Biopharmaceutical protein production by<i>Saccharomyces cerevisiae</i>: current state and future prospects

Huang, Mingtao; Bao, Jichen; Nielsen, Jens

doi:10.4155/pbp.14.8

Cited by 43 publications

(43 citation statements)

References 122 publications

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“…Eukaryal cells are often preferred cell factories for production of many pharmaceutical proteins as they can be engineered to secrete functional proteins with correct fold and modifications into the extracellular medium, which results in reduced costs for downstream purification 10 . Mammalian cells, insect cells, filamentous fungi, and yeasts are, therefore, widely used cell factories for production of recombinant proteins 11 .…”

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confidence: 99%

Efficient protein production by yeast requires global tuning of metabolism

et al. 2017

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The biotech industry relies on cell factories for production of pharmaceutical proteins, of which several are among the top-selling medicines. There is, therefore, considerable interest in improving the efficiency of protein production by cell factories. Protein secretion involves numerous intracellular processes with many underlying mechanisms still remaining unclear. Here, we use RNA-seq to study the genome-wide transcriptional response to protein secretion in mutant yeast strains. We find that many cellular processes have to be attuned to support efficient protein secretion. In particular, altered energy metabolism resulting in reduced respiration and increased fermentation, as well as balancing of amino-acid biosynthesis and reduced thiamine biosynthesis seem to be particularly important. We confirm our findings by inverse engineering and physiological characterization and show that by tuning metabolism cells are able to efficiently secrete recombinant proteins. Our findings provide increased understanding of which cellular regulations and pathways are associated with efficient protein secretion.

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Section: Introductionmentioning

confidence: 99%

Efficient protein production by yeast requires global tuning of metabolism

et al. 2017

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“…The key for success in this industry is constant improvement of the protein production capacity of the applied cell factories. Although many organisms can be used as cell factories for protein production, some expression systems, such as Escherichia coli, the yeast Saccharomyces cerevisiae, and Chinese hamster ovary cells, are preferred in consideration of quality, quantity, and cost of produced proteins (3,4).…”

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Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production

Huang

Wang

Qin

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Baker’s yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.

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“…T he yeast Saccharomyces cerevisiae is a widely used cell factory for the production of recombinant proteins (1). The advantages of S. cerevisiae are fast growth, easy cultivation, and ability to perform posttranslational modifications and secrete proteins to the extracellular medium, which facilitates the purification of the protein (2,3).…”

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Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Bao

Huang

Petranović

et al. 2017

Appl Environ Microbiol

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The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous ␣-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased ␣-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous ␣-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-␣-glucosidase, indicating that this mechanism is of general relevance.IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future. KEYWORDS protein secretion, mitochondria, ERES, ROS, SEC16T he yeast Saccharomyces cerevisiae is a widely used cell factory for the production of recombinant proteins (1). The advantages of S. cerevisiae are fast growth, easy cultivation, and ability to perform posttranslational modifications and secrete proteins to the extracellular medium, which facilitates the purification of the protein (2, 3). However, S. cerevisiae naturally secretes only a few proteins, such as aspartyl protease, invertase, and ␣-factor pheromone (4, 5); therefore, it has evolved a secretory pathway with a relatively low capacity. Many different strategies have been used to engineer this pathway with the objective of improving heterologous protein production (6-8). When expressing a protein that has a secretory signal peptide, the protein is cotranslationally translocated into the endoplasmic reticulum (ER) lumen, where disulfide bond formation occurs with the initial glycosylation (9). The secretion of recombinant proteins is affected by their signal peptide sequences (10). A synthetic leader sequence increases human insulin precursor production, whereas the use of the ␣-factor leader sequence increased ␣-amylase production (7). Excessive mannose glycosylation of recombinant proteins can reduce the efficiency of protein secretion, and it was recently found that

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Biopharmaceutical protein production bySaccharomyces cerevisiae: current state and future prospects

Cited by 43 publications

References 122 publications

Efficient protein production by yeast requires global tuning of metabolism

Efficient protein production by yeast requires global tuning of metabolism

Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

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