Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

Robinson, Anne S.; Bockhaus, Julie A.; Voegler, Anne C.; Wittrup, K. Dane

doi:10.1074/jbc.271.17.10017

Cited by 89 publications

(65 citation statements)

References 56 publications

(81 reference statements)

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“…Overexpression of chaperones that increase folding capacity has successfully been used to increase protein secretion [6,48]. For particularly large or difficult to fold proteins this may not be adequate.…”

Section: Discussionmentioning

confidence: 99%

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

et al. 2012

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BackgroundThe protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).ResultsHAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.ConclusionsApparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

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Section: Discussionmentioning

confidence: 99%

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

et al. 2012

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“…Although more useful in yeast basic research than in industrial applications, the activity of an inducible promoter can be fine-tuned by modifying its regulatory properties or accurately titrating its inducer (143,295). For example, gene expression driven by the GAL1 promoter has been modulated by several promoter deletion variants (232) or by finely controlling the concentration of the repressor/inducer molecule galactose (150).…”

Section: Fine-tuning Gene Expressionmentioning

confidence: 99%

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Nevoigt

2008

Microbiol Mol Biol Rev

526

333

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SUMMARY The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

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“…In other expression systems, this approach has in some cases been successful (19,41), although examples can also be found where overexpression of chaperones and/or foldases had no or a negative effect on protein secretion (8,13,21,35). Overexpression of chaperones or foldases in filamentous fungi has so far failed to increase the extracellular levels of the heterologous proteins analyzed (11,28,33,48).…”

mentioning

confidence: 99%

Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

Conesa

Jeenes

Archer

et al. 2002

Appl Environ Microbiol

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Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four-to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.

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Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

Cited by 89 publications

References 56 publications

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

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