distribution of cells with an intracellular protein level that is similar to that in the secreting populations (dark pink, Figure 5). A consequence of this upregulated activity, however, is that the median rate of secretion also decreases. This outcome is comparable with our experimental results, where the median amount of protein inside the non-secreting populations of pGAPDH or pAOX1 eGFP-producing strains is similar to that found in secreting populations (Figure 3). In fact, non-secreting populations of cells in strains using either promoter contain statistically lower amounts of intracellular eGFP. This observation is consistent with a mechanism in which cells under stress switch to an “off” state of secretion wherein excess folded protein is depleted from the ER and the flux of incoming proteins slows prior to restarting secretion.

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