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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Single-cell analysis of P. pastoris secreting heterologous proteins.(A) Schematic illustration of process for measuring the distributions in rates of secretion of heterologous proteins by single cells. Yeast cells cultivated by shake-flask fermentation for ∼12–24 h at 25°C were deposited onto an array of microwells at a density of ∼1 cell per well. Microengraving was performed to create a protein microarray comprising the secreted proteins captured from occupants of each individual well. Imaging cytometry was performed to determine number of cells per well. Integration of the data yielded distributions in rates of secretion for single cells; the distributions are represented as heatmaps where the gradient in color (blue to yellow to red) indicates the relative percentage of cells producing at a specific rate. (B) and (C) Heatmap representations of the distributions of single-cell rates of secretion as a function of gene copy number obtained by microengraving for proteins produced using either (B) pGAPDH or (C) pAOX1 promoter. Data shown are representative of at least three independent measurements. The threshold for secretion was determined by the background median fluorescence intensity of each individual protein microarray+2σ. Pie charts indicate average percentages of secreting cells for each strain (red).
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pone-0037915-g001: Single-cell analysis of P. pastoris secreting heterologous proteins.(A) Schematic illustration of process for measuring the distributions in rates of secretion of heterologous proteins by single cells. Yeast cells cultivated by shake-flask fermentation for ∼12–24 h at 25°C were deposited onto an array of microwells at a density of ∼1 cell per well. Microengraving was performed to create a protein microarray comprising the secreted proteins captured from occupants of each individual well. Imaging cytometry was performed to determine number of cells per well. Integration of the data yielded distributions in rates of secretion for single cells; the distributions are represented as heatmaps where the gradient in color (blue to yellow to red) indicates the relative percentage of cells producing at a specific rate. (B) and (C) Heatmap representations of the distributions of single-cell rates of secretion as a function of gene copy number obtained by microengraving for proteins produced using either (B) pGAPDH or (C) pAOX1 promoter. Data shown are representative of at least three independent measurements. The threshold for secretion was determined by the background median fluorescence intensity of each individual protein microarray+2σ. Pie charts indicate average percentages of secreting cells for each strain (red).

Mentions: We then used microengraving to monitor secretions quantitatively from thousands of individual cells within each strain (Figure 1A). Microengraving is a soft-lithographic technique for the high-throughput analysis of secreted products from single cells [26], [27], including P. pastoris[23]. The method reveals the percentage of secreting cells (similar to an enzyme-linked immunospot assay), as well as a measure of the average rate of secretion. Single-cell analysis of strains secreting proteins under transcriptional control of a constitutive promoter, pGAPDH, following shake-flask cultivation showed that protein complexity modestly affected the rate of protein secretion, but not the percentage of secreting cells within the population (Figure 1B). Under this promoter, single cells secreted eGFP slightly faster (1.4±0.2 ng*mL−1*h−1 average median rate for the single-copy strain) than either the glycosylated (0.9±0.3 ng*mL−1*h−1) or aglycosylated Fc fragment (0.7±0.2 ng*mL−1*h−1), which were secreted at similar rates. Single-cell rates of secretion increased linearly (R2 = 0.83) with gene expression for all strains assayed (Figure S1), indicating that when proteins are produced using a constitutive promoter, the cellular capacity for secretion does not fully saturate, as expected [28].


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)

Single-cell analysis of P. pastoris secreting heterologous proteins.(A) Schematic illustration of process for measuring the distributions in rates of secretion of heterologous proteins by single cells. Yeast cells cultivated by shake-flask fermentation for ∼12–24 h at 25°C were deposited onto an array of microwells at a density of ∼1 cell per well. Microengraving was performed to create a protein microarray comprising the secreted proteins captured from occupants of each individual well. Imaging cytometry was performed to determine number of cells per well. Integration of the data yielded distributions in rates of secretion for single cells; the distributions are represented as heatmaps where the gradient in color (blue to yellow to red) indicates the relative percentage of cells producing at a specific rate. (B) and (C) Heatmap representations of the distributions of single-cell rates of secretion as a function of gene copy number obtained by microengraving for proteins produced using either (B) pGAPDH or (C) pAOX1 promoter. Data shown are representative of at least three independent measurements. The threshold for secretion was determined by the background median fluorescence intensity of each individual protein microarray+2σ. Pie charts indicate average percentages of secreting cells for each strain (red).
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Related In: Results  -  Collection

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pone-0037915-g001: Single-cell analysis of P. pastoris secreting heterologous proteins.(A) Schematic illustration of process for measuring the distributions in rates of secretion of heterologous proteins by single cells. Yeast cells cultivated by shake-flask fermentation for ∼12–24 h at 25°C were deposited onto an array of microwells at a density of ∼1 cell per well. Microengraving was performed to create a protein microarray comprising the secreted proteins captured from occupants of each individual well. Imaging cytometry was performed to determine number of cells per well. Integration of the data yielded distributions in rates of secretion for single cells; the distributions are represented as heatmaps where the gradient in color (blue to yellow to red) indicates the relative percentage of cells producing at a specific rate. (B) and (C) Heatmap representations of the distributions of single-cell rates of secretion as a function of gene copy number obtained by microengraving for proteins produced using either (B) pGAPDH or (C) pAOX1 promoter. Data shown are representative of at least three independent measurements. The threshold for secretion was determined by the background median fluorescence intensity of each individual protein microarray+2σ. Pie charts indicate average percentages of secreting cells for each strain (red).
Mentions: We then used microengraving to monitor secretions quantitatively from thousands of individual cells within each strain (Figure 1A). Microengraving is a soft-lithographic technique for the high-throughput analysis of secreted products from single cells [26], [27], including P. pastoris[23]. The method reveals the percentage of secreting cells (similar to an enzyme-linked immunospot assay), as well as a measure of the average rate of secretion. Single-cell analysis of strains secreting proteins under transcriptional control of a constitutive promoter, pGAPDH, following shake-flask cultivation showed that protein complexity modestly affected the rate of protein secretion, but not the percentage of secreting cells within the population (Figure 1B). Under this promoter, single cells secreted eGFP slightly faster (1.4±0.2 ng*mL−1*h−1 average median rate for the single-copy strain) than either the glycosylated (0.9±0.3 ng*mL−1*h−1) or aglycosylated Fc fragment (0.7±0.2 ng*mL−1*h−1), which were secreted at similar rates. Single-cell rates of secretion increased linearly (R2 = 0.83) with gene expression for all strains assayed (Figure S1), indicating that when proteins are produced using a constitutive promoter, the cellular capacity for secretion does not fully saturate, as expected [28].

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