Systematic single-cell analysis of Pichia pastoris reveals secretory capacity limits productivity.
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.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Kerry Routenberg
KR
Love
Timothy J
TJ
Politano
Vasiliki
V
Panagiotou
Bo
B
Jiang
Terrance A
TA
Stadheim
J Christopher
JC
Love
D000465:Algorithms; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055571:Secretory Pathway; D059010:Single-Cell Analysis / Q000379:methods*; D014170:Transformation, Genetic
D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins