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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Analysis of steady-state distributions of rates of secretion.(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for a and b are shown. (B) Relationship between a (burst frequency) and b (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.
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pone-0037915-g002: Analysis of steady-state distributions of rates of secretion.(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for a and b are shown. (B) Relationship between a (burst frequency) and b (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.

Mentions: Our experiments showed that the rates of protein secretion by individual cells exhibited significant heterogeneity when transcription was mediated by either pGAPDH or pAOX1. Assuming that the release of secreted proteins from cells is a Poisson process, we modeled the steady-state distributions for all strains (Figure 2A) using a probability density function of the gamma distribution (Eq. 1):(1)where the probability of secreting protein at a given rate (p(x)) depends on parameters a, the average rate of secretion events, and b, the average size of those events (proportional to the number of molecules released) [31]. This analysis for all strains indicated that variations in secretion depend on both the complexity and magnitude of gene expression of a protein (Figure 2B). As gene expression increases for strains using pGAPDH, the frequency of secretion events decreases, but the size of any given burst increases. This inverse relationship between a and b suggests that either the volumes of the vesicles transporting proteins or the total intrinsic capacity of the cell for protein export must be fixed—that is, there is a discrete set of components (membranes, proteins, etc.) available for shuttling protein from the ER through the Golgi apparatus to the cell surface. The export of protein out of the ER appears to be particularly burdened under pAOX1. Fitted distributions describing single-cell secretion rates for pAOX1 strains indicate both burst frequency and burst size both remain low, perhaps indicating inefficient recycling of protein export machinery in the presence of excess protein cargo.


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)

Analysis of steady-state distributions of rates of secretion.(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for a and b are shown. (B) Relationship between a (burst frequency) and b (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369916&req=5

pone-0037915-g002: Analysis of steady-state distributions of rates of secretion.(A) Distributions of rates of secretion of eGFP for (Top) a clone with a single copy of eGFP under transcriptional control of pGAPDH, and (Bottom) a clone with two copies of eGFP under transcriptional control of pAOX1. Red squares indicate binned single-cell secretion events following microengraving with each clone. Blue lines show the best fits using Eq. (1). Values for a and b are shown. (B) Relationship between a (burst frequency) and b (burst size) for proteins expressed using either pGAPDH (top) or pAOX1 (bottom) as a function of gene copy number and complexity. Clones secreting eGFP (green), clones secreting aglycosylated Fc fragment (blue) and clones secreting glycosylated Fc fragment (red) are shown for a single gene copy (squares), 2–3 gene copies (triangles) and 4 or more gene copies (circles). Error bars represent S.E.M. for each clone from at least three separate measurements.
Mentions: Our experiments showed that the rates of protein secretion by individual cells exhibited significant heterogeneity when transcription was mediated by either pGAPDH or pAOX1. Assuming that the release of secreted proteins from cells is a Poisson process, we modeled the steady-state distributions for all strains (Figure 2A) using a probability density function of the gamma distribution (Eq. 1):(1)where the probability of secreting protein at a given rate (p(x)) depends on parameters a, the average rate of secretion events, and b, the average size of those events (proportional to the number of molecules released) [31]. This analysis for all strains indicated that variations in secretion depend on both the complexity and magnitude of gene expression of a protein (Figure 2B). As gene expression increases for strains using pGAPDH, the frequency of secretion events decreases, but the size of any given burst increases. This inverse relationship between a and b suggests that either the volumes of the vesicles transporting proteins or the total intrinsic capacity of the cell for protein export must be fixed—that is, there is a discrete set of components (membranes, proteins, etc.) available for shuttling protein from the ER through the Golgi apparatus to the cell surface. The export of protein out of the ER appears to be particularly burdened under pAOX1. Fitted distributions describing single-cell secretion rates for pAOX1 strains indicate both burst frequency and burst size both remain low, perhaps indicating inefficient recycling of protein export machinery in the presence of excess protein cargo.

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